# Patent

## Title
DNA molecules encoding beta clamp proteins of gram positive bacteria

## Abstract

The present invention relates to beta protein-encoding genes from Gram positive bacterium. Expression systems and host cells containing these genes are also disclosed.

## Background
The present application claims benefit of U.S. Provisional Patent application Ser. No. 60/146,178 filed Jul. 29, 1999, which is hereby incorporated by reference.


The present invention was made with funding from National Institutes of Health Grant No. GM38839. The United States Government may have certain rights in this invention.


FIELD OF THE INVENTION
This invention relates to genes and proteins that replicate the chromosome of Gram positive bacteria. These proteins can be used in sequencing, amplification of DNA, and in drug discovery to screen large libraries of chemicals for identification of compounds with antibiotic activity.
BACKGROUND OF THE INVENTION
All forms of life must duplicate the genetic material to propagate the species. The process by which the DNA in a chromosome is duplicated is called replication. The replication process is performed by numerous proteins that coordinate their actions to duplicate the DNA smoothly. The main protein actors are as follows (reviewed in Kornberg et al., DNA Replication, Second Edition, New York: W.H. Freeman and Company, pp. 165-194 (1992)). A helicase uses the energy of ATP hydrolysis to unwind the two DNA strands of the double helix. Two copies of the DNA polymerase use each “daughter” strand as a template to convert them into two new duplexes. The DNA polymerase acts by polymerizing the four monomer unit building blocks of DNA (the 4 dNTPs, or deoxynucleoside triphosphates are: dATP, dCTP, dGTP, dTTP). The polymerase rides along one strand of DNA using it as a template that dictates the sequence in which the monomer blocks are to be polymerized. Sometimes the DNA polymerase makes a mistake and includes an incorrect nucleotide (e.g., A instead of G). A proofreading exonuclease examines the polymer as it is made and excises building blocks that have been improperly inserted in the polymer.
Duplex DNA is composed of two strands that are oriented antiparallel to one another, one being oriented 3′-5′ and the other 5′ to 3′. As the helicase unwinds the duplex, the DNA polymerase moves continuously forward with the helicase on one strand (called the leading strand). However, due to the fact that DNA polymerases can only extend the DNA forward from a 3′ terminus, the polymerase on the other strand extends DNA in the opposite direction of DNA unwinding (called the lagging strand). This necessitates a discontinuous ratcheting motion on the lagging strand in which the DNA is made as a series of Okazaki fragments. DNA polymerases cannot initiate DNA synthesis de novo, but require a primed site (i.e., a short duplex region). This job is fulfilled by primase, a specialized RNA polymerase, that synthesizes short RNA primers on the lagging strand. The primed sites are extended by DNA polymerase. A single-stranded DNA binding protein (“SSB”) is also needed; it operates on the lagging strand. The function of SSB is to coat single stranded DNA (“ssDNA”), thereby melting short hairpin duplexes that would otherwise impede DNA synthesis by DNA polymerase.
The replication process is best understood for the Gram negative bacterium Escherichia coli and its bacteriophages T4 and T7 (reviewed in Kelman et al., “DNA Polymerase III Holoenzyme: Structure and Function of Chromosomal Replicating Machine,” Annu. Rev. Biochem., 64:171-200 (1995); Marians, K. J., “Prokaryotic DNA Replication,” Annu. Rev. Biochem., 61:673-719 (1992); McHenry, C. S., “DNA Polymerase III Holoenzyme: Components, Structure, and Mechanism of a True Replicative Complex,” J. Bio. Chem., 266:19127-19130 (1991); Young et al., “Structure and Function of the Bacteriophage T4 DNA Polymerase Holoenzyme,” Am. Chem. Soc., 31:8675-8690 (1992)). The eukaryotic systems of yeast (Saccharomyces cerevisae) (Morrison et al., “A Third Essential DNA Polymerase in S. cerevisiae,” Cell, 62:1143-51 (1990) and humans (Bambara et al., “Reconstitution of Mammalian DNA Replication, “Prog. Nuc. Acid Res.,” 51:93-123 (1995)) have also been characterized in some detail as has herpes virus (Boehmer et al., “Herpes Simplex Virus DNA Replication,” Annu. Rev. Biochem., 66:347-384 (1997)) and vaccinia virus (McDonald et al., “Characterization of a Processive Form of the Vaccinia Virus DNA Polymerase,” Virology, 234:168-175 (1997)). The helicase of E. coli is encoded by the dnaB gene and is called the DnaB-helicase. In phage T4, the helicase is the product of the gene 41, and, in T7, it is the product of gene 4. Generally, the helicase contacts the DNA polymerase in E. coli. This contact is necessary for the helicase to achieve the catalytic efficiency needed to replicate a chromosome (Kim et al., “Coupling of a Replicative Polymerase and Helicase: A tau-DnaB Interaction Mediates Rapid Replication Fork Movement,” Cell, 84:643-650 (1996)). The identity of the helicase that acts at the replication fork in a eukaryotic cellular system is still not firm.
The primase of E. coli (product of the dnaG gene), phage T4 (product of gene 61), and T7 (gene 4) require the presence of their cognate helicase for activity. The primase of eukaryotes, called DNA polymerase alpha, looks and behaves differently. DNA polymerase alpha is composed of 4 subunits. The primase activity is associated with the two smaller subunits, and the largest subunit is the DNA polymerase which extends the product of the priming subunits. DNA polymerase alpha does not need a helicase for priming activity on single strand DNA that is not coated with binding protein.
The chromosomal replicating DNA polymerase of all these systems, prokaryotic and eukaryotic, share the feature that they are processive, meaning they remain continuously associated with the DNA template as they link monomer units (dNTPs) together. This catalytic efficiency can be manifest in vitro by their ability to extend a single primer around a circular ssDNA of over 5,000 nucleotide units in length. Chromosomal DNA polymerases will be referred to here as replicases to distinguish them from DNA polymerases that function in other DNA metabolic processes and are far less processive.
There are three types of replicases known thus far that differ in how they achieve processivity and how their subunits are organized. These will be referred to here as Types I-III. The Type I is exemplified by the phage T5 replicase, which is composed of only one subunit yet is highly processive (Das et al., “Mechanism of Primer-template Dependent Conversion of dNTP-dNMP by T7 DNA Polymerase,” J. Biol. Chem., 255:7149-7154 (1980)). It is possible that the T5 enzyme achieves processivity by having a cavity within it for binding DNA, with a domain of the protein acting as a lid that opens to accept the DNA and closes to trap the DNA inside, thereby keeping the polymerase on DNA during polymerization of dNTPs. Type II is exemplified by the replicases of phage T7, herpes simplex virus, and vaccinia virus. In these systems, the replicase is composed of two subunits, the DNA polymerase and an “accessory protein” which is needed for the polymerase to become highly efficient. It is presumed that the DNA polymerase binds the DNA in a groove and that the accessory protein forms a cap over the groove, trapping the DNA inside for processive action. Type III is exemplified by the replicases of E. coli, phage T4, yeast, and humans in which there are three separate components, a sliding clamp protein, a clamp loader protein complex, and the DNA polymerase. In these systems, the sliding clamp protein is an oligomer in the shape of a ring. The clamp loader is a multiprotein complex which uses ATP to assemble the clamp around DNA. The DNA polymerase then binds the clamp which tethers the polymerase to DNA for high processivity. The replicase of the E. coli system contains a fourth component called tau that acts as a glue to hold two polymerases and one clamp loader together into one structure called Pol III*. In this application, any replicase that uses a minimum of three components (i.e., clamp, clamp loader, and DNA polymerase) will be referred to as either a three component polymerase, a type III enzyme, or a DNA polymerase III-type replicase.
The E. coli replicase is also called DNA polymerase III holoenzyme. The holoenzyme is a single multiprotein particle that contains all the components; it is comprised of ten different proteins. This holoenzyme is suborganized into four functional components called: 1) Pol III core (DNA polymerase); 2) gamma complex or tau/gamma complex (clamp loader); 3) beta subunit (sliding clamp); and 4) tau (glue protein). The DNA polymerase III “core” is a tightly associated complex containing one each of the following three subunits: 1) the alpha subunit is the actual DNA polymerase (129 kDa); 2) the epsilon subunit (28 kDa) contains the proofreading 3′-5′ exonuclease activity; and 3) the theta subunit has an unknown function. The gamma complex is the clamp loader and contains the following subunits: gamma, delta, delta prime, chi and psi (U.S. Pat. No. 5,583,026 to O'Donnell). Tau can substitute for gamma, as can a tau/gamma heterooligomer. The beta subunit is a homodimer and forms the ring shaped sliding clamp. These components associate to form the holoenzyme and the entire holoenzyme can be assembled in vitro from 10 isolated pure subunits (U.S. Pat. No. 5,583,026 to O'Donnell; U.S. Pat. No. 5,668,004 to O'Donnell). The E. coli dnaX gene encodes both tau and gamma. Tau is the product of the full gene. Gamma is the product of the first 2⁄3 of the gene; it is truncated by an efficient translational frameshift that results in incorporation of one unique residue followed by a stop codon.
The tau subunit, encoded by the same gene that encodes gamma (dnaX), also acts as a glue to hold two cores together with one gamma complex. This subassembly is called DNA polymerase III star (Pol III*). One beta ring interacts with each core in Pol III* to form DNA polymerase III holoenzyme.
During replication, the two cores in the holoenzyme act coordinately to synthesize both strands of DNA in a duplex chromosome. At the replication fork, DNA polymerase III holoenzyme physically interacts with the DnaB helicase through the tau subunit to form a yet larger protein complex termed the “replisome” (Kim et al., “Coupling of a Replicative Polymerase and Helicase: A tau-DnaB Interaction Mediates Rapid Replication Fork Movement,” Cell, 84:643-650 (1996); Yuzhakov et al., “Replisome Assembly Reveals the Basis for Asymmetric Function in Leading and Lagging Strand Replication,” Cell, 86:877-886 (1996)). The primase repeatedly contacts the helicase during replication fork movement to synthesize RNA primers on the lagging strand (Marians, K. J., “Prokaryotic DNA Replication,” Annu. Rev. Biochem., 61:673-719 (1992)).
Intensive subtyping of prokaryotic cells has now lead to a taxonomic classification of prokaryotic cells as eubacteria (true bacteria) to distinguish them from archaebacteria. Within eubacteria are many different subcategories of cells, although they can broadly be subdivided into Gram positive- and Gram negative-like cells. Numerous complete and partial genome sequences of prokaryotes have appeared in the public databases.
In the present invention, new genes from the Gram positive bacteria, Streptococcus pyogenes (e.g., S. pyogenes) and Staphylococcus aureus (e.g., S. aureus) are identified. They are assigned names based on their nearest homology to subunits in the E. coli system. The genes encoding E. coli replication proteins are as follows: alpha (dnaE); epsilon (dnaQ); theta (holE); tau (full length dnaX); gamma (frameshift product of dnaX); delta (holA); delta prime (holB); chi (holC); psi (holD); beta (dnaN); DnaB helicase (dnaB); and primase (dnaG).
Study of the organisms for which a complete genome sequence is available reveals that no organism has identifiable homologues to all the subunits of the E. coli three component polymerase, Pol III holoenzyme (see Table 1 below). All other organisms lack the 0 subunit (holE), and all except one lack genes encoding the χ and ψ subunits (holC and holD, respectively) as judged by sequence comparison searches. Further, the α and ε subunits are fused into one large a subunit in some organisms (e.g., Gram positive cells) as detailed in (Sanjanwala et al., “DNA Polymerase III Gene of Bacillus subtilis,” Proc. Natl. Acad. Sci. USA, 86:4421-4424 (1989)). Although all organisms have homologues to τ, β, δ′ and SSB, the δ subunit has diverged significantly (either not recognized or nearly not recognized by gene searching programs), perhaps even to the point where it is no longer involved in DNA replication. The DnaX product also would appear to lack frameshift signals in most organisms. This predicts only one protein (tau) will be produced from this gene, instead of two as in E. coli. Indeed, this has been shown to be true for the Staphylococcus aureus DnaX (U.S. patent application Ser. No. 09/235,245, which is hereby incorporated by reference). Finally, genetic study of Bacillus subtilis identified two genes that do not have counterparts in E. coli (dnaB, not the helicase, and dnaH) as well as one other gene, dnaI, that is only very distantly related to E. coli dnaC (Karamata et al., “Isolation and Genetic Analysis of Temperature-Sensitive Mutants of B. subtilis Defense in DNA Synthesis,” Molec. Gen. Genet., 108:277-287 (1970); Braund et al., “Nucleotide Sequence of the Bacillus subtilis dnaD Gene,” Microb., 141:321-322 (1995); Hoshino et al., “Nucleotide Sequence of Bacillus subtilis dnaB: A Gene Essential for DNA Replication Initiation and Membrance Attachment,” Proc. Natl. Acad. Sci. USA,” 84:653-657 (1987)). Keeping in mind the apparently random, or at least unpredictable process of evolution, it is possible that these apparently new genes perform novel functions that may result in a new type of polymerase for chromosomal replication. Thus, it seems possible that new proteins may have evolved to take the place of χ, ψ, θ, the frameshift product of DnaX, and possibly δ in other eubacteria. These considerations indicate that the three component polymerase of different eubacteria may have different structures. That this may be so would not be surprising as different bacteria are often less related evolutionarily than plants are to humans. For example, the split between Gram positive and Gram negative bacteria occurred about 1.2 billion years ago. This distant split makes Gram positive cells an attractive source to examine how different other eubacterial three component polymerases are from the E. coli Pol III holoenzyme.

















TABLE 1





Organism (Order)
χ
φ
θ
ε
α
β
dnaX
δ′
δ







Escherichia coli
+
+
+
+
+
+
+
+
+



Proteobacteria


Haemophilus influenzae
+
+
−
+
+
+
+
+
+



Proteobacteria


Mycoplasma genitalium
−
−
−
−
+
+
+
+
+
(weak)


Firmicutes


Synichisystis sp.
−
−
−
−
+
+
+
+
+
(weak)


Cyanobacteria


Bacillus subtilis
−
−
−
−
+
+
+
+
+
(weak)


Firmicutes


Borrelia burgdorferi
−
−
−
−
+
+
+
+
+
(weak)


Spirochaetales


Aquifex aeolicus
−
−
−
+
+
+
+
+
+
(weak)


Aquificales


Mycobacterium tuberculosis
−
−
−
+
+
+
+
+
+
(weak)


Firmicutes & Actinobacteria


Treponema pallidum
−
−
−
+
+
+
+
+
+
(weak)


Spirochaetales


Chlamydia trachomatis
−
−
−
+
+
+
+
+
+
(weak)


Chlamydiales


Rickettsia prowazekii
−
−
−
+
+
+
+
+
+
(weak)


Proteobacteria


Helicobacter pylori
−
−
−
+
+
+
+
+
+
(weak)


Proteobacteria


Thermatoga maritima
−
−
−
−
+
+
+
+
+
(weak)


Thermotogales









The goal of this invention is to learn how to form a functional three component polymerase from an organism that is highly divergent from E. coli and whether it is as rapid and processive as the E. coli Pol III holoenzyme. Namely, from bacteria lacking χ, ψ, or θ, or having a widely divergent δ subunit, or having only one DnaX product, or an α subunit that encompasses both α and ε activities. All eubacteria for which the entire genome has been sequenced have at least one of these differences from E. coli. Many Gram negative bacteria have one or more of these differences (e.g., Haemophilus influenzae and Aquifex aeolicus). Bacteria of the Gram positive class have all of these different features. Because of the distant evolutionary split between Gram positive and Gram negative bacteria, their mechanisms of replication may have diverged significantly as well. Indeed, purification of the replication polymerase from B. subtilis, a Gram positive cell, gives only a single subunit polymerase (Barnes et al., “Purification of DNA Polymerase III of Gram-Positive Bacteria,” Methods Enzy. 262:35-42 (1995); Barnes et al., “Antibody to B. subtilis DNA Polymerase III: Use in Enzyme Purification and Examination of Homology Among Replication-specific DNA Polymerases,” Nucl. Acids Res., 6:1203-209 (1979); Barnes et al., “DNA Polymerase III of Mycoplasma pulmonis: Isolation and Characterization of the Enzyme and its Structural Gene, polC,” Mol. Microb., 13:843-854, (1994); Low et al., “Purification and Characterization of DNA Polymerase III from Bacillus subtilis,” J. Biol. Chem., 251:1311-1325 (1976)) instead of a 10 subunit assembly containing the three components of a rapidly processive machine as discussed above for Pol III holoenzyme from E. coli. This finding suggests a different structural organization of the replicase and possibly different functional characteristics as well.
Although there are many studies of replication mechanisms in eukaryotes and, specifically, the Gram negative bacterium E. coli and its bacteriophages, there is very little information about how Gram positive organisms replicate. The Gram positive class of bacteria includes some of the worst human pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, and Mycobacterium tuberculosis (Youmans et al., The Biological and Clinical Basis of Infectious Disease (1985)). Until this invention, the best characterized Gram positive organism for chromosomal DNA synthesis was Bacillus subtilis. Fractionation of B. subtilis has identified three DNA polymerases. (Gass et al., “Further Genetic and Enzymological Characterization of the Three Bacillus subtilis Deoxyribonucleic Acid Polymerases,” J. Biol. Chem., 248:7688-7700 (1973); Ganesan et al., “DNA Replication in a Polymerase I Deficient Mutant and the Identification of DNA Polymerases II and III in Bacillus subtilis,” Biochem. Biophys. Res. Commun., 50:155-163 (1973)). These polymerases are thought to be analogous to the three DNA polymerases of E. coli (DNA polymerases I, II, and III). Studies in B. subtilis have identified a polymerase that appears to be involved in chromosome replication and is termed Pol III (Ott et al., “Cloning and Characterization of the polC Region of Bacillus subtilis,” J. Bacteriol., 165:951-957 (1986); Barnes et al., “Localization of the Exonuclease and Polymerase Domains of Bacillus subtilis DNA Polymerase III,” Gene, 111:43-49 (1992); Barnes et al., “The 3′-5′ Exonuclease Site of DNA Polymerase III From Gram-positive Bacteria: Definition of a Novel Motif Structure,” Gene” 165:45-50 (1995) or Barnes et al., “Purification of DNA Polymerase III of Gram-positive Bacteria,” Methods in Enzy., 262:35-42 (1995)). The B. subtilis Pol III (encoded by polC) is larger (about 165 kDa) than the E. coli alpha subunit (about 129 kDa) and exhibits 3′-5′ exonuclease activity. The polC gene encoding this Pol III shows weak homology to the genes encoding E. coli alpha and the E. coli epsilon subunit. Hence, this long form of the B. subtilis Pol III (herein referred to as α-large or Pol III-L) essentially comprises both the alpha and epsilon subunits of the E. coli core polymerase. The S. aureus α-large has also been sequenced, expressed in E. coli, and purified; it contains DNA polymerase and 3′-5′ exonuclease activity (Pacitti et al., “Characterization and Overexpression of the Gene Encoding Staphylococcus aureus DNA Polymerase III,” Gene, 165:51-56 (1995)). Although α-large is essential to cell growth (Clements et al., “Inhibition of Bacillus subtilis Deoxyribonucleic Acid Polymerase III by Phenylhydrazinopyrimidines: Demonstration of a Drug-induced Deoxyribonucleic Acid-Enzyme Complex,” J. Biol. Chem., 250:522-526 (1975); Cozzarelli et al., “Mutational Alteraction of Bacillus subtilis DNA Polymerase III to Hydroxyphenylazopyrimidine Resistance: Polymerase III is Necessary for DNA Replication,” Biochem. And Biophy. Res. Commun., 51:151-157 (1973); Low et al., “Mechanism of Inhibition of Bacillus subtilis DNA Polymerase III by the Arylhydrazinopyrimidine Antimicrobial Agents,” Proc. Natl. Acad. Sci. USA, 71:2973-2977 (1974)), there could still be another DNA polymerase(s) that is essential to the cell, such as occurs in yeast (Morrison et al., “A Third Essential DNA Polymerase in S. cerevisiae,” Cell, 62:1143-1151 (1990)).
Purification of α-large from B. subtilis results in only this single protein without associated proteins (Barnes et al., “Localization of the Exonuclease and Polymerase Domains of Bacillus subtilis DNA Polymerase III,” Gene, 111:43-49 (1992); Barnes et al., “The 3′-5′ Exonuclease Site of DNA Polymerase III From Gram-positive Bacteria: Definition of a Novel Motif Structure,” Gene” 165:45-50 (1995) or Barnes et al., “Purification of DNA Polymerase III of Gram-positive Bacteria,” Methods in Enzymol., 262:35-42 (1995)). Hence, it is possible that α-large is a member of the Type I replicase (like T5) in which it is processive on its own without accessory proteins. B. subtilis and S. aureus also have a gene encoding a protein that has approximately 30% homology to the beta subunit of E. coli; however, the protein product has not been purified or characterized (Alonso et al., “Nucleotide Sequence of the recF Gene Cluster From Staphylococcus aureus and Complementation Analysis in Bacillus subtilis recF Mutants,” Mol. Gen. Genet., 246:680-686 (1995); Alonso et al., “Nucleotide Sequence of the recF Gene Cluster From Staphylococcus aureus and Complementation Analysis in Bacillus subtilis recF Mutants,” Mol. Gen. Genet., 248:635-636 (1995)). Whether this beta subunit has a function in replication, a ring shape, or functions as a sliding clamp was not known until recently. It was also not known whether it is functional with α-large. Recently, it was shown that S. aureus β is functional as a ring, and that it also functions with α-large (U.S. patent application Ser. No. 09/235,245, which is hereby incorporated by reference). Further, a fourth DNA polymerase was identified with greater homology to E. coli a than α-large. This polymerase, called herein α-small, is shorter than α-large and lacks the domain homologous to epsilon. This polymerase also functions with the β ring, indicating that it may participate in chromosome replication. Indeed, a recent report indicates that α-small is essential for replication in Streptomyces coelicolor A3(2) (Flett et al., “A Gram-negative type” DNA Polymerase III is Essential for Replication of the Linear Chromosome of Streptomyces Coelicolor A3(2),” Mol. Micro., 31:949-958, (1999)).
As described earlier, purification of the replicase from the Gram positive B. subtilis gives only a single subunit Pol II, instead of a multicomponent complex. Also, S. aureus dnaX has been shown to encode only one subunit (U.S. patent application Ser. No. 09/235,245, which is hereby incorporated by reference). Moreover, S. aureus and B. subtilis lack homologues to χ, ψ, θ, and the δ subunit is only weakly homologous to 8 of E. coli (only 28%). Further, they lack a homologue to dnaQ encoding E. Instead, they contain this activity (3′-5′ exonuclease) in the polC gene product which provides the α-large form of α. The ε subunit is needed for high speed and processivity of the E. coli Pol III holoenzyme; the α subunit alone is much less rapid and processive with the β ring compared to the presence of both α and β (Studwell et al., “Processive Replication is Contingent on the Exonuclease Subunit of DNA Polymerase III Holoenzyme,” J. Biol Chem, 265: 1171-1178 (1990)).
Studies using the E. coli β ring (and γ complex) show they confer onto S. aureus a quite efficient synthesis (U.S. patent application Ser. No. 09/235,245, which is hereby incorporated by reference), but the efficiency is not equal to that of E. coli αε with β (and γ complex). This may be due to use of the heterologous combination of an α subunit from one organism (S. aureus) with the β clamp from another (E. coli.). However, it is also possible that S. aureus α simply does not function with a β clamp to produce speed and processivity comparable to the E. coli polymerase. Also, as described earlier, the α-large subunit of B. subtilis purifies as a single subunit, rather than associated with accessory subunits assembled into the three components of a rapid, processive machine (i.e., like E. coli Pol III holoenzyme). The lack of two DnaX products, lack of a multicomponent structure, and lack of gene homologues encoding several subunits of the three component, Pol III, of E. coli brings into question whether other types of bacteria, such as Gram positive cells, even have an enzyme with similar structure or comparable speed and processivity to that found in the Gram negative E. coli. 
The lack of gene homologues encoding several subunits of the E. coli three component polymerase creates uncertainties with respect to reconstructing a rapid and processive polymerase from a Gram positive cell that has characteristics like the Pol III system of E. coli. 
The γ and δ′ proteins are homologous to one another, encoding C-shape proteins Long et al., “DNA Polymerase III Accessory Proteins,” J. Biol. Chem., 268:11758-11765, (1993); Guenther et al., “Crystal Structure of the 8′ Subunit of the Clamp-loader Complex of E. coli DNA Polymerase III,” Cell, 91:335-345 (1997)). The clamp loaders of yeast and humans are composed of five proteins, all of which are homologous to one another and to γ and δ′ (Cullman et al., “Characterization of the Five Replication Factor C Genes of Saccharomyces Cerevisiae,” Mol. Cell. Biol., 15:4661-4671 (1995)). This provides evidence that a clamp loader can be composed entirely of C-shape proteins. Perhaps the Gram positive DnaX-protein (hereafter referred to as τ) and δ′ are sufficient to provide function as a clamp loader. Indeed, the clamp loader of T4 phage is composed of only two different proteins, gp44/62 complex (Young et al., “Structure and Function of the Bacteriophage T4 DNA Polymerase Holoenzyme,” Biochem., 31:8675-8690 (1992)). This idea is also supported by the presence of only two RFC genes in archaebacteria, suggesting that they may utilize two C-shaped proteins for clamp loading, in contrast to yeast and humans that use five. With this consideration in mind, genes were identified and isolated and the τ protein (encoded by dnaX) and 6′ (encoded by holB) of another Gram positive organism, Streptococcus pyogenes, were expressed and purified. As was observed in S. aureus, S. pyogenes dnaX produces only a single polypeptide. The β, encoded by dnaN of S. pyogenes, was also identified, expressed, and purified, as were the α-large subunit encoded by polC and the SSB encoded by the ssb gene. These proteins were studied for interactions and characterized for their effect on α-large. However, the hypothesis was incorrect as T and δ′ did not form a τδ′ complex, nor did they assemble β onto DNA or provide stimulation of a when using β on primed and SSB coated M13 mp18 ssDNA.
In light of the inability of S. pyogenes τ protein and δ′ to function as a clamp loader, it seemed reasonable to expect that one or more other proteins are needed. The fact that E. coli has some replicase subunits that other bacteria do not, suggests that other bacteria may have some replicase subunits that E. coli does not. Indeed, genetic studies of Bacillus subtilis demonstrates that it has three genes needed for replication that E. coli does not have. Two of these novel genes, called dnaB (not the same as E. coli dnaB encoding the helicase) and dnaH, have no significant homology to genes in the E. coli genome database (Bruand et al., “Nucleotide Sequence of the Bacillus subtilis dnaD gene,” Microbiol., 141:321-322 (1995); Hoshino et al., “Nucleotide Sequence of Bacillus subtilis dnaB: A gene Essential for DNA replication Initiation and Membrane Attachment,” Proc. Natl. Acad. Sci. USA, 84:653-657 (1987)). Further, dnaI of B. subtilis is important for replication and has, at best, a very limited homology to E. coli dnaC (Karamata et al., “Isolation and Genetic Analysis of Temperature-Sensitive Mutants of B. subtilis Defective in DNA synthesis,” Molec. Gen. Genetics, 108:277-287 (1970)). Perhaps one or more of these genes encode the proteins(s) necessary to provide clamp loading activity when combined with τ and δ′, or to couple with a to provide it with speed and/or processivity as the E. coli epsilon does. The S. pyogenes homologues of B. subtilis dnaI, dnaH, and dnaB were identified, cloned, and the encoded proteins were expressed and purified. However, these proteins failed to provide activity alone or in combinations with S. pyogenes τ and δ′ in loading S. pyogenes onto DNA, or in stimulating S. pyogenes α-large in combination with β, τ, and δ′ on SSB coated primed M13 mp18 ssDNA.
Weak homology exists for the holA gene among prokaryotes. This weak homologue of holA was identified in S. pyogenes and, then, it was cloned, expressed, and the putative δ was purified. The putative δ formed an isolatable complex with τ and δ′. In fact, the τδδ′ complex loaded S. pyogenes β onto DNA, and it stimulated S. pyogenes α-large in a β dependent reaction on primed SSB coated M13 mp18 ssDNA. Hence, this protein was the only missing component necessary to provide clamp loading activity. Further, a mixture of a with τδδ′, followed by ion exchange chromatography on MonoQ, indicated formation of an ατδδ′ complex. Consistent with this, τ appeared to bind a in gel filtration analysis.
Whether the S. pyogenes three component polymerase can synthesize DNA in as rapid and processive of a fashion as the E. coli Pol III holoenzyme three component polymerase is very difficult to predict, because no other DNA polymerase known to date catalyzes synthesis at the rate or processivity of the E. coli three component polymerase. For example, the three component T4 phage polymerase travels about 400 nucleotides/s, the yeast DNA polymerase delta three component polymerase travels about 120 nucleotides/s, and the human DNA polymerase delta three component enzyme appears slower and less processive than the yeast enzyme.
The standard test for these speed and processivity characteristics is examination of a time course in extension of a primer on a very long template, such as around the 7.2 kb M13mp18 ssDNA genome coated with SSB and primed with a synthetic DNA oligonucleotide. The results of experiments of this type demonstrate that the three component S. pyogenes polymerase is indeed extremely rapid in synthesis. Surprisingly, it is just as fast as the E. coli enzyme. Extension proceeds at about 700-800 nucleotides per second, completing the entire template in about 9 seconds. The enzyme was fully processive throughout replication of the M13mp18 genome, as could be determined from the fact that some templates were not extended at all, while others were extended to completion. If the enzyme had not been processive during the entire replication reaction, then when it comes off one partially extended DNA genome it would have reassociated with the unextended DNA that remained and partially replicated it as well (and so on until the entire population of DNA became fully replicated). This did not happen. Instead, the reaction showed a mixture of completely replicated templates and templates that were still untouched starting material. This indicates that the enzyme stays with a template until it completes it before it cycles over to replicate another one (i.e., it is highly processive). Each of the five proteins, α, τ, δ, δ′ and β, are needed to obtain this rapid and processive DNA synthesis.
This invention has provided an intellectual template by which the clamp loader component of these three component polymerases can be obtained from any eubacterial prokaryotic cell and how to use it with the other components to produce a rapid and processive polymerase. All prokaryotes in the eubacterial kingdom that have been sequenced to date contain homologues of these genes. As the process of lateral gene transfer appears to be a major force in evolution, it would appear that relatedness of enzymes and enzyme machines is best judged by comparisons of their genes and proteins rather than by phylogeny of which bacteria they are in (Doolittle et al., “Archaeal Genomics: Do Archaea have a Mixed Heritage?,” Curr. Biol., 8:R209—R211 (1998)). As pointed out earlier in this application, most bacteria have genetic characteristics of replication genes/proteins of S. pyogenes rather than that of E. coli (i.e., no genes encoding χ, ψ, or θ, only a weak homolog to δ, or a dnaX gene encoding only a single protein).
The dnaX gene encoding τ and γ in E. coli encodes only one protein in some organisms, but, as this application shows, it is still functional in forming a protein complex capable of rapid and processive DNA synthesis. In addition, this application shows that the delta subunit, which is only weakly homologous among different prokaryotic organisms, is an essential functional subunit of the three component polymerase (instead of having diverged so as to fulfill an entirely different function in some other intracellular process). As mentioned earlier, several genes encoding subunits of the E. coli clamp loader (γ complex; γ, δ,δ′,χ,ψ) are not obviously present in other prokaryotes (holC and holD encoding χ and ψ). Hence, one may anticipate that other genes may have evolved to encode new subunits that replace these, and that these new subunits may have been essential to the activity of the clamp loader. For example, they may have either taken over some of the functionality of another subunit, or structurally (e.g., the physical presence of a subunit could be needed for one subunit to assume its proper and active conformation, or for one or more of the subunits to form a complex together to yield the multisubunit clamp loader assembly). In addition, this application shows that the α subunit (polC gene product) is sufficient for rapid and processive synthesis with the other two components (i.e., E. coli requires ε submit to bind to α for rapid and processive synthesis of a with the β clamp). Finally, this application shows that the S. pyogenes three component polymerase synthesizes DNA as fast as the E. coli Pol III three component polymerase. Up to this point, the E. coli Pol III three component polymerase was over twice the speed of the T4 enzyme and over 5 times the speed of others. Hence, it was possible that E. coli may have been unique among prokaryotes in having a polymerase that achieves such speed. This invention shows that this is not the case. Instead, this speed in polymerization generalizes to the Gram positive prokaryotic three component DNA polymerases. It may be presumed, now that two examples of three component polymerases in widely divergent bacteria share the characteristics of rapid, processive synthesis, that the three component polymerase of other eubacteria will also be rapid and processive.
These rapid and processive three component DNA polymerases can be applied to several important uses. DNA polymerases currently in use for DNA sequencing and DNA amplification use enzymes that are much slower and thus could be improved upon. This is especially true of amplification as the three component polymerase is capable of speed and high processivity making possible amplification of very long (tens of Kb to Mb) lengths of DNA in a time efficient manner. These three component polymerases also function in conjunction with a replicative helicase (DnaB) and, thus, are capable of amplification at ambient temperature using the helicase to melt the DNA duplex. This property could be useful in amplification reaction procedures such as in polymerase chain reaction (PCR) methodology. Finally, these three component polymerases and their associated helicase (DnaB) and primase (DnaG) are attractive targets for antibiotics due to their essential and central role in cell viability.
This application provides a three component polymerase from two human pathogens in the Gram positive class. It makes possible the production of this three component polymerase from other bacteria of the Gram positive type (e.g., Streptococci, Staphylococci, Mycoplasma) and other types of bacteria lacking χ, ψ, or θ, those having only one protein produced by their dnaX gene such as obligate intracellular parasites, Mycoplasmas (possibly evolved from Gram positives), Cyanobacteria (Synechocystis), Spirochaetes such as Borrelia and Treponemia and Chlamydia, and distant relatives of E. coli in the Gram negative class (e.g., Rickettsia and Helicobacter). These three component polymerases are useful in manipulation of nucleic acids for research and diagnostic purposes (e.g., sequencing and amplification methods) and for screening chemicals for antibiotic activity (useful in human or animal therapy and agriculture such as animal feed supplements). There are several assays described previously in U.S. patent application Ser. No. 09/235,245 to O'Donnell et al., which is hereby incorporated by reference, that use these three component polymerases (or subassemblies), as well as the DnaB and DnaG homologues, either alone or in various combinations, for the purpose of screening chemicals, such as chemical libraries, for inhibitor activity. Such inhibitors can be developed further (usually by chemical manipulation and alteration) into lead compounds and then into full fledged pharmaceuticals.
There remains a need to understand the molecular details of the process of DNA replication in other cells that are quite different from E. coli, such as in Gram positive cells. It is possible that a more detailed understanding of replication proteins will lead to discovery of new antibiotics. Therefore, a deeper understanding of replication proteins of Gram positive bacteria is especially important given the emergence of drug resistant strains of these organisms. For example, Staphylococcus aureus has successfully mutated to become resistant to all common antibiotics.
The “target” protein(s) of an antibiotic drug is generally involved in a critical cell function, such that blocking its action with a drug causes the pathogenic cell to die or no longer proliferate. Current antibiotics are directed to very few targets. These include membrane synthesis proteins (e.g., vancomycin, penicillin, and its derivatives such as ampicillin, amoxicillin, and cephalosporin), the ribosome machinery (e.g., tetracycline, chloramphenicol, azithromycin, and the aminoglycosides such as kanamycin, neomycin, gentamicin, streptomycin), RNA polymerase (e.g., rifampimycin), and DNA topoisomerases (e.g., novobiocin, quinolones, and fluoroquinolones). The DNA replication apparatus is a crucial life process and, thus, the proteins involved in this process are good targets for antibiotics.
A powerful approach to discovery of a new drug is to obtain a target protein, characterize it, and develop in vitro assays of its cellular function. Large chemical libraries can then be screened in the functional assays to identify compounds that inhibit the target protein. These candidate pharmaceuticals can then be chemically modified to optimize their potency, breadth of antibiotic spectrum, non-toxicity, performance in animal models and, finally, clinical trials. The screening of large chemical libraries requires a plentiful source of the target protein. An abundant supply of protein generally requires overproduction techniques using the gene encoding the protein. This is especially true for replication proteins as they are present in low abundance in the cell.
Selective and robust assays are needed to screen reliably a large chemical library. The assay should be insensitive to most chemicals in the concentration range normally used in the drug discovery process. These assays should also be selective and not show inhibition by antibiotics known to target proteins in processes outside of replication.
The present invention is directed to overcoming these deficiencies in the art.
SUMMARY OF THE INVENTION
The present invention relates to various isolated DNA molecules from Staphylococcus aureus and Streptococcus pyogenes, both of which are Gram positive bacteria. These include DNA molecules which include a coding region from the dnaE gene (encoding α-small), dnaX gene (encoding tau), polC gene (encoding Pol III-L or α-large), dnaN gene (encoding beta), holA gene (encoding delta), holB gene (encoding delta prime), ssb gene (encoding SSB), dnaB gene (encoding DnaB), and dnaG gene (encoding DnaG) of S. aureus and/or S. pyogenes. These DNA molecules can be inserted into an expression system and used to transform host cells. The isolated proteins or polypeptides encoded by these DNA molecules, and their ability to function when used in combination is also disclosed. The resulting actions provide assembling a ring onto DNA via a clamp loader, and polymerase activity dependent on this ring that is rapid and processive.
A further aspect of the present invention relates to a method of identifying compounds which inhibit activity of a polymerase product of polC or dnaE. This method is carried out by forming a reaction mixture comprising a primed DNA molecule, a polymerase product of polC or dnaE, a candidate compound, a dNTP, and optionally either a beta subunit, a tau complex, or both the beta subunit and the tau complex, wherein at least one of the polymerase product of polC or dnaE, the beta subunit, the tau complex, or a subunit or combination of subunits thereof is derived from a Eubacteria other than Escherichia coli; subjecting the reaction mixture to conditions effective to achieve nucleic acid polymerization in the absence of the candidate compound; analyzing the reaction mixture for the presence or absence of nucleic acid polymerization extension products; and identifying the candidate compound in the reaction mixture where there is an absence of nucleic acid polymerization extension products.
The present invention deciphers the structure and mechanism of the chromosomal replicase of Gram positive bacteria and other bacteria lacking holC, holD, holE or dnaQ genes, or having a dnaX gene that encodes only one protein. Rather than use a DNA polymerase that attains high efficiency on its own, or with one other subunit, the Gram positive bacteria replicase is a three component type of replicase (class III) that uses a sliding clamp protein. The Gram positive bacteria replicase also uses a clamp loader component that assembles the sliding clamp onto DNA. This knowledge, and the enzymes involved in the replication process, can be used for the purpose of screening for potential antibiotic drugs. Further, information about chromosomal replicases may be useful in DNA sequencing, DNA amplification, polymerase chain reaction, and other DNA polymerase related techniques.
The present invention identifies two DNA polymerases (both of Pol III type) in Gram positive bacteria that utilize the sliding clamp and clamp loader. The present invention also identifies a gene with homology to the alpha subunit of E. coli DNA polymerase III holoenzyme, the chromosomal replicase of E. coli. These DNA polymerases can extend a primer around a large circular natural template when the beta clamp has been assembled onto the primed ssDNA by the clamp loader or a primer on a linear DNA where the beta clamp may assemble by itself by sliding over an end.
The present invention shows that the clamp and clamp loader components of Gram negative cells can be exchanged for those of Gram positive cells in that the clamp, once assembled onto DNA, will function with Pol III obtained from either Gram positive and Gram negative sources. This result implies that important contacts between the polymerase and clamp have been conserved during evolution. Therefore, these “mixed systems” may provide assays for an inhibitor of this conserved interaction. Such an inhibitor may be expected to shut down replication, and since the interaction is apparently conserved across the evolutionary spectrum from Gram positive and Gram negative cells, the inhibitor may exhibit a broad spectrum of antibiotic activity.
The present invention demonstrates that Gram positive bacteria contain a beta subunit that behaves as a sliding clamp that encircles DNA. A dnaX gene sequence encoding a protein homolog of the gamma/tau subunit of the clamp loader (gamma/tau complex) E. coli DNA polymerase III holoenzyme is also identified. The presence of this gene confirms the presence of a clamp loading apparatus in Gram positive bacteria that will assemble beta clamps onto DNA for the DNA polymerases.
This application also outlines methods and assays for use of these replication proteins in drug screening processes.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the construction of the S. aureus Pol III-L expression vector. The gene encoding Pol III-L was cloned into a pET11 expression vector in a three step cloning scheme as illustrated.
FIGS. 2A-D describe the expression and purification of S. aureus Pol III-L (alpha-large). FIG. 2A compares E. coli cells that contain the pET11PolC expression vector that are either induced or uninduced for protein expression. The gel is stained with Coomassie Blue. The induced band corresponds to the expected molecular weight of the S. aureus Pol III-L, and is indicated to the right of the gel. FIG. 2B-C show the results of the MonoQ chromatography of a lysate of E. coli (pET11PolC-L) induced for Pol III-L. The fractions were analyzed in a Coomassie Blue stained gel (FIG. 2B) and for DNA synthesis (FIG. 2C). Fractions containing Pol III-L are indicated. In FIG. 2D, fractions containing Pol III-L from the MonoQ column were pooled and chromatographed on a phosphocellulose column. This shows an analysis of the column fractions from the phosphocellulose column in a Coomassie Blue stained polyacrylamide gel. The position of Pol III-L is indicated to the right.
FIG. 3 shows the S. aureus beta expression vector. The dnaN gene was amplified from S. aureus genomic DNA and cloned into the pET16 expression vector.
FIGS. 4A-C illustrate the expression and purification of S. aureus beta. FIG. 4A compares E. coli cells that contain the pET16beta expression vector that are either induced or uninduced for protein expression. The gel is stained with Coomassie Blue. The induced band corresponds to the expected molecular weight of the S. aureus beta, and is indicated to the right of the gel. The migration position of size standards are indicated to the left. FIG. 4B shows the results of MonoQ chromatography of an E. coli (pET16beta) lysate induced for beta. The fractions were analyzed in a Coomassie Blue stained gel, and fractions containing beta are indicated. In FIG. 4C, fractions containing beta from the MonoQ column were pooled and chromatographed on a phosphocellulose column. This shows an analysis of the column fractions from the phosphocellulose column in a Coomassie Blue stained polyacrylamide gel. The position of beta is indicated to the right.
FIGS. 5A-B demonstrate that the S. aureus beta stimulates S. aureus Pol III-L and E. coli Pol III core on linear DNA, but not circular DNA. In FIG. 5A, the indicated proteins were added to replication reactions containing polydA-oligodT as described in the Examples infra. Amounts of proteins added, when present, were: lanes 1,2: S. aureus Pol III-L, 7.5 ng; S. aureus beta, 6.2 μg; Lanes 3,4: E. coli Pol III core, 45 ng; S. aureus beta, 9.3 μg; Lanes 5,6: E. coli Pol III core, 45 ng; E. coli beta, 5 μg. Total DNA synthesis was: Lanes 1-6: 4.4, 30.3, 5.1, 35.5, 0.97, 28.1 pmol, respectively. In FIG. 5B, Lanes 1-3, the indicated proteins were added to replication reactions containing circular singly primed M13 mp18 ssDNA as described in the Examples infra. S. aureus beta, 0.8 μg; S. aureus Pol III-L, 300 ng (purified through MonoQ); E. coli clamp loader complex, 1.7 μg. Results in the E. coli system are shown in Lanes 4-6. Total DNA synthesis was: Lanes 1-6: 0.6, 0.36, 0.99, 2.7, 3.5, 280 pmol, respectively.
FIG. 6 shows that S. aureus Pol III-L functions with E. coli beta and clamp loader complex on circular primed DNA. It also shows that S. aureus beta does not convert Pol III-L with sufficient processivity to extend the primer all the way around a circular DNA. Replication reactions were performed on the circular singly primed M13mp18 ssDNA. Proteins added to the assay are as indicated in this figure. The amount of each protein, when present, is: S. aureus beta, 800 ng; S. aureus Pol III-L, 1500 ng (MonoQ fraction 64); E. coli Pol III core, 450 ng; E. coli beta, 100 ng; E. coli gamma complex, 1720 ng. Total DNA synthesis in each assay is indicated at the bottom of the figure.
FIGS. 7A-B show that S. aureus contains four distinct DNA polymerases. Four different DNA polymerases were partially purified from S. aureus cells. S. aureus cell lysate was separated from DNA and, then, chromatographed on a MonoQ column. Fractions were analyzed for DNA polymerase activity. Three peaks of activity were observed. The second peak was the largest and was expected to be a mixture of two DNA polymerases based on early studies in B. subtilis. Chromatography of the second peak on phosphocellulose (FIG. 7B) resolved two DNA polymerases from one another.
FIGS. 8A-B show that S. aureus has two DNA Pol III's. The four DNA polymerases partially purified from S. aureus extract, designated peaks I-IV in FIG. 7, were assayed on circular singly primed M13mp18 ssDNA coated with E. coli SSB either in the presence or absence of E. coli beta (Song) and clamp loader complex (50 ng). Each reaction contained 2 μl of the partially pure polymerase (Peak I was Mono Q fraction 24 (1.4 μg), Peak 2 was phosphocellulose fraction 26 (0.016 mg/ml), Peak 3 was phosphocellulose fraction 46 (0.18 mg/ml), and Peak 4 was MonoQ fraction 50 (1 pg). FIG. 8A shows the product analysis in an agarose gel.
FIG. 8B shows the extent of DNA synthesis in each assay.
FIG. 9 compares the homology between the polypeptide encoded by dnaE of S. aureus and other organisms. An alignment is shown for the amino acid sequence of the S. aureus dnaE product (SEQ. ID. No. 85) with the dnaE products (alpha subunits) of E. coli (SEQ. ID. No. 86) and Salmonella typhimurium (SEQ. ID. No. 87).
FIG. 10 compares the homology between the N-terminal regions of the gamma/tau polypeptides of S. aureus (SEQ. ID. No. 88), B. subtilis (SEQ. ID. No. 89), and E. coli (SEQ. ID. No. 90). The conserved ATP site and the cystines forming the zinc finger are indicated above the sequence. The organisms used in the alignment were: E. coli (GenBank); and B. subtilis. 
FIG. 11 compares the homology between the DnaB polypeptide of S. aureus (SEQ. ID. No. 91) and other organisms. The organisms used in the alignment were: E. coli (GenBank) (SEQ. ID. No. 92); B. subtilis (SEQ. ID. No. 93); Sal. Typ., (Salmonella typhimurium) (SEQ. ID. No. 94).
FIGS. 12A-B show the alignment of the delta subunit encoded by holA for E. coli (SEQ. ID. No. 95) and B. subtilis (SEQ. ID. No. 96) (FIG. 12A) and for the delta subunit of B. subtilis (SEQ. ID. No. 96) and S. pyogenes (SEQ. ID. No. 97) (FIG. 12B). FIG. 12A shows ClustalW generated alignment of B. subtilis (Gram positive) delta (SEQ. ID. No. 96) to E. coli (Gram negative) delta (SEQ. ID. No. 95). FIG. 12B shows ClustalW generated alignment of B. subtilis (Gram positive) delta (SEQ. ID. No. 96) to S. pyogenes (Gram positive) delta (SEQ. ID. No. 97).
FIG. 13 is an image of an autoradiograph of an agarose gel analysis of replication products from singly primed, SSB coated M13mp18 ssDNA using the reconstituted S. aureus Pol III holozyme. Only in the presence of the τδδ′ complex does α-large (PolC) function with β to replicate a full circular duplex DNA (RFII).
FIG. 14 shows a Comassie Blue stained SDS polyacrylamide gel of the pure S. pyogenes subunits corresponding to alpha-large, alpha-small, dnaX gene product (called tau), beta, delta, delta prime, and SSB. The first lane shows the position of molecular weight markers. Purified proteins were separated on a 15% SDS-PAGE and stained with Coommassie Brilliant Blue R-250. Each lane contains 5 microgram of each protein. Lane 1, markers; lane 2, alpha-large; lane 3, alpha-small, lane 4, tau subunit; lane 5, beta subunit; lane 6, delta subunit; lane 7, delta prime subunit; lane 8, single strand DNA binding protein.
FIGS. 15A-C document the ability to reconstitute the τδδ′ complex of S. pyogenes. Proteins were mixed and gel filtered on Superose 6, followed by analysis of the column fractions in a SDS polyacrylamide gel. FIG. 15A shows a mixture of τδδ′. FIG. 15B shows a mixture of τδ. FIG. 15C shows a mixture of τδ′.
FIGS. 16A-E show that the S. pyogenes τδδ′ complex can load the S. pyogenes beta clamp onto (circular) DNA. Loading reactions contained 500 fm nicked pBSK plasmid, 500 fin either τδδ′ complex, tau, delta, or delta prime, 1 pm 32P-labelled beta dimer, 8 mM MgCl2, 1 mM ATP. Reaction components were preincubated for 10 min at 37° C. prior to loading onto 5 ml Biogel A15M column equilibrated with buffer A containing 100 mM NaCl. FIG. 16A demonstrates the ability of τδδ′ complex to load the beta dimer onto a nicked pBSK circular plasmid. FIGS. 16B-E show the results of using either: beta alone (FIG. 16B); δδ′ plus β (FIG. 16C); τ, δ and β (FIG. 16D); τ, δ′ and β (FIG. 16E).
FIGS. 17A-C show that τ and alpha interact. FIG. 17A shows the result of gel filtration analysis of a mixture of τ with alpha-large. Gel filtration fractions are analyzed in a SDS polyacrylamide gel. FIGS. 17B and 17C show the results using only τ or only alpha-large, respectively. Comparison of the elution positions of proteins shows that the positions of alpha and tau are shifted toward a higher molecular weight complex when they are present together. The fact they do not exactly comigrate may indicate that they initially are together in a complex, but that the complex dissociates during the time of the gel filtration experiment (over one half hour).
FIGS. 18 documents the ability to reconstitute αLτδδ′ (pol III*) complex of S. pyogenes. Proteins were mixed, preincubated for 20 min at 15° C., gel filtered on Superose 6, followed by analysis of the column fractions in a SDS polyacrylamide gel (FIG. 18). Proteins were loaded on a MonoQ column, then eluted with a linear gradient of 50-500 mM NaCl, followed by analysis of the column fractions in a SDS polyacrylamide gel. The aLτδδ′ complex migrates early.
FIG. 19 illustrates the speed and processivity of the S. pyogenes aLτδδ′ (pol III*) complex. The aLτδδ′ (pol III*) complex was incubated with primed M13pm 18 ssDNA (coated with S. pyogenes SSB) and only two dNTPs, then replication was initiated upon adding the remaining two dNTPs. Reactions contained 25 fmol singly primed M13 mp18 ssDNA template, 300 fmol β2, and either 75 fmol or 250 fmol aLτδδ′. Time points were quenched with SDS/EDTA then analyzed in a neutral agarose gel followed by autoradiography. Each time point is a separate reaction. The time course of polymerization was performed at two different ratios of polymerase/primed template to assess speed and processivity of nucleotide incorporation.
FIGS. 20A-I show the extent of homology between S. pyogenes replication genes and other organisms. Due to the low homology of delta (FIG. 20D), one must “walk” from one organism to the next in order to recognize the homologue with high probability. Percent identity over regions of the indicated number of amino acid residues is shown for each match (i.e., the two organisms at the opposite ends of each line). Amino acid sequences were retrieved from either GenBank or individual unfinished genome databases.
FIG. 21A-F are images illustrating that the S. pyogenes DnaE (alpha-small) polymerase functions with β. FIGS. 21A-B illustrate the relationship between DnaE and β for association with ssDNA. Different amounts of DnaE polymerase were added to a SSB coated M13mp18 ssDNA circle primed with a single DNA oligonucleotide, and products were analyzed in a native agarose gel. Reactions were performed in the presence of τδδ′ and either the absence (FIG. 21C, panels 1-4) or presence (FIG. 21D, panels 1-4) of β. Positions of completed duplex (RFII) and initial primed template (ssDNA) are indicated. FIG. 21E shows an analysis of exonuclease activity by PolC and DnaE on a 5′-32P-DNA 30-mer. Aliquots were removed at the indicated times and analyzed in a sequencing gel. FIG. 21F shows the effect of TMAU on PolC and DnaE in the presence of τδδ′ and β. DNA products were analyzed in a native agarose gel. Positions of initial primed M13mp18 (ssDNA) and completed circular duplex (RFII) are indicated.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to various isolated nucleic acid molecules from Gram positive bacteria and other bacteria lacking holC, holD, or holE genes or having a dnaX gene encoding only one subunit. These include DNA molecules which correspond to the coding regions of the dnaE, dnaX, holA, holB, polC, dnaN, SSB, dnaB, and dnaG genes. These DNA molecules can be inserted into an expression system or used to transform host cells. The isolated proteins or polypeptides encoded by these DNA molecules and their use to form a three component polymerase are also disclosed. Also encompassed by the present invention are corresponding RNA molecules transcribed from the DNA molecules.
These DNA molecules and proteins can be derived from numerous bacteria, including Staphylococcus, Streptococcus, Enterococcus, Mycoplasma, Mycobacterium, Borrelia, Treponema, Rickettsia, Chlamydia, Helicobacter, and Thermatoga. It is particularly directed to such DNA molecules and proteins derived from Streptococcus and Staphylococcus bacteria, particularly Streptococcus pyogenes and Staphylococcus aureus (see U.S. patent application Ser. No. 09/235,245, which is hereby incorporated by reference).
The gene sequences used to obtain DNA molecules of the present invention were obtained by sequence comparisons with the E. coli counterparts, followed by detailed analysis of the raw sequence data in the contigs from the S. pyogenes database (http://dna1.chem.ou.edu/strep.html) or the S. aureus database (http://www.genome.ou.edu/staph.html) to identify the open reading frames. In many instances, nucleotide errors were observed causing frameshifts in the open reading frame thus truncating it. Therefore, upon cloning the genes via PCR, the genes were sequenced to obtain correct information. Also, the full nucleotide sequence of the ssb gene was not present in the data base. This was cloned by circular PCR and the full sequence is reported below.
The S. aureus dnaX and dnaE genes were identified by aligning genes of several organisms and designing primers for use in PCR to obtain a gene fragment, followed by steps to identify the entire gene.
One aspect of the present invention relates to a newly discovered Pol III gene (herein identified as dnaE) of S. aureus whose encoded protein is homologous to E. coli alpha (product of dnaE gene). The partial nucleotide sequence of the S. aureus dnaE gene corresponds to SEQ. ID. No. 1 as follows:








atggtggcat atttaaatat tcatacggct tatgatttgt taaattcaag cttaaaaata   60



 


gaagatgccg taagacttgc tgtgtctgaa aatgttgatg cacttgccat aactgacacc  120


 


aatgtattgt atggttttcc taaattttat gatgcatgta tagcaaataa cattaaaccg  180


 


atttttggta tgacaatata tgtgacaaat ggattaaata cagtcgaaac agttgttcta  240


 


gctaaaaata atgatggatt aaaagatttg tatcaactat catcggaaat aaaaatgaat  300


 


gcattagaac atgtgtcgtt tgaattatta aaacgatttt ctaacaatat gattatcatt  360


 


tttaaaaaag tcggtgatca acatcgtgat attgtacaag tgtttgaaac ccataatgac  420


 


acatatatgg accaccttag tatttcgatt caaggtagaa aacatgtttg gattcaaaat  480


 


gtttgttacc aaacacgtca agatgccgat acgatttctg cattagcagc tattagagac  540


 


aatacaaaat tagacttaat tcatgatcaa gaagattttg gtgcacattt tttaactgaa  600


 


aaggaaatta atcaattaga tattaaccaa gaatatttaa cgcaggttga tgttatagct  660


 


caaaagtgtg atgcagaatt aaaatatcat caatctctac ttcctcaata tgagacacct  720


 


aatgatgaat cagctaaaaa atatttgtgg cgtgtcttag ttacacaatt gaaaaaatta  780


 


gaacttaatt atgacgtcta tttagagcga ttgaaatatg agtataaagt tattactaat  840


 


atgggttttg aagattattt cttaatagta agtgatttaa tccattatgc gaaaacgaat  900


 


gatgtgatgg taggtcctgg tcgtggttct tcagctggct cactggtcag ttatttattg  960


 


ggaattacaa cgattgatcc tattaaattc aatctattat ttgaacgttt tttaaaccca 1020


 


gaacgtgtaa caatgcctga tattgatatt gactttgaag atacacgccg agaaagggtc 1080


 


attcagtacg tccaagaaaa atatggcgag ctacatgtat ctggaattgt gactttcggt 1140


 


catctgcttg caagagcagt tgctagagat gttggaagaa ttatggggtt tgatgaagtt 1200


 


acattaaatg aaatttcaag tttaatccca cataaattag gaattacact tgatgaagca 1260


 


tatcaaattg acgattttaa agagtttgta catcgaaacc atcgacatga acgctggttc 1320


 


agtatttgta aaaagttaga aggtttacca agacatacat ctacacatgc ggcaggaatt 1380


 


attattaatg accatccatt atatgaatat gcccctttaa cgaaagggga tacaggatta 1440


 


ttaacgcaat ggacaatgac tgaagccgaa cgtattgggt tattaaaaat agattttcta 1500


 


gggttgagaa acttatcgat tattcatcaa atcttaacac aagtcaaaaa agatttaggt 1560


 


attaatattg atatcgaaaa gattccgttt gatgatcaaa aagtgtttga attgttgtcg 1620


 


caaggagata cgactggcat attccaatta gagtctgacg gtgtaagaag tgtattaaaa 1680


 


aaattaaagc cggaacactt tgaagatatt gttgctgtaa cttctttgta tagaccaggt 1740


 


ccaatggaag aaattccaac ttacattaca agaagacatg atccaagcaa agttcaatat 1800


 


ttacatccgc atttagaacc tatattaaaa aatacttacg gtgttattat ttatcaagag 1860


 


caaattatgc aaatagcgag cacatttgca aacttcagtt atggtgaagc ggatatttta 1920


 


agaagagcaa tgagtaaaaa aaatagagct gttcttgaaa gtgagcgtca acattttata 1980


 


gaaggtgcaa agcaaaatgg ttatcacgaa gacattagta agcaaatatt tgatttgatt 2040


 


ctgaaacttg ctgattatgg ttttcctaga gcacatgctg tcagctattc taaaattgca 2100


 


tacattacga gctttttaaa agtccattat ccaaattatt tttacgcaaa tattttaagt 2160


 


aatgttattg gaagtgagaa gaaaactgct caaatgatag aagaagcaaa aaaacaaggt 2220


 


atcactatat tgccaccgaa cattaacgaa agtcattggt tttataaacc ttcccaagaa 2280


 


ggcatttatt tatcaattgg tacaattaaa ggtgttggtt atcaaagtgt gaaagtgatt 2340


 


gttgatgaac gttatcagaa cggcaaattt aaagatttct ttgattttgc tagacgtata 2400


 


ccgaagagag tcaaaacgag aaagttactt gaagcactga ttttagtggg agcgtttgat 2460


 


gcttttggta aaacacgttc aacgttgttg caagctattg atcaagtgtt ggatggcgat 2520


 


ttaaacactg aacaagatgg ttttttattt gatattttaa cgccaaaaca gatgtatgaa 2580


 


gataaagaag aattgcctga tgcacttatt agtcagtacg aaaaagaata tttaggattt 2640


 


tatgtttcgc aacacccagt agataaaaag tttgttgcca aacaatattt aacgatattt 2700


 


aaattgagta acgcgcagaa ttataaacct atattagtac agtttgataa agttaaacaa 2760


 


attcgaacta aaaatggtca aaatatggca ttcgtcacat taaatgatgg cattgaaact 2820


 


ttagatggtg tgattttccc taatcagttt aaaaagtacg aagagttgtt atcacataat 2880


 


gacttgttta tagttagcgg gaaatttgac catagaaagc aacaacgtca actaattata 2940


 


aatgagattc agacattago cacttttgaa gaacaaaaat tagcatttgc caaacaaatt 3000


 


ataattagaa ataaatcaca aatagatatg tttgaagaga tgattaaagc tacgaaagag 3060


 


aatgctaatg atgttgtgtt atccttttat gatgaaacga ttaaacaaat gactacttta 3120


 


ggctatatta atcaaaaaga tagtatgttt aataatttta tacaatcctt taaccctagt 3180


 


gatattaggc ttata                                                  3195






The S. aureus dnaE encoded protein, called α-small, has an amino acid sequence corresponding to SEQ. ID. No. 2 as follows:








Met Val Ala Tyr Leu Asn Ile His Thr Ala Tyr Asp Leu Len Asn Ser



  1               5                  10                  15


 


Ser Leu Lys Ile Glu Asp Ala Val Arg Len Ala Val Ser Glu Asn Val


             20                  25                  30


 


Asp Ala Leu Ala Ile Thr Asp Thr Asn Val Leu Tyr Gly Phe Pro Lys


         35                  40                  45


 


Phe Tyr Asp Ala Cys Ile Ala Asn Asn Ile Lys Pro Ile Phe Gly Met


     50                  55                  60


 


Thr Ile Tyr Val Thr Asn Gly Leu Asn Thr Val Glu Thr Val Val Leu


 65                  70                  75                  80


 


Ala Lys Asn Asn Asp Gly Leu Lys Asp Leu Tyr Gln Leu Ser Ser Glu


                 85                  90                  95


 


Ile Lys Met Asn Ala Leu Glu His Val Ser Phe Glu Leu Leu Lys Arg


            100                 105                 110


 


Phe Ser Asn Asn Met Ile Ile Ile Phe Lys Lys Val Gly Asp Gln His


        115                 120                 125


 


Arg Asp Ile Val Gln Val Phe Glu Thr His Asn Asp Thr Tyr Met Asp


    130                 135                 140


 


His Leu Ser Ile Ser Ile Gln Gly Arg Lys His Val Trp Ile Gln Asn


145                 150                 155                 160


 


Val Cys Tyr Gln Thr Arg Gln Asp Ala Asp Thr Ile Ser Ala Leu Ala


                165                 170                 175


 


Ala Ile Arg Asp Asn Thr Lys Leu Asp Leu Ile His Asp Gln Glu Asp


            180                 185                 190


 


Phe Gly Ala His Phe Leu Thr Glu Lys Glu Ile Asn Gln Leu Asp Ile


        195                 200                 205


 


Asn Gln Glu Tyr Leu Thr Gln Val Asp Val Ile Ala Gln Lys Cys Asp


    210                 215                 220


 


Ala Glu Leu Lys Tyr His Gln Ser Leu Leu Pro Gln Tyr Glu Thr Pro


225                 230                 235                 240


 


Asn Asp Glu Ser Ala Lys Lys Tyr Leu Trp Arg Val Leu Val Thr Gln


                245                 250                 255


 


Leu Lys Lys Leu Glu Leu Asn Tyr Asp Val Tyr Leu Glu Arg Leu Lys


            260                 265                 270


 


Tyr Glu Tyr Lys Val Ile Thr Asn Met Gly Phe Glu Asp Tyr Phe Leu


        275                 280                 285


 


Ile Val Ser Asp Leu Ile His Tyr Ala Lys Thr Asn Asp Val Met Val


    290                 295                 300


 


Gly Pro Gly Arg Gly Ser Ser Ala Gly Ser Leu Val Ser Tyr Leu Leu


305                     310             315                 320


 


Gly Ile Thr Thr Ile Asp Pro Ile Lys Phe Asn Leu Leu Phe Glu Arg


                325                 330                 335


 


Phe Leu Asn Pro Glu Arg Val Thr Met Pro Asp Ile Asp Ile Asp Phe


            340                 345                 350


 


Glu Asp Thr Arg Arg Glu Arg Val Ile Gln Tyr Val Gln Glu Lys Tyr


        355                 360                 365


 


Gly Glu Leu His Val Ser Gly Ile Val Thr Phe Gly His Leu Leu Ala


    370                 375                 380


 


Arg Ala Val Ala Arg Asp Val Gly Arg Ile Met Gly Phe Asp Glu Val


385                 390                 395                 400


 


Thr Leu Asn Glu Ile Ser Ser Leu Ile Pro His Lys Leu Gly Ile Thr


                405                 410                 415


 


Leu Asp Glu Ala Tyr Gln Ile Asp Asp Phe Lys Glu Phe Val His Arg


            420                 425                 430


 


Asn His Arg His Gln Arg Trp Phe Ser Ile Cys Lys Lys Leu Glu Gly


        435                 440                 445


 


Leu Pro Arg His Thr Ser Thr His Ala Ala Gly Ile Ile Ile Asn Asp


    450                 455                 450


 


His Pro Leu Tyr Glu Tyr Ala Pro Leu Thr Lys Gly Asp Thr Gly Leu


465                 470                 475                 480


 


Leu Thr Gln Trp Thr Met Thr Glu Ala Glu Arg Ile Gly Leu Leu Lys


                485                 490                 495


 


Ile Asp Phe Leu Gly Leu Arg Asn Leu Ser Ile Ile His Gln Ile Leu


            500                 505                 510


 


Thr Gln Val Lys Lys Asp Leu Gly Ile Asn Ile Asp Ile Glu Lys Ile


        515                 520                 525


 


Pro Phe Asp Asp Gln Lys Val Phe Glu Leu Leu Ser Gln Gly Asp Thr


    530                 535                 540


 


Thr Gly Ile Phe Gln Leu Glu Ser Asp Gly Val Arg Ser Val Leu Lys


545                 550                 555                 560


 


Lys Leu Lys Pro Glu His Phe Glu Asp Ile Val Ala Val Thr Ser Leu


                565                 570                 575


 


Tyr Arg Pro Gly Pro Met Glu Glu Ile Pro Thr Tyr Ile Thr Arg Arg


            580                 585                 590


 


His Asp Pro Ser Lys Val Gln Tyr Leu His Pro His Leu Glu Pro Ile


        595                 600                 605


 


Leu Lys Asn Thr Tyr Gly Val Ile Ile Tyr Gln Glu Gln Ile Met Gln


    610                 615                 620


 


Ile Ala Ser Thr Phe Ala Asn Phe Ser Tyr Gly Glu Ala Asp Ile Leu


625                 630                 635                 640


 


Arg Arg Ala Met Ser Lys Lys Asn Arg Ala Val Leu Glu Ser Glu Arg


                645                 650                 655


 


Gln His Phe Ile Glu Gly Ala Lys Gln Asn Gly Tyr His Glu Asp Ile


            660                 665                 670


 


Ser Lys Gln Ile Phe Asp Leu Ile Leu Lys Phe Ala Asp Tyr Gly Phe


        675                 680                 685


 


Pro Arg Ala His Ala Val Ser Tyr Ser Lys Ile Ala Tyr Ile Met Ser


    690                 695                 700


 


Phe Leu Lys Val His Tyr Pro Asn Tyr Phe Tyr Ala Asn Ile Leu Ser


705                 710                 715                 720


 


Asn Val Ile Gly Ser Glu Lys Lys Thr Ala Gln Met Ile Glu Glu Ala


                725                 730                 735


 


Lys Lys Gln Gly Ile Thr Ile Leu Pro Pro Asn Ile Asn Glu Ser His


            740                 745                 750


 


Trp Phe Tyr Lys Pro Ser Gln Glu Gly Ile Tyr Leu Ser Ile Gly Thr


        755                 760                 765


 


Ile Lys Gly Val Gly Tyr Gln Ser Val Lys Val Ile Val Asp Glu Arg


    770                 775                 780


 


Tyr Gln Asn Gly Lys Phe Lys Asp Phe Phe Asp Phe Ala Arg Arg Ile


785                 790                 795                 800


 


Pro Lys Arg Val Lys Thr Arg Lys Leu Leu Glu Ala Leu Ile Leu Val


                805                 810                 815


 


Gly Ala Phe Asp Ala Phe Gly Lys Thr Arg Ser Thr Leu Leu Gln Ala


            820                 825                 830


 


Ile Asp Gln Val Leu Asp Gly Asp Leu Asn Ile Glu Gln Asp Gly Phe


        835                 840                 845


 


Leu Phe Asp Ile Leu Thr Pro Lys Gln Met Tyr Glu Asp Lys Glu Glu


    850                 855                 860


 


Leu Pro Asp Ala Leu Ile Ser Gln Tyr Glu Lys Glu Tyr Leu Gly Phe


865                 870                 875                 880


 


Tyr Val Ser Gln His Pro Val Asp Lys Lys Phe Val Ala Lys Gln Tyr


                885                 890                 895


 


Leu Thr Ile Phe Lys Leu Ser Asn Ala Gln Asn Tyr Lys Pro Ile Leu


            900                 905                 910


 


Val Gln Phe Asp Lys Val Lys Gln Ile Arg Thr Lys Asn Gly Gln Asn


        915                 920                 925


 


Met Ala Phe Val Thr Leu Asn Asp Gly Ile Glu Thr Leu Asp Gly Val


    930                 935                 940


 


Ile Phe Pro Asn Gln Phe Lys Lys Tyr Glu Glu Leu Leu Ser His Asn


945                 950                 955                 960


 


Asp Leu Phe Ile Val Ser Gly Lys Phe Asp His Arg Lys Gln Gln Arg


                965                 970                 975


 


Gln Leu Ile Ile Asn Glu Ile Gln Thr Leu Ala Thr Phe Glu Glu Gln


            980                 985                 990


 


Lys Leu Ala Phe Ala Lys Gln Ile Ile Ile Arg Asn Lys Ser Gln Ile


        995                1000                1005


 


Asp Met Phe Glu Glu Met Ile Lys Ala Thr Lys Glu Asn Ala Asn Asp


   1010                1015                1020


 


Val Val Leu Ser Phe Tyr Asp Glu Thr Ile Lys Gln Met Thr Thr Leu


1025               1030                1035                1040


 


Gly Tyr Ile Asn Gln Lys Asp Ser Met Phe Asn Asn Phe Ile Gln Ser


               1045                1050                1055


 


Phe Asn Pro Ser Asp Ile Arg Leu Ile


           1060                1065






The present invention also relates to the S. aureus dnaX gene. This S. aureus dnaX gene has a partial nucleotide sequence corresponding to SEQ. ID. No. 3 as follows:








ttgaattatc aagccttata tcgtatgtac agaccccaaa gtttcgagga tgtcgtcgga   60



 


caagaacatg tcacgaagac attgcgcaat gcgatttcga aagaaaaaca gtcgcatgca  120


 


tatattttta gtggtccgag aggtacgggg aaaacgagta ttgccaaagt gtttgctaaa  180


 


gcaatcaact gtttaaatag cactgatgga gaaccttgta atgaatgtca tatttgtaaa  240


 


ggcattacgc aggggactaa ttcagatgtg atagaaattg atgctgctag taataatggc  300


 


gttgatgaaa taagaaatat tagagacaaa gttaaatatg caccaagtga atcgaaatat  360


 


aaagtttata ttatagatga ggtgcacatg ctaacaacag gtgcttttaa tgccctttta  420


 


aagacgttag aagaacctcc agcacacgct atttttatat tggcaacgac agaaccacat  480


 


aaaatccctc caacaatcat ttctagggca caacgttttg attttaaagc aattagccta  540


 


gatcaaattg ttgaacgttt aaaatttgta gcagatgcac aacaaattga atgtgaagat  600


 


gaagccttgg catttatcgc taaagcgtct gaagggggta tgcgtgatgc attaagtatt  660


 


atggatcagg ctattgcttt cggcgatggc acattgacat tacaagatgc cctaaatgtt  720


 


acgggtagcg ttcatgatga agcgttggat cacttgtttg atgatattgt acaaggtgac  780


 


gtacaagcat cttttaaaaa ataccatcag tttataacag aaggtaaaga agtgaatcgc  840


 


ctaataaatg atatgattta ttttgtcaga gatacgatta tgaataaaac atctgagaaa  900


 


gatactgagt atcgagcact gatgaactta gaattagata tgttatatca aatgattgat  960


 


cttattaatg atacattagt gtcgattcgt tttagtgtga atcaaaacgt tcattttgaa 1020


 


gtattgttag taaaattagc tgagcagatt aagggtcaac cacaagtgat tgcgaatgta 1080


 


gctgaaccag cacaaattgc ttcatcgcca aacacagatg tattgttgca acgtatggaa 1140


 


cagttagagc aagaactaaa aacactaaaa gcacaaggag tgagtgttgc tcctactcaa 1200


 


aaatcttcga aaaagcctgc gagaggtata caaaaatcta aaaatgcatt ttcaatgcaa 1260


 


caaattgcaa aagtgctaga taaagcgaat aaggcagata tcaaattgtt gaaagatcat 1320


 


tggcaagaag tgattgacca tgcccaaaac aatgataaaa aatcactcgt tagtttattg 1380


 


caaaattcgg aacctgtggc ggcaagtgaa gatcacgtcc ttgtgaaatt tgaggaagag 1440


 


atccattgtg aaatcgtcaa taaagacgac gagaaacgta gtagtataga aagtgttgta 1500


 


tgtaatatcg ttaataaaaa cgttaaagtt gttggtgtac catcagatca atggcaaaga 1560


 


gttcgaacgg agtatttaca aaatcgtaaa aacgaaggcg atgatatgcc aaagcaacaa 1620


 


gcacaacaaa cagatattgc tcaaaaagca aaagatcttt tcggtgaaga aactgtacat 1680


 


gtgatagatg aagagtga                                               1698






The S. aureus dnaX encoded protein (i.e., the tau subunit) has a partial amino acid sequence corresponding to SEQ. ID. No. 4 as follows:








Leu Asn Tyr Gln Ala Leu Tyr Arg Met Tyr Arg Pro Gln Ser Phe Glu



1               5                   10                  15


 


Asp Val Val Gly Gln Glu His Val Thr Lys Thr Leu Arg Asn Ala Ile


            20                  25                  30


 


Ser Lys Glu Lys Gln Ser His Ala Tyr Ile Phe Ser Gly Pro Arg Gly


        35                  40                  45


 


Thr Gly Lys Thr Ser Ile Ala Lys Val Phe Ala Lys Ala Ile Asn Cys


    50                  55                  60


 


Leu Asn Ser Thr Asp Gly Glu Pro Cys Asn Glu Cys His Ile Cys Lys


65                  70                  75                  80


 


Gly Ile Thr Gln Gly Thr Asn Ser Asp Val Ile Glu Ile Asp Ala Ala


                85                  90                  95


 


Ser Asn Asn Gly Val Asp Glu Ile Arg Asn Ile Arg Asp Lys Val Lys


            100                 105                 110


 


Tyr Ala Pro Ser Glu Ser Lys Tyr Lys Val Tyr Ile Ile Asp Glu Val


        115                 120                 125


 


His Met Leu Thr Thr Gly Ala Phe Asn Ala Leu Leu Lys Thr Leu Glu


    130                 135                 140


 


Glu Pro Pro Ala His Ala Ile Phe Ile Leu Ala Thr Thr Glu Pro His


145                 150                 155                 160


 


Lys Ile Pro Pro Thr Ile Ile Ser Arg Ala Gln Arg Phe Asp Phe Lys


                165                 170                 175


 


Ala Ile Ser Leu Asp Gln Ile Val Glu Arg Leu Lys Phe Val Ala Asp


            180                 185                 190


 


Ala Gln Gln Ile Glu Cys Glu Asp Glu Ala Leu Ala Phe Ile Ala Lys


        195                 200                 205


 


Ala Ser Glu Gly Gly Met Arg Asp Ala Leu Ser Ile Met Asp Gln Ala


    210                 215                 220


 


Ile Ala Phe Gly Asp Gly Thr Leu Thr Leu Gln Asp Ala Leu Asn Val


225                 230                 235                 240


 


Thr Gly Ser Val His Asp Glu Ala Leu Asp His Leu Phe Asp Asp Ile


                245                 250                 255


 


Val Gln Gly Asp Val Gln Ala Ser Phe Lys Lys Tyr His Gln Phe Ile


            260                 265                 270


 


Thr Glu Gly Lys Glu Val Asn Arg Leu Ile Asn Asp Met Ile Tyr Phe


        275                 280                 285


 


Val Arg Asp Thr Ile Met Asn Lys Thr Ser Glu Lys Asp Thr Glu Tyr


    290                 295                 300


 


Arg Ala Leu Met Asn Leu Glu Leu Asp Met Leu Tyr Gln Met Ile Asp


305                 310                 315                 320


 


Leu Ile Asn Asp Thr Leu Val Ser Ile Arg Phe Ser Val Asn Gln Asn


                325                 330                 335


 


Val His Phe Glu Val Leu Leu Val Lys Leu Ala Glu Gln Ile Lys Gly


            340                 345                 350


 


Gln Pro Gln Val Ile Ala Asn Val Ala Glu Pro Ala Gln Ile Ala Ser


        355                 360                 365


 


Ser Pro Asn Thr Asp Val Leu Leu Gln Arg Met Glu Gln Leu Glu Gln


    370                 375                 380


 


Glu Leu Lys Thr Leu Lys Ala Gln Gly Val Ser Val Ala Pro Thr Gln


385                 390                 395                 400


 


Lys Ser Ser Lys Lys Pro Ala Arg Gly Ile Gln Lys Ser Lys Asn Ala


                405                 410                 415


 


Phe Ser Met Gln Gln Ile Ala Lys Val Leu Asp Lys Ala Asn Lys Ala


            420                 425                 430


 


Asp Ile Lys Leu Leu Lys Asp His Trp Gln Glu Val Ile Asp His Ala


        435                 440                 445


 


Gln Asn Asn Asp Lys Lys Ser Leu Val Ser Leu Leu Gln Asn Ser Glu


    450                 455                 460


 


Pro Val Ala Ala Ser Glu Asp His Val Leu Val Lys Phe Glu Glu Glu


465                 470                 475                 480


 


Ile His Cys Glu Ile Val Asn Lys Asp Asp Glu Lys Arg Set Ser Ile


                485                 490                 495


 


Glu Ser Val Val Cys Asn Ile Val Asn Lys Asn Val Lys Val Val Gly


            500                 505                 510


 


Val Pro Ser Asp Gln Trp Gln Arg Val Arg Thr Glu Tyr Leu Gln Asn


        515                 520                 525


 


Arg Lys Asn Glu Gly Asp Asp Met Pro Lys Gln Gln Ala Gln Gln Thr


    530                 535                 540


 


Asp Ile Ala Gln Lys Ala Lys Asp Leu Phe Gly Glu Glu Thr Val His


545                 550                 555                 560


 


Val Ile Asp Glu Glu Glx


                565






The tau subunit of S. aureus functions as does both the tau subunit and the gamma subunit of E. coli. 

This invention also relates to the partial nucleotide sequence of the S. aureus dnaB gene. The partial nucleotide sequence of this dnaB gene corresponds to SEQ. ID. No. 5 as follows:







atggatagaa tgtatgagca aaatcaaatg ccgcataaca atgaagctga acagtctgtc   60



 


ttaggttcaa ttattataga tccagaattg attaatacta ctcaggaagt tttgcttcct  120


 


gagtcgtttt ataggggtgc ccatcaacat attttccgtg caatgatgca cttaaatgaa  180


 


gataataaag aaattgatgt tgtaacattg atggatcaat tatcgacgga aggtacgttg  240


 


aatgaagcgg gtggcccgca atatcttgca gagttatcta caaatgtacc aacgacgcga  300


 


aatgttcagt attatactga tatcgtttct aagcatgcat taaaacgtag attgattcaa  360


 


actgcagata gtattgccaa tgatggatat aatgatgaac ttgaactaga tgcgatttta  420


 


agtgatgcag aacgtcgaat tttagagcta tcatcttctc gtgaaagcga tggctttaaa  480


 


gacattcgag acgtcttagg acaagtgtat gaaacagctg aagagcttga tcaaaatagt  540


 


ggtcaaacac caggtatacc tacaggatat cgagatttag accaaatgac agcagggttc  600


 


aaccgaaatg atttaattat ccttgcagcg cgtccatctg taggtaagac tgcgttcgca  660


 


cttaatattg cacaaaaagt tgcaacgcat gaagatatgt atacagttgg tattttctcg  720


 


ctagagatgg gtgctgatca gttagccaca cgtatgattt gtagttctgg aaatgttgac  780


 


tcaaaccgct taagaacggg tactatgact gaggaagatt ggagtcgttt tactatagcg  840


 


gtaggtaaat tatcacgtac gaagattttt attgatgata caccgggtat tcgaattaat  900


 


gatttacgtt ctaaatgtcg tcgattaaag caagaacatg gcttagacat gattgtgatt  960


 


gactacttac agttgattca aggtagtggt tcacgtgcgt ccgataacag acaacaggaa 1020


 


gtttctgaaa tctctcgtac attaaaagca ttagcccgtg aattaaaatg tccagttatc 1080


 


gcattaagtc agttatctcg tggtgttgaa caacgacaag ataaacgtcc aatgatgagt 1140


 


gatattcgtg aatctggttc gattgagcaa gatgccgata tcgttgcatt cttataccgt 1200


 


gatgattact ataaccgtgg cggcgatgaa gatgatgacg atgatggtgg tttcgagcca 1260


 


caaacgaatg atgaaaacgg tgaaattgaa attatcattg ctaagcaacg taacggtcca 1320


 


acaggcacag ttaagttaca ttttatgaaa caatataata aatttaccga tatcgattat 1380


 


gcacatgcag atatgatg                                               1398






The amino acid sequence of S. aureus DnaB encoded by the dnaB gene corresponds to SEQ. ID. No. 6 as follows:








Met Asp Arg Met Tyr Glu Gln Asn Gln Met Pro His Asn Asn Glu Ala



  1               5                  10                  15


 


Glu Gln Ser Val Leu Gly Ser Ile Ile Ile Asp Pro Glu Leu Ile Asn


             20                  25                  30


 


Thr Thr Gln Glu Val Leu Leu Pro Glu Ser Phe Tyr Arg Gly Ala His


         35                  40                  45


 


Gln His Ile Phe Arg Ala Met Met His Leu Asn Glu Asp Asn Lys Glu


     50                  55                  60


 


Ile Asp Val Val Thr Leu Met Asp Gln Leu Ser Thr Glu Gly Thr Leu


 65                  70                  75                  80


 


Asn Glu Ala Gly Gly Pro Gln Tyr Leu Ala Glu Leu Ser Thr Asn Val


                 85                  90                  95


 


Pro Thr Thr Arg Asn Val Gln Tyr Tyr Thr Asp Ile Val Ser Lys His


            100                 105                 110


 


Ala Leu Lys Arg Arg Leu Ile Gln Thr Ala Asp Ser Ile Ala Asn Asp


        115                 120                 125


 


Gly Tyr Asn Asp Glu Leu Glu Leu Asp Ala Ile Leu Ser Asp Ala Glu


    130                 135                 140


 


Arg Arg Ile Leu Glu Leu Ser Ser Ser Arg Glu Ser Asp Gly Phe Lys


145                 150                 155                 160


 


Asp Ile Arg Asp Val Leu Gly Gln Val Tyr Glu Thr Ala Glu Glu Leu


                165                 170                 175


 


Asp Gln Asn Ser Gly Gln Thr Pro Gly Ile Pro Thr Gly Tyr Arg Asp


            180                 185                 190


 


Leu Asp Gln Met Thr Ala Gly Phe Asn Arg Asn Asp Leu Ile Ile Leu


        195                 200                 205


 


Ala Ala Arg Pro Ser Val Gly Lys Thr Ala Phe Ala Leu Asn Ile Ala


    210                 215                 220


 


Gln Lys Val Ala Thr His Glu Asp Met Tyr Thr Val Gly Ile Phe Ser


225                 230                 235                 240


 


Leu Glu Met Gly Ala Asp Gln Leu Ala Thr Arg Met Ile Cys Ser Ser


                245                 250                 255


 


Gly Asn Val Asp Ser Asn Arg Leu Arg Thr Gly Thr Met Thr Glu Glu


            260                 265                 270


 


Asp Trp Ser Arg Phe Thr Ile Ala Val Gly Lys Leu Ser Arg Thr Lys


        275                 280                 285


 


Ile Phe Ile Asp Asp Thr Pro Gly Ile Arg Ile Asn Asp Leu Arg Ser


    290                 295                 300


 


Lys Cys Arg Arg Leu Lys Gln Glu His Gly Leu Asp Met Ile Val Ile


305                 310                 315                 320


 


Asp Tyr Leu Gln Leu Ile Gln Gly Ser Gly Ser Arg Ala Ser Asp Asn


                325                 330                 335


 


Arg Gln Gln Glu Val Ser Glu Ile Ser Arg Thr Leu Lys Ala Leu Ala


            340                 345                 350


 


Arg Glu Leu Lys Cys Pro Val Ile Ala Leu Ser Gln Leu Ser Arg Gly


        355                 360                 365


 


Val Glu Gln Arg Gln Asp Lys Arg Pro Met Met Ser Asp Ile Arg Glu


    370                 375                 380


 


Ser Gly Ser Ile Glu Gln Asp Ala Asp Ile Val Ala Phe Leu Tyr Arg


385                 390                 395                 400


 


Asp Asp Tyr Tyr Asn Arg Gly Gly Asp Glu Asp Asp Asp Asp Asp Gly


                405                 410                 415


 


Gly Phe Glu Pro Gln Thr Asn Asp Glu Asn Gly Glu Ile Glu Ile Ile


            420                 425                 430


 


Ile Ala Lys Gln Arg Asn Gly Pro Thr Gly Thr Val Lys Leu His Phe


        435                 440                 445


 


Met Lys Gln Tyr Asn Lys Phe Thr Asp Ile Asp Tyr Ala His Ala Asp


    450                 455                 460


 


Met Met


465






The present invention also relates to the S. aureus polC gene (encoding Pol III-L or α-large). The partial nucleotide sequence of this polC gene corresponds to SEQ. ID. No. 7 as follows:








atgacagagc aacaaaaatt taaagcgctt gctgatcaaa ttaaaatttc aaatcaatta   60



 


gatgctgaaa ttttaaattc aggtgaactg acacgtatag atgtttctaa caaaaacaga  120


 


acatgggaat ttcatattac attaccacaa ttcttagctc atgaagatta tttattattt  180


 


ataaatgcaa tagagcaaga gtttaaagat atcgccaacg ttacatgtcg ttttacggta  240


 


acaaatggca cgaatcaaga tgaacatgca attaaatact ttgggcactg tattgaccaa  300


 


acagctttat ctccaaaagt taaaggtcaa ttgaaacaga aaaagcttat tatgtctgga  360


 


aaagtattaa aagtaatggt atcaaatgac actgaacgta atcattttga taaggcatgt  420


 


aatggaagtc ttatcaaagc gtttagaaat tgtggttttg atatcgataa aatcatattc  480


 


gaaacaaatg ataatgatca agaacaaaac ttagcttctt tagaagcaca tattcaagaa  540


 


gaagacgaac aaagtgcacg attggcaaca gagaaacttg aaaaaatgaa agctgaaaaa  600


 


gcgaaacaac aagataacaa cgaaagtgct gtcgataagt gtcaaattgg taagccgatt  660


 


caaattgaaa atattaaacc aattgaatct attattgagg aagagtttaa agttgcaata  720


 


gagggtgtca tttttgatat aaacttaaaa gaacttaaaa gtggtcgcca tatcgtagaa  780


 


attaaagtga ctgactatac ggactcttta gttttaaaaa tgtttactcg taaaaacaaa  840


 


gatgatttag aacattttaa agcgctaagt gttggtaaat gggttagggc tcaaggtcgt  900


 


attgaagaag atacatttat tagagattta gttatgatga tgtctgatat tgaagagatt  960


 


aaaaaagcga caaaaaaaga taaggctgaa gaaaagcgtg tagaactcca cttgcatact 1020


 


gcaatgagcc aaatggatgg tatacccaat attggtgcgt atgttaaaca ggcagcagac 1080


 


tggggacatc cagccattgc ggttacagac cataatgttg tgcaagcatt tccagatgct 2140


 


cacgcagcag cggaaaaaca tggcattaaa atgatatacg gtatggaagg tatgttagtt 1200


 


gatgatggtg ttccgattgc atacaaacca caagatgtcg tattaaaaga tgctacttat 1260


 


gttgtgttcg acgttgagac aactggttta tcaaatcagt atgataaaat catcgagctt 1320


 


gcagctgtga aagttcataa cggtgaaatc atcgataagt ttgaaaggtt tagtaatccg 1380


 


catgaacgat tatcggaaac gattatcaat ttgacgcata ttactgarga tatgttagta 1440


 


gatgcccctg agattgaaga agtacttaca gagtttaaag aatgggttgg cgatgcgata 1500


 


ttcgtagcgc acaatgcttc gtttgatatg ggcttcatcg atacgggata tgaacgtctt 1560


 


gggttcggac caccaacgaa tggtgttatc gatactttag aattatcccg tacgattaat 1620


 


actgaatatg gtaaacatgg tttgaatttc ttggctaaaa aatatggcgt agaattaacg 1680


 


caacatcacc gcgccattta tgatacagaa gcaacagctt acattttcac aaaaatggtt 1740


 


caacaaatga aagaatcagg cgtattaaat cataacgaaa tcaacaaaaa actcagtaat 1800


 


gaagatgcat ataaacgtgc aagacctagt catgtcacat taattgtaca aaaccaacaa 1860


 


ggtcttaaaa atctattcaa aattgtaagt gcatcattgg tgaagtactc ctaccgtaca 1920


 


cctcgaattc cacgttcacc gttagatgaa tatcgcgagg gattattggc aggtacagcg 1980


 


cgtgatgaag gtgaattatt tacggcagtt atgcagaagg accagagtca agttgaaaaa 2040


 


attgccaaat attatgattt tattgaaatt caaccaccgg cactttatca agatttaatt 2100


 


gatagagagc ttattagaga tactgaaaca ttacatgaaa tttatcaacg tttaatacat 2160


 


gcaggtgaca cagcgggtat acctgttatt gcgacaggaa atgcacacta tttgtttgaa 2220


 


catgatggta tcgcacgtaa aattttaata gcatcacaac ccggcaatcc acttaatcgc 2280


 


tcaactttac cggaagcaca ttttagaact acagatgaaa tgttaaacga gtttcatttt 2340


 


ttaggtgaag aaaaagcgca tgaaattgtt gtgaaaaata caaacgaatt agcagatcga 2400


 


attgaacgtg ttgttcctat taaagatgaa ttatacacac cgcgtatgga aggtgctaac 2460


 


gaagaaatta gagaactaag ttatgcaaat gcgcgtaaac tgtatggtga agacctgcct 2520


 


caaatcgtaa ttgatcgatt agaaaaagaa ttaaaaagta ttatcggtaa tggatttgcg 2580


 


gtaatttact taatttcgca acgtttagtt aaaaaatcat tagatgatgg atacttagtt 2640


 


ggttcccgtg gttcagtagg ttctagtttt gtagcgacaa tgactgagat cactgaagta 2700


 


aacccgttac cgccacacta tatttgtccg aactgtaaaa cgagtgaatt tttcaatgat 2760


 


ggttcagtag gatcaggatt tgatttacct gataagacgt gtgaaacttg tggagcgcca 2820


 


cttattaaag aaggacaaga tattccgttt gaaacatttt taggatttaa gggagataaa 2880


 


gttcctgata tcgacttaaa ctttagtggt gaatatcaac cgaatgccca taactacaca 2940


 


aaagtattat ttggtgagga taaagtattc cgtgcaggta caattggtac tgttgctgaa 3000


 


aagactgctt ttggttatgt taaaggttat ttgaatgatc aaggtatcca caaaagaggt 3060


 


gctgaaatag atcgactcgt taaaggatgt acaggtgtta aacgtacaac tggacagcat 3120


 


ccagggggta ttattgtagt acctgattac atggatattt atgattttac gccgatacaa 3180


 


tatcctgccg atgatcaaaa ttcagcatgg atgacgacac attttgattt ccattctatt 3240


 


catgataatg tattaaaact tgatatactt ggacacgatg atccaacaat gattcgtatg 3300


 


cttcaagatt tatcaggaat tgatccaaaa acaatacctg tagatgataa agaagttatg 3360


 


cagatattta gtacacctga aagtttgggt gttactgaag atgaaatttt atgtaaaaca 3420


 


ggtacatttg gggtaccaga attcggtaca ggattcgtgc gtcaaatgtt agaagataca 3480


 


aagccaacaa cattttctga attagttcaa atctcaggat tatctcatgg tacagatgtg 3540


 


tggttaggca atgctcaaga attaattaaa accggtatat gtgatttatc aagtgtaatt 3600


 


ggttgtcgtg atgatatcat ggtttattta atgtatgctg gtttagaacc atcaatggct 3660


 


tttaaaataa tggagtcagt acgtaaaggt aaaggtttaa ctgaagaaat gattgaaacg 3720


 


atgaaagaaa atgaagtgcc agattggtat ttagattcat gtcttaaaat taagtacatg 3780


 


ttccctaaag cccatgcagc agcatacgtt ttaatggcag tacgtatcgc atatttcaaa 3840


 


gtacatcatc cactttatta ctatgcatct tactttacaa ttcgtgcgtc agactttgat 3900


 


ttaatcacga tgattaaaga taaaacaagc attcgaaata ctgtaaaaga catgtattct 3960


 


cgctatatgg atctaggtaa aaaagaaaaa gacgtattaa cagtcttgga aattatgaat 4020


 


gaaatggcgc atcgaggtta tcgaatgcaa ccgattagtt tagaaaagag tcaggcgttc 4080


 


gaatttatca ttgaaggcga tacacttatt ccgccgttca tatcagtgcc tgggcttggc 4140


 


gaaaacgttg cgaaacgaat tgttgaagct cgtgacgatg gcccattttt atcaaaagaa 4200


 


gatttaaaca aaaaagctgg attatctcag aaaattattg agtatttaga tgagttaggc 4260


 


tcattaccga atttaccaga taaagctcaa ctttcgatat ttgatatg              4308






The amino acid sequence of the S. aureus polC gene product, a-large, corresponds to SEQ. ID. No. 8 as follows:








Met Thr Glu Gln Gln Lys Phe Lys Val Leu Ala Asp Gln Ile Lys Ile



  1               5                  10                  15


 


Ser Asn Gln Leu Asp Ala Glu Ile Leu Asn Ser Gly Glu Leu Thr Arg


             20                  25                  30


 


Ile Asp Val Ser Asn Lys Asn Arg Thr Trp Glu Phe His Ile Thr Leu


         35                  40                  45


 


Pro Gln Phe Leu Ala His Glu Asp Tyr Leu Leu Phe Ile Asn Ala Ile


     50                  55                  60


 


Glu Gln Glu Phe Lys Asp Ile Ala Asn Val Thr Cys Arg Phe Thr Val


 65                  70                  75                  80


 


Thr Asn Gly Thr Asn Gln Asp Glu His Ala Ile Lys Tyr Phe Gly His


                 85                  90                  95


 


Cys Ile Asp Gln Thr Ala Leu Ser Pro Lys Val Lys Gly Gln Leu Lys


            100                 105                 110


 


Gln Lys Lys Leu Ile Met Ser Gly Lys Val Leu Lys Val Met Val Ser


        115                 120                 125


 


Asn Asp Ile Glu Arg Asn His Phe Asp Lys Ala Cys Asn Gly Ser Leu


    130                 135                 140


 


Ile Lys Ala Phe Arg Asn Cys Gly Phe Asp Ile Asp Lys Ile Ile Phe


145                 150                 155                 160


 


Glu Thr Asn Asp Asn Asp Gln Glu Gln Asn Leu Ala Ser Leu Glu Ala


                165                 170                 175


 


His Ile Gln Glu Glu Asp Glu Gln Ser Ala Arg Leu Ala Thr Glu Lys


            180                 185                 190


 


Leu Glu Lys Met Lys Ala Glu Lys Ala Lys Gln Gln Asp Asn Lys Gln


        195                 200                 205


 


Ser Ala Val Asp Lys Cys Gln Ile Gly Lys Pro Ile Gln Ile Glu Asn


    210                 215                 220


 


Ile Lys Pro Ile Glu Ser Ile Ile Glu Glu Glu Phe Lys Val Ala Ile


225                 230                 235                 240


 


Glu Gly Val Ile Phe Asp Ile Asn Leu Lys Glu Leu Lys Ser Gly Arg


                245                 250                 255


 


His Ile Val Glu Ile Lys Val Thr Asp Tyr Thr Asp Ser Leu Val Leu


            260                 265                 270


 


Lys Met Phe Thr Arg Lys Asn Lys Asp Asp Leu Glu His Phe Lys Ala


        275                 280                 285


 


Leu Ser Val Gly Lys Trp Val Arg Ala Gln Gly Arg Ile Glu Glu Asp


    290                 295                 300


 


Thr Phe Ile Arg Asp Leu Val Met Met Met Ser Asp Ile Glu Glu Ile


305                 310                 315                 320


 


Lys Lys Ala Thr Lys Lys Asp Lys Ala Glu Glu Lys Arg Val Glu Phe


                325                 330                 335


 


His Leu His Thr Ala Met Ser Gln Met Asp Gly Ile Pro Asn Ile Gly


            340                 345                 350


 


Ala Tyr Val Lys Gln Ala Ala Asp Trp Gly His Pro Ala Ile Ala Val


        355                 360                 365


 


Thr Asp His Asn Val Val Gln Ala Phe Pro Asp Ala His Ala Ala Ala


    370                 375                 380


 


Glu Lys His Gly Ile Lys Met Ile Tyr Gly Met Glu Gly Met Leu Val


385                 390                 395                 400


 


Asp Asp Gly Val Pro Ile Ala Tyr Lys Pro Gln Asp Val Val Leu Lys


                405                 410                 415


 


Asp Ala Thr Tyr Val Val Phe Asp Val Glu Thr Thr Gly Leu Ser Asn


            420                 425                 430


 


Gln Tyr Asp Lys Ile Ile Glu Leu Ala Ala Val Lys Val His Asn Gly


        435                 440                 445


 


Glu Ile Ile Asp Lys Phe Glu Arg Phe Ser Asn Pro His Glu Arg Leu


    450                 455                 460


 


Ser Glu Thr Ile Ile Asn Leu Thr His Ile Thr Asp Asp Met Leu Val


465                 470                 475                 480


 


Asp Ala Pro Glu Ile Glu Glu Val Leu Thr Glu Phe Lys Glu Trp Val


                485                 490                 495


 


Gly Asp Ala Ile Phe Val Ala His Asn Ala Ser Phe Asp Met Gly Phe


            500                 505                 510


 


Ile Asp Thr Gly Tyr Glu Arg Leu Gly Phe Gly Pro Ser Thr Asn Gly


        515                 520                 525


 


Val Ile Asp Thr Leu Glu Leu Ser Arg Thr Ile Asn Thr Glu Tyr Gly


    530                 535                 540


 


Lys His Gly Leu Asn Phe Leu Ala Lys Lys Tyr Gly Val Glu Leu Thr


545                 550                 555                 560


 


Gln His His Arg Ala Ile Tyr Asp Thr Glu Ala Thr Ala Tyr Ile Phe


                565                 570                 575


 


Ile Lys Met Val Gln Gln Met Lys Glu Leu Gly Val Leu Asn His Asn


            580                 585                 590


 


Glu Ile Asn Lys Lys Leu Ser Asn Glu Asp Ala Tyr Lys Arg Ala Arg


        595                 600                 605


 


Pro Ser His Val Thr Leu Ile Val Gln Asn Gln Gln Gly Leu Lys Asn


    610                 615                 620


 


Leu Phe Lys Ile Val Ser Ala Ser Leu Val Lys Tyr Phe Tyr Arg Thr


625                 630                 635                 640


 


Pro Arg Ile Pro Arg Ser Leu Leu Asp Glu Tyr Arg Glu Gly Leu Leu


                645                 650                 655


 


Val Gly Thr Ala Cys Asp Glu Gly Glu Leu Phe Thr Ala Val Met Gln


                660             665                 670


 


Lys Asp Gln Ser Gln Val Glu Lys Ile Ala Lys Tyr Tyr Asp Phe Ile


        675                 680                 685


 


Glu Ile Gln Pro Pro Ala Leu Tyr Gln Asp Leu Ile Asp Arg Glu Leu


    690                 695                 700


 


Ile Arg Asp Thr Glu Thr Leu His Glu Ile Tyr Gln Arg Leu Ile His


705                 710                 715                 720


 


Ala Gly Asp Thr Ala Gly Ile Pro Val Ile Ala Thr Gly Asn Ala His


                725                 730                 735


 


Tyr Leu Phe Glu His Asp Gly Ile Ala Arg Lys Ile Leu Ile Ala Ser


            740                 745                 750


 


Gln Pro Gly Asn Pro Leu Asn Arg Ser Thr Leu Pro Glu Ala His Phe


        755                 760                 765


 


Arg Thr Thr Asp Glu Met Leu Asn Glu Phe His Phe Leu Gly Glu Glu


    770                 775                 780


 


Lys Ala His Glu Ile Val Val Lys Asn Thr Asn Glu Leu Ala Asp Arg


785                 790                 795                 800


 


Ile Glu Arg Val Val Pro Ile Lys Asp Glu Leu Tyr Thr Pro Arg Met


                805                 810                 815


 


Glu Gly Ala Asn Glu Glu Ile Arg Glu Leu Ser Tyr Ala Asn Ala Arg


            820                 825                 830


 


Lys Leu Tyr Gly Glu Asp Leu Pro Gln Ile Val Ile Asp Arg Leu Glu


        835                 840                 845


 


Lys Glu Leu Lys Ser Ile Ile Gly Asn Gly Phe Ala Val Ile Tyr Leu


    850                 855                 860


 


Ile Ser Gln Arg Leu Val Lys Lys Ser Leu Asp Asp Gly Tyr Leu Val


865                 870                 875                 880


 


Gly Ser Arg Gly Ser Val Gly Ser Ser Phe Val Ala Thr Met Thr Glu


                885                 890                 895


 


Ile Thr Glu Val Asn Pro Leu Pro Pro His Tyr Ile Cys Pro Asn Cys


            900                 905                 910


 


Lys Thr Ser Glu Phe Phe Asn Asp Gly Ser Val Gly Ser Gly Phe Asp


        915                 920                 925


 


Leu Pro Asp Lys Thr Cys Glu Thr Cys Gly Ala Pro Leu Ile Lys Glu


    930                 935                 940


 


Gly Gln Asp Ile Pro Phe Glu Lys Phe Leu Gly Phe Lys Gly Asp Lys


945                 950                 955                 960


 


Val Pro Asp Ile Asp Leu Asn Phe Ser Gly Glu Tyr Gln Pro Asn Ala


                965                 970                 975


 


His Asn Tyr Thr Lys Val Leu Phe Gly Glu Asp Lys Val Phe Arg Ala


            980                 985                 990


 


Gly Thr Ile Gly Thr Val Ala Glu Lys Thr Ala Phe Gly Tyr Val Lys


        995                1000                1005


 


Gly Tyr Leu Asn Asp Gln Gly Ile His Lys Arg Gly Ala Glu Ile Asp


   1010                1015                1020


 


Arg Leu Val Lys Gly Cys Thr Gly Val Lys Ala Thr Thr Gly Gln His


1025               1030                1035                1040


 


Pro Gly Gly Ile Ile Val Val Pro Asp Tyr Met Asp Ile Tyr Asp Phe


               1045                1050                1055


 


Thr Pro Ile Gln Tyr Pro Ala Asp Asp Gln Asn Ser Ala Trp Met Thr


           1060                1065                1070


 


Thr His Phe Asp Phe His Ser Ile His Asp Asn Val Leu Lys Leu Asp


           1075            1080                1085


 


Ile Leu Gly His Asp Asp Pro Thr Met Ile Arg Met Leu Gln Asp Leu


   1090                1095                1100


 


Ser Gly Ile Asp Pro Lys Thr Ile Pro Val Asp Asp Lys Glu Val Met


1105               1110                1115                1120


 


Gln Ile Phe Ser Thr Pro Glu Ser Leu Gly Val Thr Glu Asp Glu Ile


               1125                1130                1135


 


Leu Cys Lys Thr Gly Thr Phe Gly Val Pro Asn Ser Asp Arg Ile Arg


           1140                1145                1150


 


Arg Gln Met Leu Glu Asp Thr Lys Pro Thr Thr Phe Ser Glu Leu Val


       1155                1160                1165


 


Gln Ile Ser Gly Leu Ser His Gly Thr Asp Val Trp Leu Gly Asn Ala


   1170                1175                1180


 


Gln Gln Leu Ile Lys Thr Gly Ile Cys Asp Leu Ser Ser Val Ile Gly


1185               1190                1195                1200


 


Cys Arg Asp Asp Ile Met Val Tyr Leu Met Tyr Ala Gly Leu Glu Pro


               1205                1210                1215


 


Ser Met Ala Phe Lys Ile Met Gln Ser Val Arg Lys Gly Lys Gly Leu


           1220                1225                1230


 


Thr Glu Gln Met Ile Glu Thr Met Lys Gln Asn Glu Val Pro Asp Trp


       1235                1240                1245


 


Tyr Leu Asp Ser Cys Leu Lys Ile Lys Tyr Ile Phe Pro Lys Ala His


   1250                1255                1260


 


Ala Ala Ala Tyr Val Leu Met Ala Val Arg Ile Ala Tyr Phe Lys Val


1265               1270                1275                1280


 


His His Pro Leu Tyr Tyr Tyr Ala Ser Tyr Phe Thr Ile Arg Ala Ser


               1285                1290                1295


 


Asp Phe Asp Leu Ile Thr Met Ile Lys Asp Lys Thr Ser Ile Arg Asn


           1300                1305                1310


 


Thr Val Lys Asp Met Tyr Ser Arg Tyr Met Asp Leu Gly Lys Lys Glu


       1315                1320                1325


 


Lys Asp Val Leu Thr Val Leu Gln Ile Met Asn Glu Met Ala His Arg


   1330                1335                1340


 


Gly Tyr Arg Met Gln Pro Ile Ser Leu Glu Lys Ser Gln Ala Phe Glu


1345               1350                1355                1360


 


Phe Ile Ile Glu Gly Asp Thr Leu Ile Pro Pro Phe Ile Ser Val Pro


               1365                1370                1375


 


Gly Leu Gly Glu Asn Val Ala Lys Arg Ile Val Gln Ala Arg Asp Asp


           1380                1385                1390


 


Gly Pro Phe Leu Ser Lys Glu Asp Leu Asn Lys Lys Ala Gly Leu Tyr


           1395            1400                1405


 


Gln Lys Ile Ile Gln Tyr Leu Asp Glu Leu Gly Ser Leu Pro Asn Leu


   1410                1415                1420


 


Pro Asp Lys Ala Gln Leu Ser Ile Phe Asp Met


1425               1430                1435






This invention also relates to the S. aureus dnaN gene encoding the beta subunit. The partial nucleotide sequence of this dnaN gene corresponds to SEQ. ID. No. 9 as follows:








atgatggaat tcactattaa aagagattat tttattacac aattaaatga cacattaaaa   60



 


gctatttcac caagaacaac attacctata ttaactggta tcaaaatcga tgcgaaagaa  120


 


catgaagtta tattaactgg ttcagactct gaaatttcaa tagaaatcac tattcctaaa  180


 


actgtagatg gcgaagatat tgtcaatatt tcagaaacag gctcagtagt acttcctgga  240


 


cgattctttg ttgatattat aaaaaaatta cctggtaaag atgttaaatt atctacaaat  300


 


gaaeaattcc agacattaat tacatcaggt cattctgaat ttaatttgag tggcttagat  360


 


ccagatcaat atcctttatt acctcaagtt tctagagatg acgcaattca attgtcggta  420


 


aaagtactta aaaacgtgat tgcacaaacg aattttgcag tgtccacctc agaaacacgc  480


 


ccagtactaa ctggtgtgaa ctggcttata caagaaaatg aattaatatg cacagcgact  540


 


gattcacacc gcttggctgt aagaaagttg cagttagaag atgtttctga aaacaaaaat  600


 


gtcatcattc caggtaaggc tttagctgaa ttaaataaaa ttatgtctga caatgaagaa  660


 


gacattgata tcttctttgc ttcaaaccaa gttttattta aagttggaaa tgtgaacttt  720


 


atttctcgat tattagaagg acattatcct gatacaacac gtttattccc tgaaaactat  780


 


gaaattaaat taagtataga caatggggag ttttatcatg cgattgatcg tgcctcttta  840


 


ttagcacgtg aaggtggtaa taacgttatt aaattaagta caggtgatga cgttgttgaa  900


 


ttatcttcta catcaccaga aattggtact gtaaaagaag aagttgatgc aaacgatgtt  960


 


gaaggtggta gcctgaaaat ttcattcaac tctaaatata tgatggatgc tttaaaagca 1020


 


atcgataatg atgaggttga agttgaattc ttcggtacaa tgaaaccatt tattctaaaa 1080


 


ccaaaaggtg acgactcggt aacgcaatta attttaccaa tcagaactta ctaa       1134






This amino acid sequence of S. aureus beta subunit is as follows (SEQ. ID. No. 10):








Met Met Glu Phe Thr Ile Lys Arg Asp Tyr Phe Ile Thr Gln Leu Asn



1               5                   10                  15


 


Asp Thr Leu Lys Ala Ile Ser Pro Arg Thr Thr Leu Pro Ile Leu Thr


            20                  25                  30


 


Gly Ile Lys Ile Asp Ala Lys Glu His Glu Val Ile Leu Thr Gly Ser


        35                  40                  45


 


Asp Ser Glu Ile Ser Ile Glu Ile Thr Ile Pro Lys Thr Val Asp Gly


    50                  55                  60


 


Glu Asp Ile Val Asn Ile Ser Glu Thr Gly Ser Val Val Leu Pro Gly


65                  70                  75                  80


 


Arg Phe Phe Val Asp Ile Ile Lys Lys Leu Pro Gly Lys Asp Val Lys


                85                  90                  95


 


Leu Ser Thr Asn Glu Gln Phe Gln Thr Leu Ile Thr Ser Gly His Ser


            100                 105                 110


 


Glu Phe Asn Leu Ser Gly Leu Asp Pro Asp Gln Tyr Pro Leu Leu Pro


        115                 120                 125


 


Gln Val Ser Arg Asp Asp Ala Ile Gln Leu Ser Val Lys Val Leu Lys


    130                 135                 140


 


Asn Val Ile Ala Gln Thr Asn Phe Ala Val Ser Thr Ser Glu Thr Arg


145                 150                 155                 160


 


Pro Val Leu Thr Gly Val Asn Trp Leu Ile Gln Glu Asn Glu Leu Ile


                165                 170                 175


 


Cys Thr Ala Thr Asp Ser His Arg Leu Ala Val Arg Lys Leu Gln Leu


            180                 185                 190


 


Glu Asp Val Ser Glu Asn Lys Asn Val Ile Ile Pro Gly Lys Ala Leu


        195                 200                 205


 


Ala Glu Leu Asn Lys Ile Met Ser Asp Asn Glu Glu Asp Ile Asp Ile


    210                 215                 220


 


Phe Phe Ala Ser Asn Gln Val Leu Phe Lys Val Gly Asn Val Asn Phe


225                 230                 235                 240


 


Ile Ser Arg Leu Leu Glu Gly His Tyr Pro Asp Thr Thr Arg Leu Phe


                245                 250                 255


 


Pro Glu Asn Tyr Glu Ile Lys Leu Ser Ile Asp Asn Gly Glu Phe Tyr


            260                 265                 270


 


His Ala Ile Asp Arg Ala Ser Leu Leu Ala Arg Glu Gly Gly Asn Asn


        275                 280                 285


 


Val Ile Lys Leu Ser Thr Gly Asp Asp Val Val Glu Leu Ser Ser Thr


    290                 295                 300


 


Ser Pro Glu Ile Gly Thr Val Lys Glu Glu Val Asp Ala Asn Asp Val


305                 310                 315                 320


 


Glu Gly Gly Ser Leu Lys Ile Ser Phe Asn Ser Lys Tyr Met Met Asp


                325                 330                 335


 


Ala Leu Lys Ala Ile Asp Asn Asp Glu Val Glu Val Glu Phe Phe Gly


            340                 345                 350


 


Thr Met Lys Pro Phe Ile Leu Lys Pro Lys Gly Asp Asp Ser Val Thr


        355                 360                 365


 


Gln Leu Ile Leu Pro Ile Arg Thr Tyr


    370                 375






This invention also relates to the S. aureus holA gene encoding the delta subunit. The partial nucleotide sequence of this holA gene corresponds to SEQ. ID. No. 11 as follows:








atggatgaac agcaacaatt gacgaatgca tatcattcaa ataaattatc gcatgcctat  60



 


ttatttgaag gtgatgatgc acaaacgatg aaacaagttg cgattaattt tgcaaagctt 120


 


attttatgtc aaacagatag tcaatgtgaa acaaaggtta gtacatataa tcatccagac 180


 


tttatgtata tatcaacaac tgagaatgca attaagaaag aacaagttga acaacttgtg 240


 


cgtcatatga atcaacttcc tatagaaagc acaaataaag tgtacatcat cgaagacttt 300


 


gaagactttg aaaagttaac tgttcaaggg gaaaacagta tcttgaaatt tcttgaagaa 360


 


ccaccggaca atacgattgc tattttattg tctacaaaac ctgagcaaat tttagacaca 420


 


atccattcaa ggtgtcagca tgtatatttc aagcctattg ataaagaaaa gtttataaat 480


 


agattagttg aacaaaacat gtctaagcca gtagctgaaa tgattagtac ttatactacg 540


 


caaatagata atgcaatggc tttaaatgaa gaatttgatt tattagcatt aaggaaatca 600


 


gttatacgtt gggaattgtt gcttactaat aagccaatgg cacttatagg tattattgat 660


 


ttattgaaac aggctaaaaa taaaaaactg caatctttaa ctattgcagc tgtgaatggt 720


 


ttcttcgaag atatcataca tacaaaggta aatgtagagg ataaacaaat atatagtgat 780


 


ttaaaaaatg atattgatca atatgcgcaa aagttgtcgt ttaatcaatt aattttgatg 840


 


tttgatcaac tgacggaagc acataagaaa ttgaatcaaa atgtaaatcc aacgcttgta 900


 


tttgaacaaa tcgtaattaa gggtgtgagt                                  930






The amino acid sequence of the delta subunit encoded by S. aureus holA corresponds to SEQ. ID. No. 12 as follows:








Met Asp Glu Gln Gln Gln Leu Thr Asn Ala Tyr His Ser Asn Lys Leu



  1                   5              10                  15


 


Ser His Ala Tyr Leu Phe Glu Gly Asp Asp Ala Gln Thr Met Lys Gln


             20                  25                  30


 


Val Ala Ile Asn Phe Ala Lys Leu Ile Leu Cys Gln Thr Asp Ser Gln


         35                  40                  45


 


Cys Glu Thr Lys Val Ser Thr Tyr Asn His Pro Asp Phe Met Tyr Ile


     50                  55                  60


 


Ser Thr Thr Glu Asn Ala Ile Lys Lys Glu Gln Val Glu Gln Leu Val


 65                  70                  75                  80


 


Arg His Met Asn Gln Leu Pro Ile Glu Ser Thr Asn Lys Val Tyr Ile


                 85                  90                  95


 


Ile Glu Asp Phe Glu Asp Phe Glu Lys Leu Thr Val Gln Gly Glu Asn


            100                 105                 110


 


Ser Ile Leu Lys Phe Leu Glu Glu Pro Pro Asp Asn Thr Ile Ala Ile


        115                 120                 125


 


Leu Leu Ser Thr Lys Pro Glu Gln Ile Leu Asp Thr Ile His Ser Arg


    130                 135                 140


 


Cys Gln His Val Tyr Phe Lys Pro Ile Asp Lys Glu Lys Phe Ile Asn


145                 150                 155                 160


 


Arg Leu Val Glu Gln Asn Met Ser Lys Pro Val Ala Glu Met Ile Ser


                165                 170                 175


 


Thr Tyr Thr Thr Gln Ile Asp Asn Ala Met Ala Leu Asn Glu Glu Phe


            180                 185                 190


 


Asp Leu Leu Ala Leu Arg Lys Ser Val Ile Arg Trp Glu Leu Leu Leu


        195                 200                 205


 


Thr Asn Lys Pro Met Ala Leu Ile Gly Ile Ile Asp Leu Leu Lys Gln


    210                 215                 220


 


Ala Lys Asn Lys Lys Leu Gln Ser Leu Thr Ile Ala Ala Val Asn Gly


225                 230                 235                 240


 


Phe Phe Glu Asp Ile Ile His Thr Lys Val Asn Val Glu Asp Lys Gln


                245                 250                 255


 


Ile Tyr Ser Asp Leu Lys Asn Asp Ile Asp Gln Tyr Ala Gln Lys Leu


            260                 265                 270


 


Ser Phe Asn Gln Leu Ile Leu Met Phe Asp Gln Leu Thr Glu Ala His


        275                 280                 285


 


Lys Lys Leu Asn Gln Asn Val Asn Pro Thr Leu Val Phe Glu Gln Ile


    290                 295                 300


 


Val Ile Lys Gly Val Ser


305                 310






This invention also relates to the S. aureus holB gene encoding the delta prime subunit. The partial nucleotide sequence of this holB gene corresponds to SEQ. ID. No. 13 as follows:








atgagcgaca atattgtagc tatttatgga gatgtgcctg aattggttga aaaacaaagt  60



 


gcagaaatca tatcacaatt tttgaaaagt gatagagatg actttaactt tgtgaaatat 120


 


aatttatacg aaacagagat tgcaccaatt gttgaagaaa cattaacatt gcctttcttt 180


 


tcagataaaa aagcaatttt ggttaaaaat gcatatatat ttacaggtga aaaagcgcca 240


 


aaagatatgg ctcataatgt agaccaatta atagaattta ttgaaaaata tgatggcgaa 300


 


aatttgattg tctttgagat atatcaaaat aaacttgatg aaagaaaaaa gttaactaaa 360


 


actctaaaaa agcatgcaag gcttaaaaaa atagagcaga tgtcggagga gatcaagtgg 420


 


attcaaaaaa aagaacaagc gattgatttt gtaaaagatc ttataacaat gaaagaagaa 480


 


ccaattaaac ttcttgcact tacatcaaat tatagacttt tttatcaatg taaaattctt 540


 


tcacaaaaag gttatagtgg tcaacaaatt gcaaaaacaa taggtgttca tccatataga 600


 


gtgaaacttg cacttggtca agtgagacat tatcaacttg atgaacttct taatattatt 660


 


gatgcatgtg cagaaacaga ttataaactt aaatcatcat atatggataa acaacttatt 720


 


cttgaacttt ttattctttc actt                                        744






The amino acid sequence of the delta prime subunit encoded by S. aureus holB corresponds to SEQ. ID. No. 14 as follows:








Met Ser Asp Asn Ile Val Ala Ile Tyr Gly Asp Val Pro Glu Leu Val



  1               5                  10                  15


 


Glu Lys Gln Ser Ala Glu Ile Ile Ser Gln Phe Leu Lys Ser Asp Arg


             20                  25                  30


 


Asp Asp Phe Asn Phe Val Lys Tyr Asn Leu Tyr Glu Thr Glu Ile Ala


         35                  40                  45


 


Pro Ile Val Glu Glu Thr Leu Thr Leu Pro Phe Phe Ser Asp Lys Lys


     50                  55                  60


 


Ala Ile Leu Val Lys Asn Ala Tyr Ile Phe Thr Gly Glu Lys Ala Pro


 65                  70                  75                  80


 


Lys Asp Met Ala His Asn Val Asp Gln Leu Ile Glu Phe Ile Glu Lys


                 85                  90                  95


 


Tyr Asp Gly Glu Asn Leu Ile Val Phe Glu Ile Tyr Gln Asn Lys Leu


            100                 105                 110


 


Asp Glu Arg Lys Lys Leu Thr Lys Thr Leu Lys Lys His Ala Arg Leu


        115                 120                 125


 


Lys Lys Ile Glu Gln Met Ser Glu Glu Ile Lys Trp Ile Gln Lys Lys


    130                 135                 140


 


Glu Gln Ala Ile Asp Phe Val Lys Asp Leu Ile Thr Met Lys Glu Glu


145                 150                 155                 160


 


Pro Ile Lys Leu Leu Ala Leu Thr Ser Asn Tyr Arg Leu Phe Tyr Gln


                165                 170                 175


 


Cys Lys Ile Leu Ser Gln Lys Gly Tyr Ser Gly Gln Gln Ile Ala Lys


            180                 185                 190


 


Thr Ile Gly Val His Pro Tyr Arg Val Lys Leu Ala Leu Gly Gln Val


        195                 200                 205


 


Arg His Tyr Gln Leu Asp Glu Leu Leu Asn Ile Ile Asp Ala Cys Ala


    210                 215                 220


 


Glu Thr Asp Tyr Lys Leu Lys Ser Ser Tyr Met Asp Lys Gln Leu Ile


225                 230                 235                 240


 


Leu Glu Leu Phe Ile Leu Ser Leu


                245






This invention also relates to the S. aureus dnaG gene encoding a primase. The partial nucleotide sequence of this dnaG gene corresponds to SEQ. ID. No. 15 as follows:








atgataggtt tgtgtccttt tcatgatgaa aagacacctt catttacagt ttctgaagat   60



 


aaacaaatct gtcattgttt tggttgtaaa aaaggtggca atgtttttca atttactcaa  120


 


gaaattaaag acatatcatt tgttgaagcg gttaaagaat taggtgatag agttaatgtt  180


 


gctgtagata ttgaggcaac acaatctaac tcaaatgttc aaattgcttc tgatgattta  240


 


caaatgattg aaatgcatga gttaatacaa gaattttatt attacgcttt aacaaagaca  300


 


gtcgaaggcg aacaagcatt aacatactta caagaacgtg gttttacaga tgcgcttatt  360


 


aaagagcgag gcattggctt tgcacccgat agctcacatt tttgtcatga ttttcttcaa  420


 


aaaaagggtt acgatattga attagcatat gaagccggac tattatcacg taacgaagaa  480


 


aatttcagtt attacgatag atttcgaaat cgtattatgt ttcctttgaa aaatgcgcaa  540


 


ggaagaattg ttggatattc aggtcgaaca tataccggtc aagaaccaaa atacctaaat  600


 


agtcctgaaa cgcctatctt tcaaaaaaga aagttgttat ataacttaga taaagcacgt  660


 


aaatcaatta gaaaattaga tgaaattgta ttactagaag gttttatgga tgttataaaa  720


 


tctgatactg ctggcttgaa aaacgttgtt gcaacaatgg gtacacagtt gtcagatgaa  780


 


catattacct ttatacgaaa gttaacatca aatataacat taatgtttga tggggatttt  840


 


gcgggtagtg aagcaacact taaaacaggt caacatttgt tacagcaagg gctaaatgta  900


 


tttgttatac aattgccatc tggcatggat ccggatgaat acattggtaa gtatggcaac  960


 


gacgcattta ctacttttgt aaaaaatgac aaaaagtcat ttgcacatta taaagtaagt 1020


 


atattaaaag atgaaattgc acataatgac ctttcatatg aacgttattt gaaagaactg 1080


 


agtcatgaca tttcacttat gaagtcatca attctgcaac aaaaggctat aaatgatgtt 1140


 


gcgccatttt tcaatgttag tcctgagcag ttagctaacg aaatacaatt caatcaagca 1200


 


ccagccaatt attatccaga agatgagtat ggcggttatg atgagtatgg cggttatatt 1260


 


gaacctgagc caattggtat ggcacaattt gacaatttga gccgtcgaga aaaagcggag 1320


 


cgagcatttt taaaacattt aatgagagat aaagatacat ttttaaatta ttatgaaagt 1380


 


gttgataagg ataacttcac aaatcagcat tttaaatatg tattcgaagt cttacatgat 1440


 


ttttatgcgg aaaatgatca atataatatc agtgatgctg tgcagtatgt taattcaaat 1500


 


gagttgagag aaacactaat tagcttagaa caatataatt tgaatggcga accatatgaa 1560


 


aatgaaattg atgattatgt caatgttatt aatgaaaaag gacaagaaac aattgagtca 1620


 


ttgaatcata aattaaggga agctacaagg attggcgatg tagaattaca aaaatactat 1680


 


ttacagcaaa ttgttgctaa gaataaagaa cgcatgtag                        1719






The amino acid sequence of primase encoded by S. aureus dnaG corresponds to SEQ. ID. No. 16 as follows:








Met Ile Gly Leu Cys Pro Phe His Asp Glu Lys Thr Pro Ser Phe Thr



1               5                   10                  15


 


Val Ser Glu Asp Lys Gln Ile Cys His Cys Phe Gly Cys Lys Lys Gly


            20                  25                  30


 


Gly Asn Val Phe Gln Phe Thr Gln Glu Ile Lys Asp Ile Ser Phe Val


        35                  40                  45


 


Glu Ala Val Lys Glu Leu Gly Asp Arg Val Asn Val Ala Val Asp Ile


    50                  55                  60


 


Glu Ala Thr Gln Ser Asn Ser Asn Val Gln Ile Ala Ser Asp Asp Leu


65                  70                  75                  80


 


Gln Met Ile Glu Met His Glu Leu Ile Gln Glu Phe Tyr Tyr Tyr Ala


                85                  90                  95


 


Leu Thr Lys Thr Val Glu Gly Glu Gln Ala Leu Thr Tyr Leu Gln Glu


            100                 105                 110


 


Arg Gly Phe Thr Asp Ala Leu Ile Lys Glu Arg Gly Ile Gly Phe Ala


        115                 120                 125


 


Pro Asp Ser Ser His Phe Cys His Asp Phe Leu Gln Lys Lys Gly Tyr


    130                 135                 140


 


Asp Ile Glu Leu Ala Tyr Glu Ala Gly Leu Leu Ser Arg Asn Glu Glu


145                 150                 155                 160


 


Asn Phe Ser Tyr Tyr Asp Arg Phe Arg Asn Arg Ile Met Phe Pro Leu


                165                 170                 175


 


Lys Asn Ala Gln Gly Arg Ile Val Gly Tyr Ser Gly Arg Thr Tyr Thr


            180                 185                 190


 


Gly Gln Glu Pro Lys Tyr Leu Asn Ser Pro Glu Thr Pro Ile Phe Gln


        195                 200                 205


 


Lys Arg Lys Leu Leu Tyr Asn Leu Asp Lys Ala Arg Lys Ser Ile Arg


    210                 215                 220


 


Lys Leu Asp Glu Ile Val Leu Leu Glu Gly Phe Met Asp Val Ile Lys


225                 230                 235                 240


 


Ser Asp Thr Ala Gly Leu Lys Asn Val Val Ala Thr Met Gly Thr Gln


                245                 250                 255


 


Leu Ser Asp Glu His Ile Thr Phe Ile Arg Lys Leu Thr Ser Asn Ile


            260                 265                 270


 


Thr Leu Met Phe Asp Gly Asp Phe Ala Gly Ser Glu Ala Thr Leu Lys


        275                 280                 285


 


Thr Gly Gln His Leu Leu Gln Gln Gly Leu Asn Val Phe Val Ile Gln


    290                 295                 300


 


Leu Pro Ser Gly Met Asp Pro Asp Glu Tyr Ile Gly Lys Tyr Gly Asn


305                 310                 315                 320


 


Asp Ala Phe Thr Thr Phe Val Lys Asn Asp Lys Lys Ser Phe Ala His


                325                 330                 335


 


Tyr Lys Val Ser Ile Leu Lys Asp Glu Ile Ala His Asn Asp Leu Ser


            340                 345                 350


 


Tyr Glu Arg Tyr Leu Lys Glu Leu Ser His Asp Ile Ser Leu Met Lys


        355                 360                 365


 


Ser Ser Ile Leu Gln Gln Lys Ala Ile Asn Asp Val Ala Pro Phe Phe


    370                 375                 380


 


Asn Val Ser Pro Glu Gln Leu Ala Asn Glu Ile Gln Phe Asn Gln Ala


385                 390                 395                 400


 


Pro Ala Asn Tyr Tyr Pro Glu Asp Glu Tyr Gly Gly Tyr Asp Glu Tyr


                405                 410                 415


 


Gly Gly Tyr Ile Glu Pro Glu Pro Ile Gly Met Ala Gln Phe Asp Asn


            420                 425                 430


 


Leu Ser Arg Arg Glu Lys Ala Glu Arg Ala Phe Leu Lys His Leu Met


        435                 440                 445


 


Arg Asp Lys Asp Thr Phe Leu Asn Tyr Tyr Glu Ser Val Asp Lys Asp


    450                 455                 460


 


Asn Phe Thr Asn Gln His Phe Lys Tyr Val Phe Glu Val Leu His Asp


465                 470                 475                 480


 


Phe Tyr Ala Glu Asn Asp Gln Tyr Asn Ile Ser Asp Ala Val Gln Tyr


                485                 490                 495


 


Val Asn Ser Asn Gln Leu Arg Glu Thr Len Ile Ser Leu Glu Gln Tyr


            500                 505                 510


 


Asn Leu Asn Gly Glu Pro Tyr Glu Ann Glu Ile Asp Asp Tyr Val Asn


        515                 520                 525


 


Val Ile Asn Glu Lys Gly Gln Glu Thr Ile Glu Ser Leu Asn His Lys


    530                 535                 540


 


Leu Arg Glu Ala Thr Arg Ile Gly Asp Val Glu Len Gln Lys Tyr Tyr


545                 550                 555                 560


 


Leu Gln Gln Ile Val Ala Lys Ann Lys Glu Arg Met


                565                 570






This invention also relates to the polC gene of Streptococcus pyogenes encoding the α-large subunit. The partial nucleotide sequence of polC (α-large) corresponds to SEQ. ID. No. 17 as follows:








atgtcagatt tattcgctaa attgatggac cagatagaaa tgccacttga catgagacgt   60



 


tcaagtgcct tttcatctgc tgatattatc gaggtaaagg tacattcggt gtcacgcttg  120


 


tgggaatttc attttgcctt tgcagcggtc ttaccgattg caacttatcg tgaattgcat  180


 


gatcgtttga taagaacttt tgaggcggct gacattaagg taacctttga catccaagct  240


 


gctcaggtgg attattcaga tgatctgctt caagcttatt accaagaagc ttttgagcat  300


 


gcaccgtgta atagtgctag ttttaaatct tctttctcaa agctcaaagt gacttatgag  360


 


gatgacaaac tcattattgc agcgccaggt tttgtgaata acgatcattt tagaaacaat  420


 


catctgccta atctggtcaa gcaattagaa gcctttggct ttggcatctt gaccatagat  480


 


atggtgtcag atcaggaaat gactgagcat ttgaccaaga attttgtttc cagtcgtcag  540


 


gctcttgtga aaaaggctgt gcaggataat ttggaagccc aaaaatctct tgaagccatg  600


 


atgccaccag ttgaggaagc cacacctgct cctaagtttg actacaagga acgagcagct  660


 


aagcgtcagg cagggtttga aaaagcaacc atcacaccaa tgattgagat tgagaccgaa  720


 


gaaaaccgga ttgtctttga gggtatggtt tttgacgtgg agcgtaaaac gactaggaca  780


 


ggtcgccata tcatcaactt taaaatgaca gactatacct cctcgtttgc tctccaaaaa  840


 


tgggctaaag acgatgagga gctccgtaaa tttgatatga ttgctaaggg agcttggtta  900


 


cgggtacaag ggaatattga gaccaatcct tttacgaaga gtctcaccat gaatgtccag  960


 


caggtcaaag aaattgtccg tcatgagcgc aaagacctga tgccagaagg gcaaaagcgg 1020


 


gtcgaacttc atgcccacac caatatgtct accatggatg ccttaccgac agtagaaagc 1080


 


ttgattgata cggcagccaa gtggggacac aaggcgattg ctatcaccga ccatgctaat 1140


 


gtgcaaagtt ttcctcatgg ctaccatagg gctcgcaaag ctgggattaa ggctattttt 1200


 


ggcctagaag ccaatattgt tgaggacaag gtgcctattt cttatgaacc tgttgatatg 1260


 


gatttgcacg aagccaccta tgtggtcttt gacgtggaaa ccacaggtct atctgctatg 1320


 


aataatgacc tgattcagat tgcggcttcc aaaatgttta aaggaaatat tgtagagcag 1380


 


tttgatgaat ccatcgatcc tgggcatccc ctttcagcct ttaccaccga attgacagga 1440


 


attaccgata agcatttgca gggcgccaag ccattggtta ctgtcctaaa agcttttcag 1500


 


gacttttgca aagatagtac cttggttgcc cacaacgcca gttttgacgt gggctttatg 1560


 


aacgccaatt atgaacgcca cgacttgccc aaaatcacac agcctgtgat tgatacctta 1620


 


gaatttgcta gaaacttgta tcctgagtac aagcgtcacg gtttgggacc gctcaccaag 1680


 


cgtttccaag tgagtctaga ccaccatcat atggccaatt acgacgcgga agccacagga 1740


 


cgtcttttgt ttatttttct aaaagatgcc agagaaaagc atggcatcaa aaatcttttg 1800


 


caactcaata cagatttggt ggctgaggat tcttacaaaa aagcgcggat taagcatgcg 1860


 


actatctatg tgcaaaatca ggttggtctt aaaaatatgt ttaagttggt cagcctttcc 1920


 


aatatcaaat attttgaagg ggtgccgcgt attccaagaa ccgtcttaga tgctcacaga 1980


 


gagggtttgt tacttggtac agcttgttct gacggcgagg tttttgatgc cgttctgact 2040


 


aaaggaattg atgcagcggt tgatttggct aggtattatg attttatcga aatcatgcca 2100


 


ccagccattt accagccatt ggttgtccgt gaattaatca aagatcaagc aggtattgag 2160


 


caggtgattc gtgacctcat tgaagtaggg aaacgagcta agaaacctgt gcttgccact 2220


 


gggaatgtgc attatctaga gcctgaagaa gagatttacc gtgaaattat tgtgcgtagt 2280


 


cttggtcagg gtgccatgat taatagaaca atcggccgtg gggaaggggc acagcctgct 2340


 


cctctaccta aagcgcactt tagaacaacc aatgaaatgc tggatgagtt tgcctttctt 2400


 


ggaaaagacc tcgcttatca agtagttgtg caaaatactc aggattttgc ggaccgtatt 2460


 


gaggaagtgg aagtggttaa gggcgacctt tacaccccgt atattgataa ggccgaagag 2520


 


acggttgccg aattaaccta tcaaaaagcc tttgaaattt atggtaatcc tctcccagat 2580


 


attattgatt tacgcattga aaaagagtta acctctatct tggggaacgg ttttgctgtg 2640


 


atttatctcg cttcccaaat gcttgttaac cggtcaaatg agcgaggcta cctagttggt 2700


 


tctaggggat ctgtagggtc tagctttgtc gccaccatga ttgggattac tgaggttaat 2760


 


cctatgccgc ctcactacgt ttgcccgtcc tgccaacatt ctgaatttat cacagatggg 2820


 


tcagttggat ctggctatga tttgcctaat aaaccctgtc cgaaatgtgg caccccttat 2880


 


caaaaagatg ggcaagacat tccctttgag acctttcttg ggtttgatgg ggataaggtg 2940


 


cccgatattg atttgaactt ctctggtgat gaccagccca gtgcccattt ggatgtccga 3000


 


gatatttttg gtgacgaata cgcctttcgt gctggaacag ttggtaccgt agcagaaaaa 3060


 


acagcttatg gatttgtcaa aggctatgaa cgcgactatg gcaagttcta tcgtgatgct 3120


 


gaggtggatc gtctagcagc aggtgctgct ggtgtgaaac gaacgactgg gcagcaccct 3180


 


ggggggattg ttgttattcc taattacatg gatgtttatg attttacccc cgtgcaatat 3240


 


ccagccgatg atgtaacggc ttcttggcag acaactcact ttaacttcca tgatattgat 3300


 


gaaaacgtct tgaaacttga tatcctaggg catgatgatc cgaccatgat tcgtaaactt 3360


 


caggatttat cgggcattga tcctattact attcctgctg atgatccggg agttatggct 3420


 


ctcttttctg ggacagaggt tttgggcgtt accccggaac aaattgggac accgactggt 3480


 


atgctaggca ttccagaatt tggaaccaac tttgttcgcg gcatggttaa tgagacgcat 3540


 


ccgaccactt ttgcggagct tttgcagctg tctggactat ctcatggaac cgatgtttgg 3600


 


cttggtaatg cacaagattc gattaaagaa ggcattgcaa ccctaaaaac cgttatcggc 3660


 


tgtcgtgacg acatcatggt ttacctcatg cacgcaggct tagaaccaaa aatggccttt 3720


 


accattatgg agcgtgtgcg taagggatta tggctaaaaa tttctgagga agaacgtaat 3780


 


ggctatattg atgccatgcg agaaaacaat gtgcccgact ggtacattga atcgtgtgga 3840


 


aaaatcaagt acatgttccc taaagcccat gcggcagctt atgttttgat ggcccttcgg 3900


 


gtggcttatt tcaaggtgca ccaccccatt atgtattatt gtgcttattt ctctattcgt 3960


 


gcgaaggctt ttgaattaaa aaccatgagt ggtggtttag atgctgttaa agcaagaatg 4020


 


gaagatatta ctataaaacg taaaaataat gaagccacca atgtggaaaa tgacctcttt 4080


 


acaaccttgg agattgtcaa cgaaatgtta gaacgcggct ttaagtttgg caaattagac 4140


 


ctttacaaaa gtgatgctat agaattccaa atcaaaggag atacccttat ccctccattt 4200


 


atagcgctag aaggtctggg tgaaaacgtg gccaagcaaa tcgttaaagc tcgtcaagaa 4260


 


ggcgaattcc tctctaaaat ggaattgcgt aaacgaggcg gggcatcgtc aacgctcgtt 4320


 


gagaaaatgg atgagacggg tattttagga aatatgccag aagataatca attaagtctt 4380


 


tttgatgact ttttc                                                  4395






The encoded α-large subunit has an amino acid sequence corresponding to SEQ. ID. No. 18 as follows:









Met Ser Asp Leu Phe Ala Lys Leu Met Asp Gln Ile Glu Met Pro Leu



  1               5                  10                  15


 


Asp Met Arg Arg Ser Ser Ala Phe Ser Ser Ala Asp Ile Ile Glu Val


             20                  25                  30


 


Lys Val His Ser Val Ser Arg Leu Trp Glu Phe His Phe Ala Phe Ala


         35                  40                  45


 


Ala Val Leu Pro Ile Ala Thr Tyr Arg Glu Leu His Asp Arg Leu Ile


     50                  55                  60


 


Arg Thr Phe Glu Ala Ala Asp Ile Lys Val Thr Phe Asp Ile Gln Ala


 65                  70                  75                  80


 


Ala Gln Val Asp Tyr Ser Asp Asp Leu Leu Gln Ala Tyr Tyr Gln Glu


                 85              90                      95


 


Ala Phe Glu His Ala Pro Cys Asn Ser Ala Ser Phe Lys Ser Ser Phe


            100                 105                 110


 


Ser Lys Leu Lys Val Thr Tyr Glu Asp Asp Lys Leu Ile Ile Ala Ala


        115                 120                 125


 


Pro Gly Phe Val Asn Asn Asp His Phe Arg Asn Asn His Leu Pro Asn


    130                 135                 140


 


Leu Val Lys Gln Leu Glu Ala Phe Gly Phe Gly Ile Leu Thr Ile Asp


145                 150                 155                 160


 


Met Val Ser Asp Gln Glu Met Thr Glu His Leu Thr Lys Asn Phe Val


                165                 170                 175


 


Ser Ser Arg Gln Ala Leu Val Lys Lys Ala Val Gln Asp Asn Leu Glu


            180                 185                 190


 


Ala Gln Lys Ser Leu Glu Ala Met Met Pro Pro Val Glu Glu Ala Thr


        195                 200                 205


 


Pro Ala Pro Lys Phe Asp Tyr Lys Glu Arg Ala Ala Lys Arg Gln Ala


    210                 215                 220


 


Gly Phe Glu Lys Ala Thr Ile Thr Pro Met Ile Glu Ile Glu Thr Glu


225                 230                 235                 240


 


Glu Asn Arg Ile Val Phe Glu Gly Met Val Phe Asp Val Glu Arg Lys


                245                 250                 255


 


Thr Thr Arg Thr Gly Arg His Ile Ile Asn Phe Lys Met Thr Asp Tyr


            260                 265                 270


 


Thr Ser Ser Phe Ala Leu Gln Lys Trp Ala Lys Asp Asp Glu Glu Leu


        275                 280                 285


 


Arg Lys Phe Asp Met Ile Ala Lys Gly Ala Trp Leu Arg Val Gln Gly


    290                 295                 300


 


Asn Ile Glu Thr Asn Pro Phe Thr Lys Ser Leu Thr Met Asn Val Gln


305                 310                 315                 320


 


Gln Val Lys Glu Ile Val Arg His Glu Arg Lys Asp Leu Met Pro Glu


                325                 330                 335


 


Gly Gln Lys Arg Val Glu Leu His Ala His Thr Asn Met Ser Thr Met


            340                 345                 350


 


Asp Ala Leu Pro Thr Val Glu Ser Leu Ile Asp Thr Ala Ala Lys Trp


        355                 360                 365


 


Gly His Lys Ala Ile Ala Ile Thr Asp His Ala Asn Val Gln Ser Phe


    370                 375                 380


 


Pro His Gly Tyr His Arg Ala Arg Lys Ala Gly Ile Lys Ala Ile Phe


385                 390                 395                 400


 


Gly Leu Glu Ala Asn Ile Val Glu Asp Lys Val Pro Ile Ser Tyr Glu


                405                 410                 415


 


Pro Val Asp Met Asp Leu His Glu Ala Thr Tyr Val Val Phe Asp Val


            420                 425                 430


 


Glu Thr Thr Gly Leu Ser Ala Met Asn Asn Asp Leu Ile Gln Ile Ala


        435                 440                 445


 


Ala Ser Lys Met Phe Lys Gly Asn Ile Val Glu Gln Phe Asp Glu Phe


    450                 455                 460


 


Ile Asp Pro Gly His Pro Leu Ser Ala Phe Thr Thr Glu Leu Thr Gly


465                 470                 475                 480


 


Ile Thr Asp Lys His Leu Gln Gly Ala Lys Pro Leu Val Thr Val Leu


                485                 490                 495


 


Lys Ala Phe Gln Asp Phe Cys Lys Asp Ser Ile Leu Val Ala His Asn


            500                 505                 510


 


Ala Ser Phe Asp Val Gly Phe Met Asn Ala Asn Tyr Glu Arg His Asp


        515                 520                 525


 


Leu Pro Lys Ile Thr Gln Pro Val Ile Asp Thr Leu Glu Phe Ala Arg


    530                 535                 540


 


Asn Leu Tyr Pro Glu Tyr Lys Arg His Gly Leu Gly Pro Leu Thr Lys


545                 550                 555                 560


 


Arg Phe Gln Val Ser Leu Asp His His His Met Ala Asn Tyr Asp Ala


                565                 570                 575


 


Glu Ala Thr Gly Arg Leu Leu Phe Ile Phe Leu Lys Asp Ala Arg Glu


            580                 585                 590


 


Lys His Gly Ile Lys Asn Leu Leu Gln Leu Asn Thr Asp Leu Val Ala


        595                 600                 605


 


Glu Asp Ser Tyr Lys Lys Ala Arg Ile Lys His Ala Thr Ile Tyr Val


    610                 615                 620


 


Gln Asn Gln Val Gly Leu Lys Asn Met Phe Lys Leu Val Ser Leu Ser


625                 630                 635                 640


 


Asn Ile Lys Tyr Phe Glu Gly Val Pro Arg Ile Pro Arg Thr Val Leu


                645                 650                 655


 


Asp Ala His Arg Glu Gly Leu Leu Leu Gly Thr Ala Cys Ser Asp Gly


            660                 665                 670


 


Glu Val Phe Asp Ala Val Leu Thr Lys Gly Ile Asp Ala Ala Val Asp


        675                 680                 685


 


Leu Ala Arg Tyr Tyr Asp Phe Ile Glu Ile Met Pro Pro Ala Ile Tyr


    690                 695                 700


 


Gln Pro Leu Val Val Arg Glu Leu Ile Lys Asp Gln Ala Gly Ile Glu


705                 710                 715                 720


 


Gln Val Ile Arg Asp Leu Ile Glu Val Gly Lys Arg Ala Lys Lys Pro


                725                 730                 735


 


Val Leu Ala Thr Gly Asn Val His Tyr Leu Glu Pro Glu Glu Glu Ile


            740                 745                 750


 


Tyr Arg Glu Ile Ile Val Arg Ser Leu Gly Gln Gly Ala Met Ile Asn


        755                 760                 765


 


Arg Thr Ile Gly Arg Gly Glu Gly Ala Gln Pro Ala Pro Leu Pro Lys


    770                 775                 780


 


Ala His Phe Arg Thr Thr Asn Glu Met Leu Asp Glu Phe Ala Phe Leu


785                 790                 795                 800


 


Gly Lys Asp Leu Ala Tyr Gln Val Val Val Gln Asn Thr Gln Asp Phe


                805                 810                 815


 


Ala Asp Arg Ile Glu Glu Val Glu Val Val Lys Gly Asp Leu Tyr Thr


            820                 825                 830


 


Pro Tyr Ile Asp Lys Ala Glu Glu Thr Val Ala Glu Leu Thr Tyr Gln


        835                 840                 845


 


Lys Ala Phe Glu Ile Tyr Gly Asn Pro Leu Pro Asp Ile Ile Asp Leu


    850                 855                 860


 


Arg Ile Glu Lys Glu Leu Thr Ser Ile Leu Gly Asn Gly Phe Ala Val


865                 870                 875                 880


 


Ile Tyr Leu Ala Ser Gln Met Leu Val Asn Arg Ser Asn Glu Arg Gly


                885                 890                 895


 


Tyr Leu Val Gly Ser Arg Gly Ser Val Gly Ser Ser Phe Val Ala Thr


            900                 905                 910


 


Met Ile Gly Ile Thr Glu Val Asn Pro Met Pro Pro His Tyr Val Cys


        915                 920                 925


 


Pro Ser Cys Gln His Ser Glu Phe Ile Thr Asp Gly Ser Val Gly Ser


    930                 935                 940


 


Gly Tyr Asp Leu Pro Asn Lys Pro Cys Pro Lys Cys Gly Thr Pro Tyr


945                 950                 955                 960


 


Gln Lys Asp Gly Gln Asp Ile Pro Phe Glu Thr Phe Leu Gly Phe Asp


                965                 970                 975


 


Gly Asp Lys Val Pro Asp Ile Asp Leu Asn Phe Ser Gly Asp Asp Gln


            980                 985                 990


 


Pro Ser Ala His Leu Asp Val Arg Asp Ile Phe Gly Asp Glu Tyr Ala


        995                1000                1005


 


Phe Arg Ala Gly Thr Val Gly Thr Val Ala Glu Lys Thr Ala Tyr Gly


   1010                1015                1020


 


Phe Val Lys Gly Tyr Glu Arg Asp Tyr Gly Lys Phe Tyr Arg Asp Ala


1025               1030                1035                1040


 


Glu Val Asp Arg Leu Ala Ala Gly Ala Ala Gly Val Lys Arg Thr Thr


               1045                1050                1055


 


Gly Gln His Pro Gly Gly Ile Val Val Ile Pro Asn Tyr Met Asp Val


           1060            1065                    1070


 


Tyr Asp Phe Thr Pro Val Gln Tyr Pro Ala Asp Asp Val Thr Ala Ser


       1075                1080                1085


 


Trp Gln Thr Thr His Phe Asn Phe His Asp Ile Asp Glu Asn Val Leu


   1090                1095                1100


 


Lys Leu Asp Ile Leu Gly His Asp Asp Pro Thr Met Ile Arg Lys Leu


1105               1110                1115                1120


 


Gln Asp Leu Ser Gly Ile Asp Pro Ile Thr Ile Pro Ala Asp Asp Pro


               1125                1130                1135


 


Gly Val Met Ala Leu Phe Ser Gly Thr Glu Val Leu Gly Val Thr Pro


           1140                1145                1150


 


Glu Gln Ile Gly Thr Pro Thr Gly Met Leu Gly Ile Pro Glu Phe Gly


       1155                1160                1165


 


Thr Asn Phe Val Arg Gly Met Val Asn Glu Thr His Pro Thr Thr Phe


   1170                1175                1180


 


Ala Glu Leu Leu Gln Leu Ser Gly Leu Ser His Gly Thr Asp Val Trp


1185               1190                1195                1200


 


Leu Gly Asn Ala Gln Asp Leu Ile Lys Glu Gly Ile Ala Thr Leu Lys


               1205                1210                1215


 


Thr Val Ile Gly Cys Arg Asp Asp Ile Met Val Tyr Leu Met His Ala


           1220                1225                1230


 


Gly Leu Glu Pro Lys Met Ala Phe Thr Ile Met Glu Arg Val Arg Lys


       1235                1240                1245


 


Gly Leu Trp Leu Lys Ile Ser Glu Glu Glu Arg Asn Gly Tyr Ile Asp


   1250                1255                1260


 


Ala Met Arg Glu Asn Asn Val Pro Asp Trp Tyr Ile Glu Ser Cys Gly


1265               1270                1275                1280


 


Lys Ile Lys Tyr Met Phe Pro Lys Ala His Ala Ala Ala Tyr Val Leu


               1285                1290                1295


 


Met Ala Leu Arg Val Ala Tyr Phe Lys Val His His Pro Ile Met Tyr


           1300                1305                1310


 


Tyr Cys Ala Tyr Phe Ser Ile Arg Ala Lys Ala Phe Glu Leu Lys Thr


       1315                1320                1325


 


Met Ser Gly Gly Leu Asp Ala Val Lys Ala Arg Met Glu Asp Ile Thr


   1330                1335                1340


 


Ile Lys Arg Lys Asn Asn Glu Ala Thr Asn Val Glu Asn Asp Leu Phe


1345               1350                1355                1360


 


Thr Thr Leu Glu Ile Val Asn Glu Met Leu Glu Arg Gly Phe Lys Phe


               1365                1370                1375


 


Gly Lys Leu Asp Leu Tyr Lys Ser Asp Ala Ile Glu Phe Gln Ile Lys


           1380                1385                1390


 


Gly Asp Thr Leu Ile Pro Pro Phe Ile Ala Leu Glu Gly Leu Gly Glu


       1395                1400                1405


 


Asn Val Ala Lys Gln Ile Val Lys Ala Arg Gln Glu Gly Glu Phe Leu


   1410                1415                1420


 


Ser Lys Met Glu Leu Arg Lys Arg Gly Gly Ala Ser Ser Thr Leu Val


1425               1430                1435                1440


 


Glu Lys Met Asp Glu Met Gly Ile Leu Gly Asn Met Pro Glu Asp Asn


               1445                1450                1455


 


Gln Leu Ser Leu Phe Asp Asp Phe Phe


           1460                1465






The present invention also relates to the dnaE gene of Streptococcus pyogenes encoding the α-small subunit. The partial nucleotide sequence of the dnaE gene corresponds to SEQ. ID. No. 19 as follows:








atgtttgctc aacttgatac taaaactgta tactcattta tggatagttt aattgactta   60



 


aatcattatt ttgaacgagc aaagcaattt ggttaccaca ccataggaat catggataag  120


 


gataatcttt atggtgctta ccattttatt aaaggttgtc aaaaaaatgg actgcagcca  180


 


gttttaggtt tggaaataga gattctctat caagagcggc aggtgctcct taacttaatc  240


 


gcccagaata cacaaggcta tcatcagctt ttaaaaattt ccacggcaaa aatgtctggc  300


 


aagcttcata tggattactt ctgccaacat ttggaaggga tagcggttat tattcctagt  360


 


aagggttgga gcgatacatt agtggtccct tttgactact atatgggtgt tgatcagtat  420


 


actgatttat ctcatatgga ttctaagagg cagcttatac ccctaaggac agttcgttat  480


 


tttgcgcaag atgatatgga aaccctgcac atgttgcatg ccattcgaga taacctcagt  540


 


ctggcagaga cccctgtggt agaaagtgat caagagttag cagattgtca acaactaacc  600


 


gccttctatc aaacacactg ccctcaagct ctacagaatt tagaagactt agtgtcagga  660


 


atctattatg atttcgatac aaatttaaaa ttgcctcatt ttaatagaga taagtctgcc  720


 


aagcaagaat tgcaagactt gactgaggct ggtttgaagg aaaaaggatt gtggaaagag  780


 


ccttatcaat cgcgcttact acatgaactg gtcattattt ctgacatggg ctttgatgat  840


 


tattttttga ttgtgtggga tttacttcgc tttggacgca gtaaaggcta ttatatggga  900


 


atgggacgtg gctcggcggc aggtagtcta gtggcttatg ctctgaacat tacagggatt  960


 


gatccagttc aacatgattt gctatttgag cgctttttaa acaaagaacg ttatagcatg 1020


 


cctgatattg atatcgatct tccagatatt taccgttcag aatttctacg gtatgtccga 1080


 


aatcgttatg gtagcgacca ttcggcgcaa attgtgacct tttcaacctt tggccaggct 1140


 


attcgtgatg ttttcaaacg gttcggggtt ccagaatacg aactgactaa tctcactaaa 1200


 


aaaattggtt ttaaagatag cttggctact gtctatgaaa agtcaatctc ttttaggcag 1260


 


gttattaata gtagaactga atttcaaaag gcttttgcca ttgccaagcg tatcgaagga 1320


 


aatccaagac aaacgtccat tcacgcagct ggtattgtga tgagtgatga tgccttgacc 1380


 


aatcatattc ctctaaaatc gggcgatgac atgatgatca cccagtatga tgctcatgcg 1440


 


gtcgaagcta atggcctgtt aaaaatggat tttttggggt taagaaattt gacctttgtt 1500


 


caaaaaatgc aagagaaggt tgctaaagac tacgggtgtc agattgatat tacagccatt 1560


 


gatttagaag acccgcaaac gttggcactt tttgctaaag gggataccaa gggaattttc 1620


 


caatttgaac aaaatggtgc tattaatctt ttaaaacgga ttaagccaca acgttttgaa 1680


 


gaaattgttg ccactaccag tctaaataga ccaggggcaa gtgactatac cactaatttc 1740


 


attaaacgaa gagaaggaca agaaaaaatt gatttgattg atcctgtgat tgctcccatt 1800


 


ttagagccaa cttacggtat tatgctttat caagaacaag ttatgcagat tgcacaggtt 1860


 


tatgctggtt ttacgttagg caaggccgac ttgttaaggc gtgccatgtc taaaaaaaat 1920


 


ctacaagaaa tgcaaaaaat ggaagaagac tttattgctt ctgctaagca cctagggaga 1980


 


gctgaagaaa cagctagagg actttttaaa cggatggaaa aatttgcagg ttatggtttt 2040


 


aaccgcagcc atgcctttgc ctattcagct ttagcttttc aattggctta tttcaaagcc 2100


 


cattacccgg ctgtttttta cgatatcatg atgaattatt ctagcagtga ctatatcaca 2160


 


gatgctctag aatcagattt tcaagtagcg caagttacca ttaatagtat tccttacact 2220


 


gataaaattg aagctagcaa gatttacatg gggctgaaaa atattaaggg gttgccaagg 2280


 


gattttgctt attggattat cgagcaaaga ccatttaata gcgtagagga ttttctcact 2340


 


agaactccag aaaaatatca aaaaaaggtt ttccttgagc ctctgataaa aataggtctg 2400


 


tttgattgct ttgagcctaa ccgtaaaaaa attctggaca atttggatgg tttactggta 2460


 


tttgttaacg agcttggttc tcttttttca gattcttcct ttagttgggt agatacgaaa 2520


 


gattactcag taactgaaaa atattctttg gaacaggaga tcgttggagt tggcatgagc 2580


 


aagcatcctt taattgatat tgctgagaaa agtacccaaa cttttactcc tatttcacag 2640


 


ttagtcaaag aaagcgaagc agtcgtactg attcaaatag atagcattag gatcattaga 2700


 


accaaaacaa gtgggcagca aatggctttt ttaagtgtga atgacactaa gaaaaagctc 2760


 


gatgtcacac tttttccaca agagtatgcc atttataaag accaattaaa agaaggagaa 2820


 


ttctattact taaaaggtag aataaaagaa agagaccatc gactgcagat ggtgtgtcag 2880


 


caagtgcaaa tggctattag tcaaaaatat tggttattag ttgaaaacca tcagtttgat 2940


 


tcccaaattt ctgagatttt aggtgccttt ccaggaacga ctccagttgt tattcactat 3000


 


caaaaaaata aggaaacaat tgcattaact aagattcagg ttcatgtaac agagaattta 3060


 


aaggaaaaac ttcgtccttt tgttctgaaa acggtttttc ga                    3102






The encoded α-small subunit has an amino acid sequence corresponding to SEQ. ID. No. 20 as follows:









Met Phe Ala Gln Leu Asp Thr Lys Thr Val Tyr Ser Phe Met Asp Ser



  1               5                  10                  15


 


Leu Ile Asp Leu Asn His Tyr Phe Glu Arg Ala Lys Gln Phe Gly Tyr


             20                  25                  30


 


His Thr Ile Gly Ile Met Asp Lys Asp Asn Leu Tyr Gly Ala Tyr His


         35                  40                  45


 


Phe Ile Lys Gly Cys Gln Lys Asn Gly Leu Gln Pro Val Leu Gly Leu


     50                  55                  60


 


Glu Ile Glu Ile Leu Tyr Gln Glu Arg Gln Val Leu Leu Asn Leu Ile


 65                  70                  75                  80


 


Ala Gln Asn Thr Gln Gly Tyr His Gln Leu Leu Lys Ile Ser Thr Ala


                 85                  90                  95


 


Lys Met Ser Gly Lys Leu His Met Asp Tyr Phe Cys Gln His Leu Glu


            100                 105                 110


 


Gly Ile Ala Val Ile Ile Pro Ser Lys Gly Trp Ser Asp Thr Leu Val


        115                 120                 125


 


Val Pro Phe Asp Tyr Tyr Met Gly Val Asp Gln Tyr Thr Asp Leu Ser


    130                 135                 140


 


His Met Asp Ser Lys Arg Gln Leu Ile Pro Leu Arg Thr Val Arg Tyr


145                 150                 155                 160


 


Phe Ala Gln Asp Asp Met Glu Thr Leu His Met Leu His Ala Ile Arg


                165                 170                 175


 


Asp Asn Leu Ser Leu Ala Glu Thr Pro Val Val Glu Ser Asp Gln Glu


            180                 185                 190


 


Leu Ala Asp Cys Gln Gln Leu Thr Ala Phe Tyr Gln Thr His Cys Pro


        195                 200                 205


 


Gln Ala Leu Gln Asn Leu Glu Asp Leu Val Ser Gly Ile Tyr Tyr Asp


    210                 215                 220


 


Phe Asp Thr Asn Leu Lys Leu Pro His Phe Asn Arg Asp Lys Ser Ala


225                 230                 235                 240


 


Lys Gln Glu Leu Gln Asp Leu Thr Glu Ala Gly Leu Lys Glu Lys Gly


                245                 250                 255


 


Leu Trp Lys Glu Pro Tyr Gln Ser Arg Leu Leu His Glu Leu Val Ile


            260                 265                 270


 


Ile Ser Asp Met Gly Phe Asp Asp Tyr Phe Leu Ile Val Trp Asp Leu


        275                 280                 285


 


Leu Arg Phe Gly Arg Ser Lys Gly Tyr Tyr Met Gly Met Gly Arg Gly


    290                 295                 300


 


Ser Ala Ala Gly Ser Leu Val Ala Tyr Ala Leu Asn Ile Thr Gly Ile


305                 310                 315                 320


 


Asp Pro Val Gln His Asp Leu Leu Phe Glu Arg Phe Leu Asn Lys Glu


                325                 330                 335


 


Arg Tyr Ser Met Pro Asp Ile Asp Ile Asp Leu Pro Asp Ile Tyr Arg


            340                 345                 350


 


Ser Glu Phe Leu Arg Tyr Val Arg Asn Arg Tyr Gly Ser Asp His Ser


        355                 360                 365


 


Ala Gln Ile Val Thr Phe Ser Thr Phe Gly Pro Lys Gln Ala Ile Arg


    370                 375                 380


 


Asp Val Phe Lys Arg Phe Gly Val Pro Glu Tyr Glu Leu Thr Asn Leu


385                 390                 395                 400


 


Thr Lys Lys Ile Gly Phe Lys Asp Ser Leu Ala Thr Val Tyr Glu Lys


                405                 410                 415


 


Ser Ile Ser Phe Arg Gln Val Ile Asn Ser Arg Thr Glu Phe Gln Lys


            420                 425                 430


 


Ala Phe Ala Ile Ala Lys Arg Ile Glu Gly Asn Pro Arg Gln Thr Ser


        435                 440                 445


 


Ile His Ala Ala Gly Ile Val Met Ser Asp Asp Ala Leu Thr Asn His


    450                 455                 460


 


Ile Pro Leu Lys Ser Gly Asp Asp Met Met Ile Thr Gln Tyr Asp Ala


465                 470                 475                 480


 


His Ala Val Glu Ala Asn Gly Leu Leu Lys Met Asp Phe Leu Gly Leu


                485                 490                 495


 


Arg Asn Leu Thr Phe Val Gln Lys Met Gln Glu Lys Val Ala Lys Asp


            500                 505                 510


 


Tyr Gly Cys Gln Ile Asp Ile Thr Ala Ile Asp Leu Glu Asp Pro Gln


        515                 520                 525


 


Thr Leu Ala Leu Phe Ala Lys Gly Asp Thr Lys Gly Ile Phe Gln Phe


    530                 535                 540


 


Glu Gln Asn Gly Ala Ile Asn Leu Leu Lys Arg Ile Lys Pro Gln Arg


545                 550                 555                 560


 


Phe Glu Glu Ile Val Ala Thr Thr Ser Leu Asn Arg Pro Gly Ala Ser


                565                 570                 575


 


Asp Tyr Thr Thr Asn Phe Ile Lys Arg Arg Glu Gly Gln Glu Lys Ile


            580                 585                 590


 


Asp Leu Ile Asp Pro Val Ile Ala Pro Ile Leu Glu Pro Thr Tyr Gly


        595                 600                 605


 


Ile Met Leu Tyr Gln Glu Gln Val Met Gln Ile Ala Gln Val Tyr Ala


    610                 615                 620


 


Gly Phe Thr Leu Gly Lys Ala Asp Leu Leu Arg Arg Ala Met Ser Lys


625                 630                 635                 640


 


Lys Asn Leu Gln Glu Met Gln Lys Met Glu Glu Asp Phe Ile Ala Ser


                645                 650                 655


 


Ala Lys His Leu Gly Arg Ala Glu Glu Thr Ala Arg Gly Leu Phe Lys


            660                 665                 670


 


Arg Met Glu Lys Phe Ala Gly Tyr Gly Phe Asn Arg Ser His Ala Phe


        675                 680                 685


 


Ala Tyr Ser Ala Leu Ala Phe Gln Leu Ala Tyr Phe Lys Ala His Tyr


    690                 695                 700


 


Pro Ala Val Phe Tyr Asp Ile Met Met Asn Tyr Ser Ser Ser Asp Tyr


705                 710                 715                 720


 


Ile Thr Asp Ala Leu Glu Ser Asp Phe Gln Val Ala Gln Val Thr Ile


                725                 730                 735


 


Asn Ser Ile Pro Tyr Thr Asp Lys Ile Glu Ala Ser Lys Ile Tyr Met


            740                 745                 750


 


Gly Leu Lys Asn Ile Lys Gly Leu Pro Arg Asp Phe Ala Tyr Trp Ile


        755                 760                 765


 


Ile Glu Gln Arg Pro Phe Asn Ser Val Glu Asp Phe Leu Thr Arg Thr


    770                 775                 780


 


Pro Glu Lys Tyr Gln Lys Lys Val Phe Leu Glu Pro Leu Ile Lys Ile


785                 790                 795                 800


 


Gly Leu Phe Asp Cys Phe Glu Pro Asn Arg Lys Lys Ile Leu Asp Asn


                805                 810                 815


 


Leu Asp Gly Leu Leu Val Phe Val Asn Glu Leu Gly Ser Leu Phe Ser


            820                 825                 830


 


Asp Ser Ser Phe Ser Trp Val Asp Thr Lys Asp Tyr Ser Val Thr Glu


        835                 840                 845


 


Lys Tyr Ser Leu Glu Gln Glu Ile Val Gly Val Gly Met Ser Lys His


    850                 855                 860


 


Pro Leu Ile Asp Ile Ala Glu Lys Ser Thr Gln Thr Phe Thr Pro Ile


865                 870                 875                 880


 


Ser Gln Leu Val Lys Glu Ser Glu Ala Val Val Leu Ile Gln Ile Asp


                885                 890                 895


 


Ser Ile Arg Ile Ile Arg Thr Lys Thr Ser Gly Gln Gln Met Ala Phe


            900                 905                 910


 


Leu Ser Val Asn Asp Thr Lys Lys Lys Leu Asp Val Thr Leu Phe Pro


        915                 920                 925


 


Gln Glu Tyr Ala Ile Tyr Lys Asp Gln Leu Lys Glu Gly Glu Phe Tyr


    930                 935                 940


 


Tyr Leu Lys Gly Arg Ile Lys Glu Arg Asp His Arg Leu Gln Met Val


945                 950                 955                 960


 


Cys Gln Gln Val Gln Met Ala Ile Ser Gln Lys Tyr Trp Leu Leu Val


                965                 970                 975


 


Glu Asn His Gln Phe Asp Ser Gln Ile Ser Glu Ile Leu Gly Ala Phe


            980                 985                 990


 


Pro Gly Thr Thr Pro Val Val Ile His Tyr Gln Lys Asn Lys Glu Thr


        995                1000                1005


 


Ile Ala Leu Thr Lys Ile Gln Val Thr Glu Asn Leu Lys Glu Lys Leu


   1010                1015                1020


 


Arg Pro Phe Val Leu Lys Thr Val Phe Arg


1025               1030






The present invention also relates to the holA gene of Streptococcus pyogenes encoding the δ subunit. The holA gene has a nucleotide sequence which corresponds to SEQ. ID. No. 21 as follows:








atgattgcga tagaaaagat tgaaaaactg agtaaagaaa atttgggtct tataaccctt   60



 


gtcacaggag atgacattgg tcagtatagc cagttgaaat cccgcttaat ggagcagatt  120


 


gcttttgata aggatgattt ggcctattct tactttgata tgtctgaggc cgcttatcag  180


 


gatgcagaaa tggatctagt gagcctaccc ttctttgctg agcagaaggt ggttattttt  240


 


gaccatttgt tagatatcac gaccaataaa aaaagtttct taaaagaaaa agacctaaag  300


 


gcctttgaag cctatttaga aaatccctta gagactactc gactaattat ctttgctcca  360


 


ggtaaattgg atagtaagag acggcttgtt aagcttttga aacgtgatgc ccttgtttta  420


 


gaagccaacc ctctgaaaga agcagagcta agaacttatt ttcaaaaata cagtcatcaa  480


 


ctgggtttag gtttcgagag tggtgccttt gaccaattac ttttgaaatc aaacgatgat  540


 


tttagtcaaa tcatgaaaaa catggccttt ttaaaagcct ataaaaaaac gggaaatatt  600


 


agcctaactg atattgagca agccattcct aaaagtttac aagataatat tttcgatctg  660


 


actagacttg tcctaggagg taaaattgat gcggctagag atttgattca tgatttacgg  720


 


ttatctggag aagatgacat taaattaatc gctatcatgc taggceaatt tcgcttattt  780


 


ttgcagctga ctattcttgc tagagatgta aaaaacgagc aacaactagt gattagttta  840


 


tcagatattc ttgggcggcg ggttaatcct taccaggtca agtatgcgtt aaaggattct  900


 


aggaccttat ctcttgcctt tctaacagga gcggtgaaaa ccttgattga gacagattac  960


 


cagataaaaa caggacttta tgagaagagt tatctagttg atattgctct cttaaaaatc 1020


 


atgactcact ctcaaaaa                                               1038






The encoded δ subunit has an amino acid sequence corresponding to SEQ. ID. No. 22 as follows:









Met Ile Ala Ile Glu Lys Ile Glu Lys Leu Ser Lys Glu Asn Leu Gly



  1               5                  10                  15


 


Leu Ile Thr Leu Val Thr Gly Asp Asp Ile Gly Gln Tyr Ser Gln Leu


             20                  25                  30


 


Lys Ser Arg Leu Met Glu Gln Ile Ala Phe Asp Lys Asp Asp Leu Ala


         35                  40                  45


 


Tyr Ser Tyr Phe Asp Met Ser Glu Ala Ala Tyr Gln Asp Ala Glu Met


     50                  55                  60


 


Asp Leu Val Ser Leu Pro Phe Phe Ala Glu Gln Lys Val Val Ile Phe


 65                  70                  75                  80


 


Asp His Leu Leu Asp Ile Thr Thr Asn Lys Lys Ser Phe Leu Lys Glu


                 85                  90                  95


 


Lys Asp Leu Lys Ala Phe Glu Ala Tyr Leu Glu Asn Pro Leu Glu Thr


            100                 105                 110


 


Thr Arg Leu Ile Ile Phe Ala Pro Gly Lys Leu Asp Ser Lys Arg Arg


        115                 120                 125


 


Leu Val Lys Leu Leu Lys Arg Asp Ala Leu Val Leu Glu Ala Asn Pro


    130                 135                 140


 


Leu Lys Glu Ala Glu Leu Arg Thr Tyr Phe Gln Lys Tyr Ser His Gln


145                 150                 155                 160


 


Leu Gly Leu Gly Phe Glu Ser Gly Ala Phe Asp Gln Leu Leu Leu Lys


                165                 170                 175


 


Ser Asn Asp Asp Phe Ser Gln Ile Met Lys Asn Met Ala Phe Leu Lys


            180                 185                 190


 


Ala Tyr Lys Lys Thr Gly Asn Ile Ser Leu Thr Asp Ile Glu Gln Ala


        195                 200                 205


 


Ile Pro Lys Ser Leu Gln Asp Asn Ile Phe Asp Leu Thr Arg Leu Val


    210                 215                 220


 


Leu Gly Gly Lys Ile Asp Ala Ala Arg Asp Leu Ile His Asp Leu Arg


225                 230                 235                 240


 


Leu Ser Gly Glu Asp Asp Ile Lys Leu Ile Ala Ile Met Leu Gly Gln


                245                 250                 255


 


Phe Arg Leu Phe Leu Gln Leu Thr Ile Leu Ala Arg Asp Val Lys Asn


            260                 265                 270


 


Glu Gln Gln Leu Val Ile Ser Leu Ser Asp Ile Leu Gly Arg Arg Val


        275                 280                 285


 


Asn Pro Tyr Gln Val Lys Tyr Ala Leu Lys Asp Ser Arg Thr Leu Ser


    290                 295                 300


 


Leu Ala Phe Leu Thr Gly Ala Val Lys Thr Leu Ile Glu Thr Asp Tyr


305                 310                 315                 320


 


Gln Ile Lys Thr Gly Leu Tyr Glu Lys Ser Tyr Leu Val Asp Ile Ala


                325                 330                 335


 


Leu Leu Lys Ile Met Thr His Ser Gln Lys


            340                 345






The present invention also relates to the holB gene of Streptococcus pyogenes encoding the δ′ subunit. The holB gene has a nucleotide sequence which corresponds to SEQ. ID. No. 23 as follows:








atggatttag cgcaaaaagc tcctaacgtt tatcaagctt ttcagacaat tttaaagaaa  60



 


gaccgtctga atcatgctta tcttttttcg ggtgattttg ctaatgaaga aatggctctt 120


 


tttttagcta aggtcatctt ttgtgaacag aaaaaggatc agacgccctg cgggcattgt 180


 


cgctcttgtc aattgattga acaaggagat tttgccgatg tgacggtatt ggaaccaaca 240


 


gggcaagtga ttaaaacgga tgtggtcaaa gaaatgatgg ctaacttttc tcagacagga 300


 


tatgaaaaca aacgacaagt ttttattatc aaagattgtg acaaaatgca tatcaatgcc 360


 


gctaatagct tgctaaaata cattgaggag cctcagggag aagcttacat atttttattg 420


 


accaatgatg ataacaaagt gcttccgacc attaaaagtc ggacacaggt ttttcagttt 480


 


cctaaaaacg aagcctatct ttaccaattg gcacaagaaa agggattatt aaaccatcag 540


 


gctaagctag tagccaaact tgccacaaac accagtcatc tagaacgtct gttgcaaacg 600


 


agcaagcttt tagaactgat aactcaagca gagcgttttg tatctatttg gctgaaagat 660


 


cagttgcagg catatttagc gttgaaccgt ctggtacagt tagcaactga aaaagaagaa 720


 


caagatttag ttttgaccct tttgaccttg ctcttggcaa gagagcgtgc gcaaacgcct 780


 


ttgacacaat tggaggctgt ctatcaggct aggctcatgt ggcagagcaa tgttaatttt 840


 


caaaacacat tagaatatat ggtgatgtca gaa                              873






The encoded δ′ subunit has an amino acid sequence corresponding to SEQ. ID. No. 24 as follows:









Met Asp Leu Ala Gln Lys Ala Pro Asn Val Tyr Gln Ala Phe Gln Thr



  1               5                  10                  15


 


Ile Leu Lys Lys Asp Arg Leu Asn His Ala Tyr Leu Phe Ser Gly Asp


             20                  25                  30


 


Phe Ala Asn Glu Glu Met Ala Leu Phe Leu Ala Lys Val Ile Phe Cys


         35                  40                  45


 


Glu Gln Lys Lys Asp Gln Thr Pro Cys Gly His Cys Arg Ser Cys Gln


     50                  55                  60


 


Leu Ile Glu Gln Gly Asp Phe Ala Asp Val Thr Val Leu Glu Pro Thr


 65                  70                  75                  80


 


Gly Gln Val Ile Lys Thr Asp Val Val Lys Glu Met Met Ala Asn Phe


                 85                  90                  95


 


Ser Gln Thr Gly Tyr Glu Asn Lys Arg Gln Val Phe Ile Ile Lys Asp


            100                 105                 110


 


Cys Asp Lys Met His Ile Asn Ala Ala Asn Ser Leu Leu Lys Tyr Ile 


        115                 120                 125


 


Glu Glu Pro Gln Gly Glu Ala Tyr Ile Phe Leu Leu Thr Asn Asp Asp


    130                 135                 140


 


Asn Lys Val Leu Pro Thr Ile Lys Ser Arg Thr Gln Val Phe Gln Phe


145                 150                 155                 160


 


Pro Lys Asn Glu Ala Tyr Leu Tyr Gln Leu Ala Gln Glu Lys Gly Leu


                165                 170                 175


 


Leu Asn His Gln Ala Lys Leu Val Ala Lys Leu Ala Thr Asn Thr Ser


            180                 185                 190


 


His Leu Glu Arg Leu Leu Gln Thr Ser Lys Leu Leu Glu Leu Ile Thr


        195                 200                 205


 


Gln Ala Glu Arg Phe Val Ser Ile Trp Leu Lys Asp Gln Leu Gln Ala


    210                 215                 220


 


Tyr Leu Ala Leu Asn Arg Leu Val Gln Leu Ala Thr Glu Lys Glu Glu


225                 230                 235                 240


 


Gln Asp Leu Val Leu Thr Leu Leu Thr Leu Leu Leu Ala Arg Glu Arg


                245                 250                 255


 


Ala Gln Thr Pro Leu Thr Gln Leu Glu Ala Val Tyr Gln Ala Arg Leu


            260                 265                 270


 


Met Trp Gln Ser Asn Val Asn Phe Gln Asn Thr Leu Glu Tyr Met Val


        275                 280                 285


 


Met Ser Glu


    290






The present invention also relates to the dnaX gene of Streptococcus pyogenes encoding the τ subunit. The dnaX gene has a nucleotide sequence which corresponds to SEQ. ID. No. 25 as follows:








atgtatcaag ctctttatcg gaaataccgg agccaaacgt ttgaegaaat ggtgggacaa   60



 


tcggttattt ccacaacttt aaagcaggca gttgaatctg gcaagattag ccatgcttat  120


 


cttttttcag gtcctagagg gactgggaaa acaagtgcgg caaagatttt tgcaaaggcc  180


 


atgaattgtc ctaaccaagt cgatggtgaa ccctgtaatc aatgcgatat ttgccgagat  240


 


atcacgaatg gaagcttgga agatgtgatt gaaattgatg ctgcctcgaa taatggtgtt  300


 


gatgaaattc gtgacattcg agacaaatca acctatgcgc caagtcgtgc gacttacaag  360


 


gtttatatta ttgatgaggt tcacatgtta tcaacagggg cttttaatgc gcttttgaaa  420


 


actttggaag aaccgacaga atgttgtctt tatcttggca acaacggaat gcataaaatt  480


 


ccagccacta ttttatctcg tgtgcaacgc tttgaattca aagctattaa gcaaaaagct  540


 


attcgagagc atttagcctg ggttttggac aaagaaggta ttgcctatga ggtggatgct  600


 


ttaaatctca ttgcaaggcg agcagaagga ggcatgcgtg atgctttatc tattttagat  660


 


caggctttga gcttgtcacc agataatcag gtcgccattg caattgccga agaaattaca  720


 


ggttctattt ccatacttgc tctgggtgac tatgttcgat atgtctccca agaacaggct  780


 


acgcaagctc tggcagcctt agagaccatt tatgatagtg ggaagagcat gagccgcttt  840


 


gcgacagatt tattgaccta tctgcgtgat ttattggtgg ttaaagctgg cggcgacaat  900


 


caacgtcagt cagctgtttt tgataccaat ttgtctctct cgatagatcg tatattccaa  960


 


atgataacag ttgttactag tcatctccct gaaatcaaaa agggaaccca tcctcggatt 1020


 


tatgccgaaa tgatgactat ccaattagct cagaaagagc agattttgtc ccaagtaaac 1080


 


ttgtcaggag agttaatctc agagattgaa acgctcaaaa atgagttggc acaacttaaa 1140


 


caacaattgt cgcagctcca atcgcgtcct gattcactgg caagatctga taaaacgaaa 1200


 


cctaaaacca caagctacag ggttgatcgg gttaccattt tgaaaatcat ggaagaaacg 1260


 


gttcgaaata gccaacaatc tcgacaatat ctagatgctc taaaaaatgc ttggaatgaa 1320


 


attctagata acatttctgc ccaagacaga gccttattga tgggctctga gcctgtctta 1380


 


gcaaatagtg agaatgcgat tttggctttc gaggctgcct ttaatgcaga acaagtcatg 1440


 


agccgaaata atcttaatga tatgtttggt aacattatga gtaaagctgc tggtttttct 1500


 


cccaatattc tggcagtacc aaggacagat tttcagcata ttcgtaagga atttgctcag 1560


 


caaatgaaat cgcaaaaaga cagtgttcaa gaagaacaag aagtagcgct tgatattcca 1620


 


gaagggtttg attttttgct cgataaaata aatactattg acgac                 1665






The encoded τ subunit has an amino acid sequence corresponding to SEQ. ID. No. 26 as follows:









Met Tyr Gln Ala Leu Tyr Arg Lys Tyr Arg Ser Gln Thr Phe Asp Glu



  1               5                  10                  15


 


Met Val Gly Gln Ser Val Ile Ser Thr Thr Leu Lys Gln Ala Val Glu


             20                  25                  30


 


Ser Gly Lys Ile Ser His Ala Tyr Leu Phe Ser Gly Pro Arg Gly Thr


         35                  40                  45


 


Gly Lys Thr Ser Ala Ala Lys Ile Phe Ala Lys Ala Met Asn Cys Pro


     50                  55                  60


 


Asn Gln Val Asp Gly Glu Pro Cys Asn Gln Cys Asp Ile Cys Arg Asp


 65                  70                  75                  80


 


Ile Thr Asn Gly Set Leu Glu Asp Val Ile Glu Ile Asp Ala Ala Ser


                 85                  90                  95


 


Asn Asn Gly Val Asp Glu Ile Arg Asp Ile Arg Asp Lys Ser Thr Tyr


            100                 105                 110


 


Ala Pro Ser Arg Ala Thr Tyr Lys Val Tyr Ile Ile Asp Glu Val His


        115                 120                 125


 


Met Leu Ser Thr Gly Ala Phe Asn Ala Leu Leu Lys Thr Leu Glu Glu


    130                 135                 140


 


Pro Thr Glu Asn Val Phe Ile Leu Ala Thr Thr Glu Leu His Lys Ile 


145                 150                 155                 160


 


Pro Ala Thr Ile Leu Set Arg Val Gln Arg Phe Glu Phe Lys Ala Ile 


                165                 170                 175


 


Lys Gln Lys Ala Ile Arg Glu His Leu Ala Trp Val Leu Asp Lys Glu


            180                 185                 190


 


Gly Ile Ala Tyr Glu Val Asp Ala Leu Asn Leu Ile Ala Arg Arg Ala


        195                 200                 205


 


Glu Gly Gly Met Arg Asp Ala Leu Set Ile Leu Asp Gln Ala Leu Set


    210                 215                 220


 


Leu Ser Pro Asp Asn Gln Val Ala Ile Ala Ile Ala Glu Glu Ile Thr


225                 230                 235                 240


 


Gly Ser Ile Set Ile Leu Ala Leu Gly Asp Tyr Val Arg Tyr Val Ser


                245                 250                 255


 


Gln Glu Gln Ala Thr Gln Ala Leu Ala Ala Leu Glu Thr Ile Tyr Asp


            260                 265                 270


 


Ser Gly Lys Set Met Set Arg Phe Ala Thr Asp Leu Leu Thr Tyr Leu


        275                 280                 285


 


Arg Asp Leu Leu Val Val Lys Ala Gly Gly Asp Asn Gln Arg Gln Ser


    290                 295                 300


 


Ala Val Phe Asp Thr Asn Leu Ser Leu Ser Ile Asp Arg Ile Phe Gln


305                 310                 315                 320


 


Met Ile Thr Val Val Thr Set His Leu Pro Glu Ile Lys Lys Gly Thr


                325                 330                 335


 


His Pro Arg Ile Tyr Ala Glu Met Met Thr Ile Gln Leu Ala Gln Lys


            340                 345                 350


 


Glu Gln Ile Leu Ser Gln Val Asn Leu Ser Gly Glu Leu Ile Ser Glu


        355                 360                 365


 


Ile Glu Thr Leu Lys Asn Glu Leu Ala Gln Leu Lys Gln Gln Leu Ser


    370                 375                 380


 


Gln Leu Gln Ser Arg Pro Asp Ser Leu Ala Arg Ser Asp Lys Thr Lys


385                 390                 395                 400


 


Pro Lys Thr Thr Ser Tyr Arg Val Asp Arg Val Thr Ile Leu Lys Ile 


                405                 410                 415


 


Met Glu Glu Thr Val Arg Asn Ser Gln Gln Ser Arg Gln Tyr Leu Asp


            420                 425                 430


 


Ala Leu Lys Asn Ala Trp Asn Glu Ile Leu Asp Asn Ile Ser Ala Gln


        435                 440                 445


 


Asp Arg Ala Leu Leu Met Gly Ser Glu Pro Val Leu Ala Asn Ser Glu


    450                 455                 460


 


Asn Ala Ile Leu Ala Phe Glu Ala Ala Phe Asn Ala Glu Gln Val Met


465                 470                 475                 480


 


Ser Arg Asn Asn Leu Asn Asp Met Phe Gly Asn Ile Met Ser Lys Ala


                485                 490                 495


 


Ala Gly Phe Ser Pro Asn Ile Leu Ala Val Pro Arg Thr Asp Phe Gln


            500                 505                 510


 


His Ile Arg Lys Glu Phe Ala Gln Gln Met Lys Ser Gln Lys Asp Ser


        515                 520                 525


 


Val Gln Glu Glu Gln Glu Val Ala Leu Asp Ile Pro Glu Gly Phe Asp


    530                 535                 540


 


Phe Leu Leu Asp Lys Ile Asn Thr Ile Asp Asp


545                 550                 555






The present invention also relates to the dnaN gene of Streptococcus pyogenes encoding the β subunit. The dnaN gene has a nucleotide sequence which corresponds to SEQ. ID. No. 27 as follows:








atgattcaat tttcaattaa tcgcacatta tttattcatg ctttaaatac aactaaacgt   60



 


gctattagca ctaaaaatgc cattcctatt ctttcatcaa taaaaattga agtcacttct  120


 


acaggagtaa ctttaacagg gtctaacggt caaatatcaa ttgaaaacac tattcctgta  180


 


agtaatgaaa atgctggttt gctaattacc tctccaggag ctattttatt agaagctagt  240


 


ttttttatta atattatttc aagtttgcca gatattagta taaatgttaa agaaattgaa  300


 


caacaccaag ttgttttaac cagtggtaaa tcagagatta ccttaaaagg aaaagatgtt  360


 


gaccagtatc ctcgtctaca agaagtatca acagaaaatc ctttgatttt aaaaacaaaa  420


 


ttattgaagt ctattattgc tgaaacagct tttgcagcca gtttacaaga aagtcgtcct  480


 


attttaacag gagttcatat tgtattaagt aatcataaag attttaaagc agtagcgact  540


 


gactctcatc gtatgagcca acgtttaatc actttggaca atacttcagc agatttgatg  600


 


gtagttcttc caagtaaatc tttgagagaa ttttcagcag tatttacaga tgatattgag  660


 


accgttgagg tatttttctc accaagccaa atcttgttca gaagtgaaca catttctttt  720


 


tatacacgcc tcttagaagg aaattatccc gatacagacc gtttattaat gacagaattt  780


 


gagacggagg ttgttttcaa tacccaatcc cttcgccacg ctatggaacg tgccttcttg  840


 


acttccaatg ctactcaaaa tggtactgtt aagcttgaga ttactcaaaa tcatatttca  900


 


gctcatgtta actcacctga ggttggtaag gtaaacgagg atttagatac tgttagtcag  960


 


tctggtagtg atttaactat cagcttcaat ccaacttacc ttattgagtc tttaaaagcc 1020


 


attaaaagtg aaacagtaaa aattcatttc ttatcaccag ttcgaccatt caccctaaca 1080


 


ccaggcgatg aggaagaaag ttttatccaa ttaattacac cagtacgaac aaac       1134






The encoded β subunit has all amino acid sequence corresponding to SEQ. ID. No. 28 as follows:









Met Ile Gln Phe Ser Ile Asn Arg Thr Leu Phe Ile His Ala Leu Asn



  1               5                  10                  15


 


Thr Thr Lys Arg Ala Ile Ser Thr Lys Asn Ala Ile Pro Ile Leu Ser


             20                  25                  30


 


Ser Ile Lys Ile Glu Val Thr Ser Thr Gly Val Thr Leu Thr Gly Ser


         35                  40                  45


 


Asn Gly Gln Ile Ser Ile Glu Asn Thr Ile Pro Val Ser Asn Glu Asn


     50                  55                  60


 


Ala Gly Leu Leu Ile Thr Ser Pro Gly Ala Ile Leu Leu Glu Ala Ser


 65                  70                  75                  80


 


Phe Phe Ile Asn Ile Ile Ser Ser Leu Pro Asp Ile Ser Ile Asn Val


                 85                  90                  95


 


Lys Gln Ile Gln Gln His Gln Val Val Leu Thr Ser Gly Lys Ser Glu


            100                 105                 110


 


Ile Thr Leu Lys Gly Lys Asp Val Asp Gln Tyr Pro Arg Leu Gln Glu


        115                 120                 125


 


Val Ser Thr Glu Asn Pro Leu Ile Leu Lys Thr Lys Leu Leu Lys Ser


    130                 135                 140


 


Ile Ile Ala Glu Thr Ala Phe Ala Ala Ser Leu Gln Glu Ser Arg Pro


145                 150                 155                 160


 


Ile Leu Thr Gly Val His Ile Val Leu Ser Asn His Lys Asp Phe Lys


                165                 170                 175


 


Ala Val Ala Thr Asp Ser His Arg Met Ser Gln Arg Leu Ile Thr Leu


            180                 185                 190


 


Asp Asn Thr Ser Ala Asp Leu Met Val Val Leu Pro Ser Lys Ser Leu


        195                 200                 205


 


Arg Glu Phe Ser Ala Val Phe Thr Asp Asp Ile Glu Thr Val Glu Val


    210                 215                 220


 


Phe Phe Ser Pro Ser Gln Ile Leu Phe Arg Ser Glu His Ile Ser Phe


225                 230                 235                 240


 


Tyr Thr Arg Leu Leu Glu Gly Asn Tyr Pro Asp Thr Asp Arg Leu Leu


                245                 250                 255


 


Met Thr Glu Phe Glu Thr Glu Val Val Phe Asn Thr Glu Ser Leu Arg


            260                 265                 270


 


His Ala Met Glu Arg Ala Phe Leu Ile Ser Asn Ala Thr Gln Asn Gly


        275                 280                 285


 


Thr Val Lys Leu Glu Ile Thr Gln Asn His Ile Ser Ala His Val Asn


    290                 295                 300


 


Ser Pro Glu Val Gly Lys Val Asn Glu Asp Leu Asp Ile Val Ser Gln


305                 310                 315                 320


 


Ser Gly Ser Asp Leu Thr Ile Ser Phe Asn Pro Thr Tyr Leu Ile Glu


                325                 330                 335


 


Ser Leu Lys Ala Ile Lys Ser Glu Thr Val Lys Ile His Phe Leu Ser


            340                 345                 350


 


Pro Val Arg Pro Phe Thr Leu Thr Pro Gly Asp Glu Glu Glu Ser Phe


        355                 360                 365


 


Ile Gln Leu Ile Thr Pro Val Arg Thr Asn


    370                 375






The present invention also relates to the ssb gene of Streptococcus pyogenes encoding the single strand-binding protein (SSB). The ssb gene has a nucleotide sequence which corresponds to SEQ. ID. No. 29 as follows:








atgattaata atgtagtact agttggtcgc atgaccaagg atgcagaact tcgttacaca  60



 


ccaagtcaag tagctgtggc taccttcaca cttgctgtta accgtacctt taaaagccaa 120


 


aatggtgaac gcgaggcaga tttcattaac tgtgtgatct ggcgtcaacc ggctgaaaat 180


 


ttagcgaact gggctaaaaa aggtgctttg atcggagtta cgggtcgtat tcatacacgt 240


 


aactacgaaa accaacaagg acaacgtgtc tatgtaacag aagttgttgc agataatttc 300


 


caaatgttgg aaagtcgtgc tacacgtgaa ggtggctcaa ctggctcatt taatggtggt 360


 


tttaacaata acacttcatc atcaaacagt tactcagcgc ctgcacaaca aacgcctaac 420


 


tttggaagag atgatagccc atttgggaac tcaaacccga tggatatctc agatgacgat 480


 


cttccattct ag                                                     492






The encoded SSB protein has an amino acid sequence corresponding to SEQ. ID. No. 30 as follows:









Met Ile Asn Asn Val Val Leu Val Gly Arg Met Thr Lys Asp Ala Glu



  1               5                  10                  15


 


Leu Arg Tyr Thr Pro Ser Gln Val Ala Val Ala Thr Phe Thr Leu Ala


             20                  25                  30


 


Val Asn Arg Thr Phe Lys Ser Gln Asn Gly Glu Arg Glu Ala Asp Phe


         35                  40                  45


 


Ile Asn Cys Val Ile Trp Arg Gln Pro Ala Glu Asn Leu Ala Asn Trp


     50                  55                  60


 


Ala Lys Lys Gly Ala Leu Ile Gly Val Thr Gly Arg Ile Gln Thr Arg


 65                  70                  75                  80


 


Asn Tyr Glu Asn Gln Gln Gly Gln Arg Val Tyr Val Thr Glu Val Val


                 85                  90                  95


 


Ala Asp Asn Phe Gln Met Leu Glu Ser Arg Ala Thr Arg Glu Gly Gly


            100                 105                 110


 


Ser Thr Gly Ser Phe Asn Gly Gly Phe Asn Asn Asn Thr Ser Ser Ser


        115                 120                 125


Asn Ser Tyr Ser Ala Pro Ala Gln Gln Thr Pro Asn Phe Gly Arg Asp


    130                 135                 140


 


Asp Ser Pro Phe Gly Asn Ser Asn Pro Met Asp Ile Ser Asp Asp Asp


145                 150                 155                 160


 


Leu Pro Phe






The present invention also relates to the dnaG gene of Streptococcus pyogenes encoding the primase. The dnaG gene has a nucleotide sequence which corresponds to SEQ. ID. No. 31 as follows:








atgggatttt tatggggagg tgacgatttg gcaattgaca aagaaatgat ttcccaagta   60



 


aaaaatagcg ttaatattgt cgatgtcatt ggagaagtgg tcaaactttc ccgatcaggg  120


 


cggcattacc tcgggctttg cccatttcat aaggaaaaga caccctcttt taatgttgtt  180


 


gaagacagac aattttttca ctgctttggc tgtggaaaat caggggatgt ttttaaattt  240


 


attgaggaat accgccaagt ccccttctta gaaagtgttc agattattgc cgataagact  300


 


ggtatgtcgc ttaatatacc gccaagtcag gcagtacttg ctagccaaca caagcaccct  360


 


aatcacgctt tgatgacact tcatgaggat gctgctaaat tttaccatgc agttttgatg  420


 


accactacca ttggtcaaga agctaggaag tacctttacc agagaggctt ggatgaccaa  480


 


ttaattgagc atttcaatat tggtttagcc ccagatgagt cagattatct ttatcaagct  540


 


ctttctaaaa aatacgagga aggtcaattg gttgcttcag gattgtttca cttgtccgat  600


 


caatccaata ccatttacga cgcctttcga aatcgtatca tgtttccctt atcagatgac  660


 


cgagggcata ttattgcctt ttcaggacgt atctggacgg cagctgatat ggaaaagaga  720


 


caggcaaagt ataaaaattc aagaggaaca gttcttttta acaaatctta tgaattgtat  780


 


catctggaca aggcaaggcc tgttattgcc aaaacccatg aagtgtttct aatggaaggg  840


 


tttatggacg tgattgccgc ttaccgttcc ggctatgaaa atgctgttgc ttcaatgggg  900


 


acggcattga ctcaagaaca tgtcaatcac cttaagcaag tcactaaaaa agttgttttg  960


 


atttatgatg gtgacgatgc tggacaacat gctattgcaa aatcactaga attgcttaaa 1020


 


gattttgtcg tcgaaattgt cagaatcccc aataaaatgg atcctgacga atttgtacaa 1080


 


cggcattccc cagaagcatt tgcagatttg cttaagcagt cacggatcag tagtgttgaa 1140


 


ttttttattg attacctaaa acctactaat gtagacaatt tgcaatcaca aattgtttat 1200


 


gtggagaaaa tggcaccatt gattgctcaa tcaccatcca tcacagctca acattcgtat 1260


 


attaacaaga ttgctgattt gttgccaaac tttgactatt ttcaagtaga acaatcagta 1320


 


aatgcattaa ggattcaaga taggcaaaaa catcaaggtc aaatagctca agccgtcagc 1380


 


aatcttgtga ccttaccaat gccaaaaagt ttgacagcta ttgctaagac agaaagtcat 1440


 


ctcatgcatc ggctcttaca tcatgactat ttattaaatg aatttcgaca tcgtgatgat 1500


 


ttttattttg atacctctac cttagaatta ctttatcaac ggctgaagca acaaggacac 1560


 


attacatctt atgatttgtc agagatgtca gaggaagtta accgtgctta ttacaatgtt 1620


 


ttagaagaaa accttcccaa agaagtagct cttggtgaga ttgatgatat tttatccaaa 1680


 


cgtgccaaac ttttagcaga gcgcgatctt cacaaacaag ggaaaaaagt tagagaatct 1740


 


agtaacaaag gcgatcatca agcggctcta gaagtactag aacattttat tgcgcagaaa 1800


 


cgaaaaatgg aatag                                                  1815






The encoded primase has an amino acid sequence corresponding to SEQ. ID. No. 32 as follows:









Met Gly Phe Leu Trp Gly Gly Asp Asp Leu Ala Ile Asp Lys Glu Met



  1               5                  10                  15


 


Ile Ser Gln Val Lys Asn Ser Val Asn Ile Val Asp Val Ile Gly Glu


             20                  25                  30


 


Val Val Lys Leu Ser Arg Ser Gly Arg His Tyr Leu Gly Leu Cys Pro


         35                  40                  45


 


Phe His Lys Glu Lys Thr Pro Ser Phe Asn Val Val Glu Asp Arg Gln


     50                  55                  60


 


Phe Phe His Cys Phe Gly Cys Gly Lys Ser Gly Asp Val Phe Lys Phe


 65                  70                  75                  80


 


Ile Glu Glu Tyr Arg Gln Val Pro Phe Leu Glu Ser Val Gln Ile Ile 


                 85                  90                  95


 


Ala Asp Lys Thr Gly Met Ser Leu Asn Ile Pro Pro Ser Gln Ala Val


            100                 105                 110


 


Leu Ala Ser Gln His Lys His Pro Asn His Ala Leu Met Thr Leu His


        115                 120                 125


 


Glu Asp Ala Ala Lys Phe Tyr His Ala Val Leu Met Thr Thr Thr Ile 


    130                 135                 140


 


Gly Gln Glu Ala Arg Lys Tyr Leu Tyr Gln Arg Gly Leu Asp Asp Gln


145                 150                 155                 160


 


Leu Ile Glu His Phe Asn Ile Gly Leu Ala Pro Asp Glu Ser Asp Tyr


                165                 170                 175


 


Leu Tyr Gln Ala Leu Ser Lys Lys Tyr Glu Glu Gly Gln Leu Val Ala


            180                 185                 190


 


Ser Gly Leu Phe His Leu Ser Asp Gln Ser Asn Thr Ile Tyr Asp Ala


        195                 200                 205


 


Phe Arg Asn Arg Ile Met Phe Pro Leu Ser Asp Asp Arg Gly His Ile 


    210                 215                 220


 


Ile Ala Phe Ser Gly Arg Ile Trp Thr Ala Ala Asp Met Glu Lys Arg


225                 230                 235                 240


 


Gln Ala Lys Tyr Lys Asn Ser Arg Gly Thr Val Leu Phe Asn Lys Ser


                245                 250                 255


 


Tyr Glu Leu Tyr His Leu Asp Lys Ala Arg Pro Val Ile Ala Lys Thr


            260                 265                 270


 


His Glu Val Phe Leu Met Glu Gly Phe Met Asp Val Ile Ala Ala Tyr


        275                 280                 285


 


Arg Ser Gly Tyr Glu Asn Ala Val Ala Ser Met Gly Thr Ala Leu Thr


    290                 295                 300


 


Gln Glu His Val Asn His Leu Lys Gln Val Thr Lys Lys Val Val Leu


305                 310                 315                 320


 


Ile Tyr Asp Gly Asp Asp Ala Gly Gln His Ala Ile Ala Lys Ser Leu


                325                 330                 335


 


Glu Leu Leu Lys Asp Phe Val Val Glu Ile Val Arg Ile Pro Asn Lys


            340                 345                 350


 


Met Asp Pro Asp Glu Phe Val Gln Arg His Ser Pro Glu Ala Phe Ala


        355                 360                 365


 


Asp Leu Leu Lys Gln Ser Arg Ile Ser Ser Val Glu Phe Phe Ile Asp


    370                 375                 380


 


Tyr Leu Lys Pro Thr Asn Val Asp Asn Leu Gln Ser Gln Ile Val Tyr


385                 390                 395                 400


 


Val Glu Lys Met Ala Pro Leu Ile Ala Gln Ser Pro Ser Ile Thr Ala


                405                 410                 415


 


Gln His Ser Tyr Ile Asn Lys Ile Ala Asp Leu Leu Pro Asn Phe Asp


            420                 425                 430


 


Tyr Phe Gln Val Glu Gln Ser Val Asn Ala Leu Arg Ile Gln Asp Arg


        435                 440                 445


 


Gln Lys His Gln Gly Gln Ile Ala Gln Ala Val Ser Asn Leu Val Thr


    450                 455                 460


 


Leu Pro Met Pro Lys Ser Leu Thr Ala Ile Ala Lys Thr Glu Ser His


465                 470                 475                 480


 


Leu Met His Arg Leu Leu His His Asp Tyr Leu Leu Asn Glu Phe Arg


                485                 490                 495


 


His Arg Asp Asp Phe Tyr Phe Asp Thr Ser Thr Leu Glu Leu Leu Tyr


            500                 505                 510


 


Gln Arg Leu Lys Gln Gln Gly His Ile Thr Ser Tyr Asp Leu Ser Glu


        515                 520                 525


 


Met Ser Glu Glu Val Asn Arg Ala Tyr Tyr Asn Val Leu Glu Glu Asn


    530                 535                 540


 


Leu Pro Lys Glu Val Ala Leu Gly Glu Ile Asp Asp Ile Leu Ser Lys


545                 550                 555                 560


 


Arg Ala Lys Leu Leu Ala Glu Arg Asp Leu His Lys Gln Gly Lys Lys


                565                 570                 575


 


Val Arg Glu Ser Ser Asn Lys Gly Asp His Gln Ala Ala Leu Glu Val


            580                 585                 590


 


Leu Glu His Phe Ile Ala Gln Lys


        595                 600






The present invention also relates to the dnaB gene of Streptococcus pyogenes encoding DnaB. The dnaB gene has a nucleotide sequence which corresponds to SEQ. ID. No. 33 as follows:








atgaggttgc ctgaagtagc tgaattacga gttcaacccc aagatttact agcagagcaa   60



 


tctgttcttg ggtcaatctt tatctcacct gataagctga ttgcagtgag agaatttatc  120


 


agtccagacg atttttataa gtacgctcat aaaattatct ttcgggcaat gattaccctc  180


 


agcgatcgta atgatgccat tgatgcaacc actataagaa caatcctaga tgatcaagat  240


 


gatctgcaaa gtattggtgg cttatcctat attgttgaac tagttaatag tgtcccaact  300


 


agtgctaatg cagaatatta tgctaaaatt gtagctgaga aagctatgtt gcgtgatatt  360


 


attgctaggt tgacagaatc tgtcaaccta gcttatgatg aaattttaaa accagaagag  420


 


gttatcgctg gagttgagag agctttaatt gaactcaatg aacatagtaa tcgtagtggg  480


 


tttcgcaaaa tttcagatgt gctaaaagtt aattacgagg ctttagaagc acgttctaag  540


 


cagacttcaa atgttacagg tttaccaact ggttttagag accttgacaa gattacaaca  600


 


ggtttacacc cagatcaatt agttatttta gctgctcggc cagcagtggg gaagactgcc  660


 


tttgttctta atattgcgca aaatgtgggg actaagcaaa aaaagactgt tgctattttt  720


 


tctttggaaa tgggtgctga aagtttagta gatcgtatgc ttgcagcaga aggaatggtt  780


 


gattcgcaca gtttaagaac agggcaactc acagatcagg attggaataa tgtaacaatt  840


 


gctcagggag ctttggcaga agcaccgatt tatattgacg atacgcccgg gattaaaatt  900


 


actgaaatcc gcgcaagatc acggaaattg tctcaagaag tggatggtgg tttaggtctc  960


 


attgtaattg actacttaca gttgattaca ggaactaaac ccgaaaatcg tcagcaagag 1020


 


gtttcagata tttcaagaca gcttaaaatc ctagctaaag aattgaaagt accagttatt 1080


 


gccctaagtc agctttctcg tggcgttgag caaaggcaag ataaacgacc agttttatca 1140


 


gatattcgtg aatcaggatc tattgagcag gatgccgata ttgtagcctt cttataccgg 1200


 


gacgattatt accgtaaaga atgtgatgat gctgaagaag ctgttgaaga taacacaatt 1260


 


gaagttatcc tcgagaaaaa tagagctggg gcgcgtggaa cagtcaaact gatgttccaa 1320


 


aaagaataca acaaattctc aagtatagcc cagtttgaag aaagataa              1368






The encoded DnaB has an amino acid sequence corresponding to SEQ. ID. No. 34 as follows:









Met Arg Leu Pro Glu Val Ala Glu Leu Arg Val Gln Pro Gln Asp Leu



  1               5                  10                  15


 


Leu Ala Glu Gln Ser Val Leu Gly Ser Ile Phe Ile Ser Pro Asp Lys


             20                  25                  30


 


Leu Ile Ala Val Arg Glu Phe Ile Ser Pro Asp Asp Phe Tyr Lys Tyr


         35                  40                  45


 


Ala His Lys Ile Ile Phe Arg Ala Met Ile Thr Leu Ser Asp Arg Asn


     50                  55                  60


 


Asp Ala Ile Asp Ala Thr Thr Ile Arg Thr Ile Leu Asp Asp Gln Asp


65                  70                  75                  80


 


Asp Leu Gln Ser Ile Gly Gly Leu Ser Tyr Ile Val Glu Leu Val Asn


                 85                  90                  95


 


Ser Val Pro Thr Ser Ala Asn Ala Glu Tyr Tyr Ala Lys Ile Val Ala


            100                 105                 110


 


Glu Lys Ala Met Leu Arg Asp Ile Ile Ala Arg Leu Thr Glu Ser Val


        115                 120                 125


 


Asn Leu Ala Tyr Asp Glu Ile Leu Lys Pro Glu Glu Val Ile Ala Gly


    130                 135                 140


 


Val Glu Arg Ala Gln Gly Ala Leu Ala Glu Ala Pro Ile Tyr Ile Asp


145                 150                 155                 160


 


Asp Thr Pro Gly Ile Lys Ile Ala Leu Ile Glu Leu Asn Glu His Ser


                165                 170                 175


 


Asn Arg Ser Gly Phe Arg Lys Ile Ser Asp Val Leu Lys Val Asn Tyr


            180                 185                 190


 


Glu Ala Leu Glu Ala Arg Ser Lys Gln Thr Ser Asn Val Thr Gly Leu


        195                 200                 205


 


Pro Thr Gly Phe Arg Asp Leu Asp Lys Ile Thr Thr Gly Leu His Pro


    210                 215                 220


 


Asp Gln Leu Val Ile Leu Ala Ala Arg Pro Ala Val Gly Lys Thr Ala


225                 230                 235                 240


 


Phe Val Leu Asn Ile Ala Gln Asn Val Gly Thr Lys Gln Lys Lys Thr


                245                 250                 255


 


Val Ala Ile Phe Ser Leu Gln Met Gly Ala Gln Ser Leu Val Asp Arg


            260                 265                 270


 


Met Leu Ala Ala Gln Gly Met Val Asp Ser His Ser Leu Arg Thr Gly


        275                 280                 285


 


Gln Leu Thr Asp Gln Asp Trp Asn Asn Val Thr Ile Thr Glu Ile Arg


    290                 295                 300


 


Ala Arg Ser Arg Lys Leu Ser Gln Gln Val Asp Gly Gly Leu Gly Leu


305                 310                 315                 320


 


Ile Val Ile Asp Tyr Leu Gln Leu Ile Thr Gly Thr Lys Pro Gln Asn


                325                 330                 335


 


Arg Gln Gln Glu Val Ser Asp Ile Ser Arg Gln Leu Lys Ile Leu Ala


            340                 345                 350


 


Lys Gln Leu Lys Val Pro Val Ile Ala Leu Ser Gln Leu Ser Arg Gly


        355                 360                 365


 


Val Gln Gln Arg Gln Asp Lys Arg Pro Val Leu Ser Asp Ile Arg Glu


    370                 375                 380


 


Ser Gly Ser Ile Glu Gln Asp Ala Asp Ile Val Ala Phe Leu Tyr Arg


385                 390                 395                 400


 


Asp Asp Tyr Tyr Arg Lys Glu Cys Asp Asp Ala Glu Glu Ala Val Glu


                405                 410                 415


 


Asp Asn Thr Ile Glu Val Ile Leu Glu Lys Asn Arg Ala Gly Ala Arg


            420                 425                 430


 


Gly Thr Val Lys Leu Met Phe Gln Lys Glu Tyr Asn Lys Phe Ser Ser


        435                 440                 445


 


Ile Ala Gln Phe Glu Glu Arg


    450                 455






Fragments of the above polypeptides or proteins are also encompassed by the present invention.
Suitable fragments can be produced by several means. In the first, subclones of the gene encoding the protein of the present invention are produced by conventional molecular genetic manipulation by subcloning gene fragments. The subclones then are expressed in vitro or in vivo in bacterial cells to yield a smaller protein or peptide that can be tested for activity according to the procedures described below.
As an alternative, fragments of replication proteins can be produced by digestion of a full-length replication protein with proteolytic enzymes like chymotrypsin or Staphylococcus proteinase A, or trypsin. Different proteolytic enzymes are likely to cleave replication proteins at different sites based on the amino acid sequence of the protein. Some of the fragments that result from proteolysis may be active and can be tested for activity as described below.
In another approach, based on knowledge of the primary structure of the protein, fragments of a replication protein gene may be synthesized by using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein. These then would be cloned into an appropriate vector for increased expression of a truncated peptide or protein.
Chemical synthesis can also be used to make suitable fragments. Such a synthesis is carried out using known amino acid sequences of replication proteins being produced. Alternatively, subjecting a full length replication protein to high temperatures and pressures will produce fragments. These fragments can then be separated by conventional procedures (e.g., chromatography, SDS-PAGE).
Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the properties, secondary structure, and hydropathic nature of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which cotranslationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.
Suitable DNA molecules are those that hybridize to a DNA molecule comprising a nucleotide sequence of at least about 20, more preferably at least about 30 to about 50, continuous bases of either SEQ. ID. Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 under stringent conditions such as those characterized by a hybridization buffer comprising 0.9 M sodium citrate (“SSC”) buffer at a temperature of about 37° C. and remaining bound when subject to washing the SSC buffer at a temperature of about 37° C.; and preferably in a hybridization buffer comprising 20% formamide in 0.9 M SSC buffer at a temperature of about 42° C. and remaining bound when subject to washing at about 42° C. with 0.2×SSC buffer. Stringency conditions can be further varied by modifying the temperature and/or salt content of the buffer, or by modifying the length of the hybridization probe.
The proteins or polypeptides of the present invention are preferably produced in purified form (preferably at least 80%, more preferably 90%, pure) by conventional techniques. Typically, the proteins or polypeptides of the present invention is secreted into the growth medium of recombinant host cells. Alternatively, the proteins or polypeptides of the present invention are produced but not secreted into growth medium. In such cases, to isolate the protein, the host cell (e.g., E. coli) carrying a recombinant plasmid is propagated, lysed by sonication, heat, or chemical treatment, and the homogenate is centrifuged to remove bacterial debris. The supernatant is then subjected to purification procedures such as ammonium sulfate precipitation, gel filtration, ion exchange chromatography, FPLC, and HPLC.
The DNA molecule encoding replication polypeptides or proteins derived from Gram positive bacteria can be incorporated in cells using conventional recombinant DNA technology. Generally, this involved inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture.
Recombinant genes may also be introduced into viruses, such as vaccina virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/− or KS +/(see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see F. W. Studier et al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology vol. 185 (1990), which is hereby incorporated by reference), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference.
A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation).
Transcription of DNA is dependent upon the presence of a promotor which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promoters differ from those of procaryotic promoters. Furthermore, eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further procaryotic promoters are not recognized and do not function in eucaryotic cells.
Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eukaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the same codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby incorporated by reference.
Promoters vary in their “strength” (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced. In certain operations, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls. Additionally, the cell may carry the gene for a heterologous RNA polymerase such as from phage T7. Thus, a promoter specific for T7 RNA polymerase is used. The T7 RNA polymerase may be under inducible control.
Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promotor, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires an SD sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, an SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
Once the isolated DNA molecule encoding a replication polypeptide or protein has been cloned into an expression system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, viruses, yeast, mammalian cells, insects, plants, and the like.
The invention provides efficient methods of identifying pharmacological agents or lead compounds for agents active at the level of a replication protein function, particularly DNA replication. Generally, these screening methods involve assaying for compounds which interfere with the replication activity. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development. Target therapeutic indications are limited only in that the target cellular function be subject to modulation, usually inhibition, by disruption of a replication activity or the formation of a complex comprising a replication protein and one or more natural intracellular binding targets. Target indications may include arresting cell growth or causing cell death resulting in recovery from the bacterial infection in animal studies.
A wide variety of assays for activity and binding agents are provided, including DNA synthesis, ATPase, clamp loading onto DNA, protein-protein binding assays, immunoassays, cell based assays, etc. The replication protein compositions, used to identify pharmacological agents, are in isolated, partially pure or pure form and are typically recombinantly produced. The replication protein may be part of a fusion product with another peptide or polypeptide (e.g., a polypeptide that is capable of providing or enhancing protein-protein binding, stability under assay conditions (e.g., a tag for detection or anchoring), etc.). The assay mixtures comprise a natural intracellular replication protein binding target such as DNA, another protein, NTP, or dNTP. For binding assays, while native binding targets may be used, it is frequently preferred to use portions (e.g., peptides, nucleic acid fragments) thereof so long as the portion provides binding affinity and avidity to the subject replication protein conveniently measurable in the assay. The assay mixture also comprises a candidate pharmacological agent. Generally, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control (i.e., at zero concentration or below the limits of assay detection). Additional controls are often present such as a positive control, a dose response curve, use of known inhibitors, use of control heterologous proteins, etc. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably they are small organic compounds and are obtained from a wide variety of sources, including libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture. These include reagents like salts, buffers, neutral proteins (e.g., albumin, detergents, etc.), which may be used to facilitate optimal binding and/or reduce nonspecific or background interactions, etc. Also reagents that otherwise improve the efficiency of the assay (e.g., protease inhibitors, nuclease inhibitors, antimicrobial agents, etc.) may be used.
The invention provides replication protein specific assays and the binding agents including natural intracellular binding targets such as other replication proteins, etc., and methods of identifying and making such agents, and their use in a variety of diagnostic and therapeutic applications, especially where disease is associated with excessive cell growth. Novel replication protein-specific binding agents include replication protein-specific antibodies and other natural intracellular binding agents identified with assays such as one- and two-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries, etc.
Generally, replication protein-specificity of the binding agent is shown by binding equilibrium constants. Such agents are capable of selectively binding a replication protein (i.e., with an equilibrium constant at least about 107 M−1, preferably, at least about 108 M−1, more preferably, at least about 109 M−1). A wide variety of cell-based and cell-free assays may be used to demonstrate replication protein-specific activity, binding, gel shift assays, immunoassays, etc.
The resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the replication protein specifically binds the cellular binding target, portion, or analog. The mixture of components can be added in any order that provides for the requisite bindings. Incubations may be performed at any temperature which facilitates optimal binding, typically between 4° C. and 40° C., more commonly between 15° C. and 40° C. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening, and are typically between 0.1 and 10 hours, preferably less than 5 hours, more preferably less than 2 hours.
After incubation, the presence or absence of activity or specific binding between the replication protein and one or more binding targets is detected by any convenient way. For cell-free activity and binding type assays, a separation step may be used to separate the activity product or the bound from unbound components. Separation may be effected by precipitation (e.g., immunoprecipitation), immobilization (e.g., on a solid substrate such as a microtiter plate), etc., followed by washing. Many assays that do not require separation are also possible such as use of europium conjugation in proximity assays or a detection system that is dependent on a product or loss of substrate.
Detection may be effected in any convenient way. For cell-free activity and binding assays, one of the components usually comprises or is coupled to a label. A wide variety of labels may be employed—essentially any label that provides for detection of DNA product, loss of DNA substrate, conversion of a nucleotide substrate, or bound protein is useful. The label may provide for direct detection such as radioactivity, fluorescence, luminescence, optical, or electron density, etc. or indirect detection such as an epitope tag, an enzyme, etc. The label may be appended to the protein (e.g., a phosphate group comprising a radioactive isotope of phosphorous), or incorporated into the DNA substrate or the protein structure (e.g., a methionine residue comprising a radioactive isotope of sulfur.) A variety of methods may be used to detect the label depending on the nature of the label and other assay components. For example, the label may be detected bound to the solid substrate, or a portion of the bound complex containing the label may be separated from the solid substrate, and thereafter the label detected. Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfer, fluorescence emission, etc. or indirectly detected with antibody conjugates, etc. For example, in the case of radioactive labels, emissions may be detected directly (e.g., with particle counters) or indirectly (e.g., with scintillation cocktails and counters).
The present invention identifies the set of proteins that together result in a three component polymerase from bacteria that are distantly related to E. coli, such as Gram positive bacteria. Specifically, these bacteria lack several genes that E. coli DNA polymerase III has, such as holD, holD or holE. Further, dnaX is believed to encode only one protein, tau. Also, holA is quite divergent in homology suggesting it may function in another process in these organisms. Gram positive cells even have replication genes that E. coli does not, implying that they may not utilize the replication strategies exemplified by E. coli. 
The present invention identifies genes and proteins that form a three component polymerase in Gram positive organisms, such as S. pyogenes and S. aureus. In S. pyogenes and S. aureus, the polymerase α-large, functions with a β clamp and a clamp loader component of τδδ′. They display high speed and processivity in synthesis of ssDNA coated with SSB and primed with a DNA oligonucleotide.
This invention also expresses and purifies a protein from a Gram positive bacteria that is homologous to the E. coli beta subunit. The invention demonstrates that it behaves like a circular protein. Further, this invention shows that a beta subunit from a Gram positive bacteria is functional with both Pol III-L α-large) from a Gram positive bacteria and with DNA polymerase III from a Gram negative bacteria. This result can be explained by an interaction between the clamp and the polymerase that has been conserved during the evolutionary divergence of Gram positive and Gram negative cells. A chemical inhibitor that would disrupt this interaction would be predicted to have a broad spectrum of antibiotic activity, shutting down replication in gram negative and gram positive cells alike. This assay, and others based on this interaction, can be devised to screen chemicals for such inhibition. Further, since all the proteins in this assay are highly overexpressed through recombinant techniques, sufficient quantities of the protein reagents can be obtained for screening hundreds of thousands of compounds.
This invention also shows that the DnaE polymerase (α-small), encoded by the dnaE gene, functions with the beta clamp and τδδ′ complex. The speed of DnaE is not significantly increased by τδδ′ and β, but the processivity of DnaE is greatly increased by τδδ′ and β. Hence, the DnaE polymerase, coupled with its β clamp on DNA (loaded by τδδ′) may also be an important target for a candidate pharmaceutical drug.
The present invention provides methods by which replication proteins from a Gram positive bacteria are used to discover new pharmaceutical agents. The function of replication proteins is quantified in the presence of different chemical compounds. A chemical compound that inhibits the function is a candidate antibiotic. Some replication proteins from a Gram positive bacteria and from a Gram negative bacteria can be interchanged for one another. Hence, they can function as mixtures. Reactions that assay for the function of enzyme mixtures consisting of proteins from Gram positive bacteria and from Gram negative bacteria can also be used to discover drugs. Suitable E. coli replication proteins are the subunits of its Pol III holoenzyme which are described in U.S. Pat. Nos. 5,583,026 and 5,668,004 to O'Donnell, which are hereby incorporated by reference.
The methods described herein to obtain genes, and the assays demonstrating activity behavior of S. pyogenes and S. aureus replication proteins are likely to generalize to all members of the Streptococcus and Staphylococcus genuses, as well as to all Gram positive bacteria. Such assays are also likely to generalize to other cells besides Gram positive bacteria which also share features in common with S. pyogenes and S. aureus that are different from E. coli (i.e., lacking holC, holD, or holE; having a dnaX gene encoding a single protein; or having a weak homology to holA encoding delta).
The present invention describes a method of identifying compounds which inhibit the activity of a polymerase product of polC or dnaE. This method is carried out by forming a reaction mixture that includes a primed DNA molecule, a polymerase product of polC or dnaE, a candidate compound, a dNTP, and optionally either a beta subunit, a tau complex, or both the beta subunit and the tau complex, wherein at least one of the polymerase product of polC or dnaE, the beta subunit, the tau complex, or a subunit or combination of subunits thereof is derived from a Eubacteria other than Escherichia coli; subjecting the reaction mixture to conditions effective to achieve nucleic acid polymerization in the absence of the candidate compound; analyzing the reaction mixture for the presence or absence of nucleic acid polymerization extension products; and identifying the candidate compound in the reaction mixture where there is an absence of nucleic acid polymerization extension products. Preferably, the polymerase product of polC or dnaE, the beta subunit, the tau complex, or the subunit or combination of subunits thereof is derived from a Gram positive bacterium, more preferably a Streptococcus bacterium such as S. pyogenes or a Staphylococcus bacterium such as S. aureus. 
The present invention describes a method to identify chemicals that inhibit the activity of the three component polymerase. This method involves contacting primed DNA with the DNA polymerase in the presence of the candidate pharmaceutical, and dNTPs (or modified dNTPs) to form a reaction mixture. The reaction mixture is subjected to conditions effective to achieve nucleic acid polymerization in the absence of the candidate pharmaceutical and the presence or absence of the extension product in the reaction mixture is analyzed. The candidate pharmaceutical is detected by the absence of product.
The present invention describes a method to identify candidate pharmaceuticals that inhibit the activity of a clamp loader complex and a beta subunit in stimulating the DNA polymerase. The method includes contacting a primed DNA (which may be coated with SSB) with a DNA polymerase, a beta subunit, and a tau complex (or subunit or subassembly of the tau complex) in the presence of the candidate pharmaceutical, and dNTPs (or modified dNTPs) to form a reaction mixture. The reaction mixture is subjected to conditions which, in the absence of the candidate pharmaceutical, would effect nucleic acid polymerization and the presence or absence of the extension product in the reaction mixture is analyzed. The candidate pharmaceutical is detected by the absence of product. The DNA polymerase, the beta subunit, and/or the tau complex or subunit(s) thereof are derived from a Gram positive bacterium.
The present invention describes a method to identify chemicals that inhibit the ability of a beta subunit and a DNA polymerase to interact physically. This method involves contacting the beta subunit with the DNA polymerase in the presence of the candidate pharmaceutical to form a reaction mixture. The reaction mixture is subjected to conditions under which the DNA polymerase and the beta subunit would interact in the absence of the candidate pharmaceutical. The reaction mixture is then analyzed for interaction between the beta unit and the DNA polymerase. The candidate pharmaceutical is detected by the absence of interaction between the beta subunit and the DNA polymerase. The DNA polymerase and/or the beta subunit are derived from a Gram positive bacterium.
The present invention describes a method to identify chemicals that inhibit the ability of a beta subunit and a tau complex (or a subunit or subassembly of the tau complex) to interact. This method includes contacting the beta subunit with the tau complex (or subunit or subassembly of the tau complex) in the presence of the candidate pharmaceutical to form a reaction mixture. The reaction mixture is subjected to conditions under which the tau complex (or the subunit or subassembly of the tau complex) and the beta subunit would interact in the absence of the candidate pharmaceutical. The reaction mixture is then analyzed for interaction between the beta subunit and the tau complex (or the subunit or subassembly of the tau complex). The candidate pharmaceutical is detected by the absence of interaction between the beta subunit and the tau complex (or the subunit or subassembly of the tau complex). The beta subunit and/or the tau complex or subunit thereof is derived from a Gram positive bacterium.
The present invention describes a method to identify chemicals that inhibit the ability of a tau complex (or a subassembly of the tau complex) to assemble a beta subunit onto a DNA molecule. This method involves contacting a circular primed DNA molecule (which may be coated with SSB) with the tau complex (or the subassembly thereof) and the beta subunit in the presence of the candidate pharmaceutical, and ATP or dATP to form a reaction mixture. The reaction mixture is subjected to conditions under which the tau complex (or subassembly) assembles the beta subunit on the DNA molecule absent the candidate pharmaceutical. The presence or absence of the beta subunit on the DNA molecule in the reaction mixture is analyzed. The candidate pharmaceutical is detected by the absence of the beta subunit on the DNA molecule. The beta subunit and/or the tau complex are derived from a Gram positive bacterium.
The present invention describes a method to identify chemicals that inhibit the ability of a tau complex (or a subunit(s) of the tau complex) to disassemble a beta subunit from a DNA molecule. This method comprises contacting a DNA molecule onto which the beta subunit has been assembled in the presence of the candidate pharmaceutical, to form a reaction mixture. The reaction mixture is subjected to conditions under which the tau complex (or a subunit(s) or subassembly of the tau complex) disassembles the beta subunit from the DNA molecule absent the candidate pharmaceutical. The presence or absence of the beta subunit on the DNA molecule in the reaction mixture is analyzed. The candidate pharmaceutical is detected by the presence of the beta subunit on the DNA molecule. The beta subunit and/or the tau complex are derived from a Gram positive bacterium.
The present invention describes a method to identify chemicals that disassemble a beta subunit from a DNA molecule. This method involves contacting a circular primed DNA molecule (which may be coated with SSB) upon which the beta subunit has been assembled (e.g. by action of the tau complex) with the candidate pharmaceutical. The presence or absence of the beta subunit on the DNA molecule in the reaction mixture is analyzed. The candidate pharmaceutical is detected by the absence of the beta subunit on the DNA molecule. The beta subunit is derived from a Gram positive bacterium.
The present invention describes a method to identify chemicals that inhibit the dATP/ATP binding activity of a tau complex or a tau complex subunit (e.g. tau subunit). This method includes contacting the tau complex (or the tau complex subunit) with dATP/ATP either in the presence or absence of a DNA molecule and/or the beta subunit in the presence of the candidate pharmaceutical to form a reaction. The reaction mixture is subjected to conditions in which the tau complex (or the subunit of tau complex) interacts with dATP/ATP in the absence of the candidate pharmaceutical. The reaction is analyzed to determine if dATP/ATP is bound to the tau complex (or the subunit of tau complex) in the presence of the candidate pharmaceutical. The candidate pharmaceutical is detected by the absence of hydrolysis. The tau complex and/or the beta subunit is derived from a Gram positive bacterium.
The present invention describes a method to identify chemicals that inhibit the dATP/ATPase activity of a tau complex or a tau complex subunit (e.g., the tau subunit). This method involves contacting the tau complex (or the tau complex subunit) with dATP/ATP either in the presence or absence of a DNA molecule and/or a beta subunit in the presence of the candidate pharmaceutical to form a reaction mixture. The reaction mixture is subjected to conditions in which the tau subunit (or complex) hydrolyzes dATP/ATP in the absence of the candidate pharmaceutical. The reaction is analyzed to determine if dATP/ATP was hydrolyzed. Suitable candidate pharmaceuticals are identified by the absence of hydrolysis. The tau complex and/or the beta subunit is derived from a Gram positive bacterium.
Further methods for identifying chemicals that inhibit the activity of a DNA polymerase encoded by either the dnaE gene, polC gene, or their accessory proteins (i.e., clamp loader, clamp, etc.), are as follows:
1) Contacting a primed DNA molecule with the encoded product of the dnaE gene or polC gene in the presence of the candidate pharmaceutical, and dNTPs (or modified dNTPs) to form a reaction mixture. The reaction mixture is subjected to conditions, which in the absence of the candidate pharmaceutical, affect nucleic acid polymerization and the presence or absence of the extension product in the reaction mixture is analyzed. The candidate pharmaceutical is detected by the absence of extension product. The protein encoded by the dnaE gene and PolC gene is derived from a Gram positive bacterium.
2) Contacting a linear primed DNA molecule with a beta subunit and the encoded product of dnaE or PolC in the presence of the candidate pharmaceutical, and dNTPs (or modified dNTPs) to form a reaction mixture. The reaction mixture is subjected to conditions, which in the absence of the candidate pharmaceutical, affect nucleic acid polymerization, and the presence or absence of the extension product in the reaction mixture is analyzed. The candidate pharmaceutical is detected by the absence of extension product. The protein encoded by the dnaE gene and PolC gene is derived from a Gram positive bacterium.
3) Contacting a circular primed DNA molecule (may be coated with SSB) with a tau complex, a beta subunit and the encoded product of a dnaE gene or PolC gene in the presence of the candidate pharmaceutical, and dNTPs (or modified dNTPs) to form a reaction mixture. The reaction mixture is subjected to conditions, which in the absence of the candidate pharmaceutical, affect nucleic acid polymerization, and the presence or absence of the extension product in the reaction mixture is analyzed. The candidate pharmaceutical is detected by the absence of product. The protein encoded by the dnaE gene and PolC gene, the beta subunit, and/or the tau complex are derived from a Gram positive bacterium.
4) Contacting a beta subunit with the product encoded by a dnaE gene or PolC gene in the presence of the candidate pharmaceutical to form a reaction mixture. The reaction mixture is then analyzed for interaction between the beta subunit and the product encoded by the dnaE gene or PolC gene. The candidate pharmaceutical is detected by the absence of interaction between the beta subunit and the product encoded by the dnaE gene or PolC gene. The beta subunit and/or the protein encoded by the dnaE gene and PolC gene is derived from a Gram positive bacterium.
5) The present invention discloses a method to identify chemicals that inhibit a DnaB helicase. The method includes contacting the DnaB helicase with a DNA molecule substrate that has a duplex region in the presence of a nucleoside or deoxynucleoside triphosphate energy source and a candidate pharmaceutical to form a reaction mixture. The reaction mixture is subjected to conditions that support helicase activity in the absence of the candidate pharmaceutical. The DNA duplex molecule in the reaction mixture is analyzed for whether it is converted to ssDNA. The candidate pharmaceutical is detected by the absence of conversion of the duplex DNA molecule to the ssDNA molecule. The DnaB helicase is derived from a Gram positive bacterium.
6) The present invention describes a method to identify chemicals that inhibit the nucleoside or deoxynucleoside triphosphatase activity of a DnaB helicase. The method includes contacting the DnaB helicase with a DNA molecule substrate that has a duplex region in the presence of a nucleoside or deoxynucleoside triphosphate energy source and the candidate pharmaceutical to form a reaction mixture. The reaction mixture is subjected to conditions that support nucleoside or deoxynucleoside triphosphatase activity of the DnaB helicase in the absence of the candidate pharmaceutical. The candidate pharmaceutical is detected by the absence of conversion of nucleoside or deoxynucleoside triphosphate to nucleoside or deoxynucleoside diphosphate. The DnaB helicase is derived from a Gram positive bacterium.
7) The present invention describes a method to identify chemicals that inhibit a primase. The method includes contacting primase with a ssDNA molecule in the presence of a candidate pharmaceutical to form a reaction mixture. The reaction mixture is subjected to conditions that support primase activity (e.g., the presence of nucleoside or deoxynucleoside triphosphates, appropriate buffer, presence or absence of DnaB helicase) in the absence of the candidate pharmaceutical. Suitable candidate pharmaceuticals are identified by the absence of primer formation detected either directly or indirectly. The primase is derived from a Gram positive bacterium.
8) The present invention describes a method to identify chemicals that inhibit the ability of a primase and the protein encoded by a dnaB gene to interact. This method includes contacting the primase with the protein encoded by the dnaB gene in the presence of the candidate pharmaceutical to form a reaction mixture. The reaction mixture is subjected to conditions under which the primase and the protein encoded by the dnaB gene interact in the absence of the candidate pharmaceutical. The reaction mixture is then analyzed for interaction between the primase and the protein encoded by the dnaB gene. The candidate pharmaceutical is detected by the absence of interaction between the primase and the protein encoded by the dnaB gene. The primase and/or the dnaB gene are derived from a Gram positive bacterium.
9) The present invention describes a method to identify chemicals that inhibit the ability of a protein encoded by a dnaB gene to interact with a DNA molecule. This method includes contacting the protein encoded by the dnaB gene with the DNA molecule in the presence of the candidate pharmaceutical to form a reaction mixture. The reaction mixture is subjected to conditions under which the DNA molecule and the protein encoded by the dnaB gene interact in the absence of the candidate pharmaceutical. The reaction mixture is then analyzed for interaction between the protein encoded by the dnaB gene and the DNA molecule. The candidate pharmaceutical is detected by the absence of interaction between the DNA molecule and the protein encoded by the dnaB gene. The dnaB gene is derived from a Gram positive bacterium.
EXAMPLES
The following examples are provided to illustrate embodiments of the present invention, but they are by no means intended to limit its scope.
Example 1
Materials
Labeled deoxy- and ribonucleoside triphosphates were from Dupont-New England Nuclear; unlabelled deoxy- and ribonucleoside triphosphates were from Pharmacia-LKB; E. coli replication proteins were purified as described, alpha, epsilon, gamma, and tau (Studwell et al., “Processive Replication is Contingent on the Exonuclease Subunit of DNA Polymerase III Holoenzyme,” J. Biol. Chem., 265:1171-1178 (1990), which is hereby incorporated by reference), beta (Kong et al., “Three Dimensional Structure of the Beta Subunit of Escherichia coli DNA Polymerase III Holoenzyme: A Sliding DNA Clamp,” Cell, 69:425-437 (1992), which is hereby incorporated by reference), delta and delta prime (Dong et al., “DNA Polymerase III Accessory Proteins. I. HolA and holB Encoding δ and δ′,” J. Biol. Chem., 268:11758-11765 (1993), which is hereby incorporated by reference), chi and psi (Xiao et al., “DNA Polymerase III Accessory Proteins. III. HolC and holD Encoding chi and psi,” J. Biol. Chem., 268:11773-11778 (1993), which is hereby incorporated by reference), theta (Studwell-Vaughan et al., “DNA Polymerase III Accessory Proteins. V. Theta Encoded by holE,” J. Biol. Chem., 268:11785-11791 (1993), which is hereby incorporated by reference), and SSB (Weiner et al., “The Deoxyribonucleic Acid Unwinding Protein of Escherichia coli,” J. Biol. Chem., 250:1972-1980 (1975), which is hereby incorporated by reference). E. coli Pol III core and clamp loader complex (composed of subunits gamma, delta, delta prime, chi, and psi) were reconstituted as described in Onrust et al., “Assembly of a Chromosomal Replication Machine: Two DNA Polymerases, a Clamp Loader and Sliding Clamps in One Holoenzyme Particle. I. Organization of the Clamp Loader,” J. Biol. Chem., 270:13348-13357 (1995), which is hereby incorporated by reference. Pol III* was reconstituted and purified as described in Onrust et al., “Assembly of a Chromosomal Replication Machine Two DNA Polymerases, a Clamp Loader and Sliding Clamps in One Holoenzyme Particle. III. Interface Between Two Polymerases and the Clamp Loader,” J. Biol. Chem., 270:13366-13377 (1995), which is hereby incorporated by reference. Protein concentrations were quantitated by the Protein Assay (Bio-Rad) method using bovine serum albumin (BSA) as a standard. DNA oligonucleotides were synthesized by Oligos etc. Calf thymus DNA was from Sigma. Buffer A is 20 mM Tris-HCl (pH=7.5), 0.5 mM EDTA, 2 mM DTT, and 20% glycerol. Replication buffer is 20 mM Tris-Cl (pH 7.5), 8 mM MgCl2, 5 mM DTT, 0.5 mM EDTA, 40 μg/ml BSA, 4% glycerol, 0.5 mM ATP, 3 mM each dCTP, dGTP, dATP, and 20 μM [α-32P]dTTP. P-cell buffer is 50 mM potassium phosphate (pH 7.6), 5 mM DTT, 0.3 mM EDTA, 20% glycerol. T.E. buffer is 10 mM Tris-HCl (pH 7.5), 1 mM EDTA. Cell lysis buffer is 50 mM Tris-HCl (pH 8.0) 10% sucrose, 1 M NaCl, 0.3 mM spermidine.
Example 2
Calf Thymus DNA Replication Assays
These assays were used in the purification of DNA polymerases from S. aureus cell extracts. Assays contained 2.5 μg activated calf thymus DNA in a final volume of 25 μl replication buffer. An aliquot of the fraction to be assayed was added to the assay mixture on ice followed by incubation at 37° C. for 5 min. DNA synthesis was quantitated using DE81 paper as described in Rowen et al., “Primase, the DnaG Protein of Escherichia coli. An Enzyme Which Starts DNA Chains,” J. Biol. Chem., 253:758-764 (1979), which is hereby incorporated by reference.
Example 3
PolydA-oligodT Replication Assays
PolydA-oligodT was prepared as follows. PolydA of average length 4500 nucleotides was purchased from SuperTecs. OligodT35 was synthesized by Oligos etc. 145 ul of 5.2 mM (as nucleotide) polydA and 22 μl of 1.75 mM (as nucleotide) oligodT were mixed in a final volume of 2100 μl T.E. buffer (ratio as nucleotide was 21:1 polydA to oligodT). The mixture was heated to boiling in a 1 ml eppendorf tube, then removed and allowed to cool to room temperature. Assays were performed in a final volume of 25 μl 20 mM Tris-Cl (pH 7.5), 8 mM MgCl2, 5 mM DTT, 0.5 mM EDTA, 40 μg/ml BSA, 4% glycerol, containing 20 μM [α-32P]dTTP and 0.36 μg polydA-oligodT. Proteins were added to the reaction on ice, then shifted to 37° C. for 5 min. DNA synthesis was quantitated using DE81 paper as described in Rowen et al., “Primase, the DnaG Protein of Escherichia coli. An Enzyme Which Starts DNA Chains,” J. Biol. Chem., 253:758-764 (1979), which is hereby incorporated by reference.
Example 4
Singly Primed M13mp18 ssDNA Replication Assays
M13mp18 was phenol extracted from phage and purified by two successive bandings (one downward and one upward) in cesium chloride gradients. M13mp18 ssDNA was singly primed with a DNA 30mer (map position 6817-6846) as described in Studwell et al. “Processive Replication is Contingent on the Exonuclease Subunit of DNA Polymerase III Holoenzyme,” J. Biol. Chem., 265:1171-1178 (1990), which is hereby incorporated by reference. Replication assays contained 72 ng of singly primed M13mp18 ssDNA in a final volume of 25 μl of replication buffer. Other proteins added to the assay, and their amounts, are indicated in the Brief Description of the Drawings. Reactions were incubated for 5 min. at 37° C. and then were quenched upon adding an equal volume of 1% SDS and 40 mM EDTA. DNA synthesis was quantitated using DE81 paper as described in Rowen et al., “Primase, the DnaG Protein of Escherichia coli. An Enzyme Which Starts DNA Chains,” J. Biol. Chem., 253:758-764 (1979), which is hereby incorporated by reference, and product analysis was performed in a 0.8% native agarose gel followed by autoradiography.
Example 5
Genomic Staphylococcus aureus DNA
Two strains of S. aureus were used. For PCR of the first fragment of the dnaX gene sequence, the strain was ATCC 25923. For all other work the strain was strain 4220 (a gift of Dr. Pat Schlievert, University of Minnisota). This strain lacks a gene needed for producing toxic shock (Kreiswirth et al., “The Toxic Shock Syndrome Exotoxin Structural Gene is Not Detectably Transmitted by a Prophage,” Nature, 305:709-712 (1996) and Balan et al., “Autocrine Regulation of Toxin Synthesis by Staphylococcus aureus,” Proc. Natl. Acad. Sci. USA, 92:1619-1623 (1995), which are hereby incorporated by reference). S. aureus cells were grown overnight at 37° C. in LB containing 0.5% glucose. Cells were collected by centrifugation (24 g wet weight). Cells were resuspended in 80 ml solution I (50 mM glucose, 10 mM EDTA, 25 mM Tris-HCL (pH 8.0)). SDS and NaOH were then added to 1% and 0.2 N, respectively, followed by incubation at 65° C. for 30 min. to lyse the cells. 68.5 ml of 3 M sodium acetate (pH 5.0) was added followed by centrifugation at 12,000 rpm for 30 min. The supernatant was discarded and the pellet was washed twice with 50 ml of 6 M urea, 10 mM Tris-HCL (pH 7.5), 1 mM EDTA using a dounce homogenizer. After each wash, the resuspended pellet was collected by centrifugation (12,000 rpm for 20 min.). After the second wash, the pellet was resuspended in 50 ml 10 mM T.E. buffer using a dounce homogenizer and then incubated for 30 min. at 65° C. The solution was centrifuged at 12,000 rpm for 20 min., and the viscous supernatant was collected. 43.46 g CsCl2 was added to the 50 ml of supernatant (density between 1.395-1.398) and poured into two 35 ml quick seal ultracentrifuge tubes (tubes were completely filled using the same density of CsCl2 in T.E.). To each tube was added 0.5 ml of a 10 mg/ml stock of ethidium bromide. Tubes were spun at 55,000 rpm for 18 h at 18° C. in a Sorvall TV860 rotor. The band of genomic DNA was extracted using a syringe and needle. Ethidium bromide was removed using two butanol extractions and then dialyzed against 4 1 of T.E. at pH 8.0 overnight. The DNA was recovered by ethanol precipitation and then resuspended in T.E. buffer (1.7 mg total) and stored at −20° C.
Example 6
Cloning and Purification of S. aureus Pol III-L
To further characterize the mechanism of DNA replication in S. aureus, large amounts of its replication proteins were produced through use of the genes. The polC gene encoding S. aureus Pol III-L (alpha-large) subunit has been sequenced and expressed in E. coli (Pacitti et al., “Characterization and Overexpression of the Gene Encoding Staphylococcus aureus DNA Polymerase III,” Gene, 165:51-56 (1995), which is hereby incorporated by reference). The previous work utilized a pBS[KS] vector for expression in which the E. coli RNA polymerase is used for gene transcription. In the earlier study, the S. aureus polC gene was precisely cloned at the 5′ end encoding the N-terminus, but the amount of the gene that remained past the 3′ end was not disclosed and the procedure for subcloning the gene into the expression vector was only briefly summarized. Furthermore, the previous study does not show the level of expression of the S. aureus Pol III-L, nor the amount of S. aureus Pol III-L that is obtained from the induced cells. Since the previously published procedure could not be repeated and the efficiency of the expression vector could not be assessed, another strategy outlined below had to be developed.
The isolated polC gene was cloned into a vector that utilizes T7 RNA polymerase for transcription as this process generally expresses a large amount of protein. Hence, the S. aureus polC gene was cloned precisely into the start codon at the NdeI site downstream of the T7 promotor in a pET vector. As the polC gene contains an internal NdeI site, the entire gene could not be amplified and placed it into the NdeI site of a pET vector. Hence, a three step cloning strategy that yielded the desired clone was devised (FIG. 1). These attempts were quite frustrating initially as no products of cloning in standard E. coli strains such as DH5α, a typical laboratory strain for preparation of DNA, could be obtained. Finally, a cell that was mutated in several genes affecting DNA stability was useful in obtaining the desired products of cloning.
In brief, the cloning strategy required use of another expression vector (called pET1137 kDa) in which the 37 kDa subunit of human RFC, the clamp loader of the human replication system, had been cloned into the pET11 vector. The gene encoding the 37 kDa subunit contains an internal NsiI site, which was needed for the precise cloning of the isolated polC gene. This three step strategy is shown in FIG. 1. In the first step, an approximately 2.3 kb section of the 5′ section of the gene (encoding the N-terminus of Pol III-L) was amplified using the polymerase chain reaction (PCR). Primers were as follows:
Upstream (SEQ. ID. No. 35)

ggtggtaatt gtcttgcata tgacagagc  29

Downstream (SEQ. ID. No. 36)

agcgattaag tggattgccg ggttgtgatg c  31

Amplification was performed using 500 ng genomic DNA, 0.5 mM EDTA, 1 μM of each primer, 1 mM MgSO4, 2 units vent DNA polymerase (New England Biolabs) in 100 μl of vent buffer (New England Biolabs). Forty cycles were performed using the following cycling scheme: 94° C., 1 min; 60° C., 1 min.; 72° C., 2.5 min. The product was digested with NdeI (underlined in the upstream primer) and NsiI (an internal site in the product) and the approximately 1.8 kb fragment was gel purified. A pET11 vector containing as an insert the 37 kDa subunit of human replication factor C (pET1137 kDa) was digested with NdeI and NsiI and gel purified. The PCR fragment was ligated into the digested pET1137 kDa vector and the ligation reaction was transformed into Epicurean coli supercompetent SURE 2 cells (Stratagene) and colonies were screened for the correct chimera (pET11PolC1) by examining minipreps for proper length and correct digestion products using NdeI and NsiI. In the second step, an approximately 2076 bp fragment containing the DNA encoding the C-terminus of Pol III-L subunit was amplified using the following sequences as primers:

Upstream (SEQ. ID. No. 37)

agcatcacaa cccggcaatc cacttaatcg c  31

Downstream (SEQ. ID. No. 38)

gactacgcca tgggcattaa ataaatacc  29

The amplification cycling scheme was as described above except the elongation step at 72° C. was for 2 min. The product was digested with BamHI (underlined in the downstream primer) and NsiI (internal to the product) and the approximately 480 bp product was gel purified and ligated into the pET11PolC1 that had been digested with NsiI/BamHI and gel purified (ligated product is pET11PolC2). To complete the expression vector, an approximately 2080 bp PCR product was amplified over the two NsiI sites internal to the gene using the following primers:

Upstream (SEQ. ID. No. 39)

gaagatgcat ataaacgtgc aagacctagt  30

Downstream (SEQ. ID. No. 40)

gtctgacgca cgaattgtaa agtaagatgc atag  34

The amplification cycling scheme was as described above except the 72° C. elongation step was 2 min. The PCR product, and the pET11PolC2 vector, were digested with NsiI and gel purified. The ligation mixture was transformed as described above and colonies were screened for the correct chimera (pET11PolC).

To express Pol III-L polymerase, the pET11PolC plasmid was transformed into E. coli strain BL21 (DE3). 24 L of E. coli BL21(DE3)pET11PolC were grown in LB media containing 50 pg/ml ampicillin at 37° C. to an OD of 0.7 and then the temperature was lowered to 15° C. Cells were then induced for Pol III-L expression upon addition of 1 mM IPTG to produce the T7 RNA polymerase needed to transcribe polC. This step was followed by further incubation at 15° C. for 18 h. Expression of S. aureus Pol III-L polymerase was so high that it could easily be visualized by Coomassie staining of a SDS polyacrylamide, gel of whole cells (FIG. 2A). The expressed protein migrated in the SDS polyacrylamide gel in a position expected for a 165 kDa polypeptide. In this procedure, it is important that cells are induced at 15° C., as induction at 37° C. produces a truncated version of Pol III-L polymerase, of approximately 130 kDa.
Cells were collected by centrifugation at 5° C. Cells (12 g wet weight) were stored at −70° C. The following steps were performed at 4° C. Cells were thawed and lysed in cell lysis buffer as described (final volume=50 ml) and were passed through a French Press (Amico) at a minimum of 20,000 psi. PMSF (2 mM) was added to the lysate as the lysate was collected from the French Press. DNA was removed and the lysate was clarified by centrifugation. The supernatent was dialyzed for 1 h against Buffer A containing 50 mM NaCl. The final conductivity was equivalent to 190 mM NaCl. Supernatent (24 ml, 208 mg) was diluted to 50 ml using Buffer A to bring the conductivity to 96 mM MgCl2, and then was loaded onto an 8 ml MonoQ column equilibrated in Buffer A containing 50 mM NaCl. The column was eluted with a 160 ml linear gradient of Buffer A from 50 mM NaCl to 500 mM NaCl. Seventy five fractions (1.3 ml each) were collected (FIG. 2B-C). Aliquots were analyzed for their ability to synthesize DNA, and 20 μl of each fraction was analyzed by Coomassie staining of an SDS polyacrylamide gel. Based on the DNA synthetic capability, and the correct size band in the gel, fractions 56-65 containing Pol III-L polymerase were pooled (22 ml, 31 mg). The pooled fractions were dialyzed overnight at 4° C. against 50 mM phosphate (pH 7.6), 5 mM DTT, 0.1 mM EDTA, 2 mM PMSF, and 20% glycerol (P-cell buffer). The dialyzed pool was loaded onto a 4.5 ml phosphocellulose column equilibrated in P-cell buffer, and then eluted with a 25 ml linear gradient of P-cell buffer from 0 M NaCl to 0.5 M NaCl. Fractions of 1 ml were collected and analyzed in a SDS polyacrylamide gel stained with Coomassie Blue (FIG. 2D). Fractions 20-36 contained the majority of the Pol III-large at a purity of greater than 90% (5 mg).
Example 7
S. aureus Pol III-L is not Processive on its Own
The Pol III-L polymerase purifies from B. subtilis as a single subunit without accessory factors (Barnes et al., “Purification of DNA Polymerase III of Gram-positive Bacteria,” Methods in Enzy., 262:35-42 (1995), which is hereby incorporated by reference). Hence, it seemed possible that it may be a Type I replicase (e.g., like T5 polymerase) and, thus, be capable of extending a single primer full length around a long singly primed template. To perform this experiment, a template M13 mp18 ssDNA primed with a single DNA oligonucleotide either in the presence or absence of SSB was used. DNA products were analyzed in a neutral agarose gel which resolved products by size. The results showed that Pol III-L polymerase was incapable of extending the primer around the DNA (to form a completed duplex circle referred to as replicative form II (“RFII”)) whether SSB was present or not. This experiment has been repeated using more enzyme and longer times, but no full length RFII products are produced. Hence, Pol III-L would appear not to follow the paradigm of the T5 system (Type I replicase) in which the polymerase is efficient in synthesis in the absence of any other protein(s).
Example 8
Cloning and Purification of S. aureus Beta Subunit
The sequence of an S. aureus homolog of the E. coli dnaN gene (encoding the beta subunit) was obtained in a study in which the large recF region of DNA was sequenced (Alonso et al., “Nucleotide Sequence of the recF Gene Cluster From Staphylococcus aureus and Complementation Analysis in Bacillus subtilis recF Mutants,” Mol. Gen. Genet., 246:680-686 (1995), Alonso et al., “Nucleotide Sequence of the recF Gene Cluster From Staphylococcus aureus and Complementation Analysis in Bacillus subtilis recF Mutants,” Mol. Gen. Genet., 248:635-636 (1995), which are hereby incorporated by reference). Sequence alignment of the S. aureus beta and E. coli beta show approximately 30% identity. Overall this level of homology is low and makes it uncertain that S. aureus beta will have the same shape and function as the E. coli beta subunit.
To obtain S. aureus beta protein, the dnaN gene was isolated and precisely cloned into a pET vector for expression in E. coli. S. aureus genomic DNA was used as template to amplify the homolog of the dnaN gene (encoding the putative beta). The upstream and downstream primers were designed to isolate the dnaN gene by PCR amplification from genomic DNA. Primers were:
Upstream (SEQ. ID. No. 41)

cgactggaag gagttttaac atatgatgga attcac  36

Downstream (SEQ. ID. No. 42)

ttatatggat ccttagtaag ttctgattgg  30

The NdeI site used for cloning into pET16b (Novagen) is underlined in the Upstream primer and the BamHI site used for cloning into pET16b is underlined in the Downstream primer. The NdeI and BamHI sites were used for directional cloning into pET16 (FIG. 3). Amplification was performed using 500 ng genomic DNA, 0.5 mM dNTPs, 1 μM of each primer, 1 mM MgSO4, 2 units vent DNA polymerase in 100 ul of vent buffer. Forty cycles were performed using the following cycling scheme: 94° C., 1 min; 60° C., 1 min.; 72° C., 1 min. 10s. The 1167 bp product was digested with NdeI and BamHI and purified in a 0.7% agarose gel. The pure digested fragment was ligated into the pET16b vector which had been digested with NdeI and BamHI and gel purified in a 0.7% agarose gel. Ligated products were transformed into E. coli competent SURE II cells (Stratagene) and colonies were screened for the correct chimera by examining minipreps for proper length and correct digestion products using NdeI and BamHI.

24 L of BL21(DE3)pETbeta cells were grown in LB containing 50 pg/ml ampicillin at 37° C. to an O.D. of 0.7, and, then, the temperature was lowered to 15C. IPTG was added to a concentration of 2 mM and after a further 18 h at 15° C. to induce expression of S. aureus beta (FIG. 4A). It is interesting to note that the beta subunit, when induced at 37° C., was completely insoluble. However, induction of cells at 15° C. provided strong expression of beta and, upon cell lysis, over 50% of the beta was present in the soluble fraction.
Cells were harvested by centrifugation (44 g wet weight) and stored at −70° C. The following steps were performed at 4° C. Cells (44 g wet weight) were thawed and resuspended in 45 ml 1× binding buffer (5 mM imidizole, 0.5 M NaCl, 20 mM Tris HCl (final pH 7.5)) using a dounce homogenizer. Cells were lysed using a French Pressure cell (Aminco) at 20,000 psi, and then 4.5 ml of 10% polyamine P (Sigma) was added. Cell debris and DNA was removed by centrifugation at 13,000 rpm for 30 min. at 4° C. The pET16beta vector places a 20 residue leader containing 10 histidine residues at the N-terminus of beta. Hence, upon lysing the cells, the S. aureus beta was greatly purified by chromatography on a nickel chelate resin (FIG. 4B). The supernatant (890 mg protein) was applied to a 10 ml HiTrap Chelating Separose column (Pharmacia-LKB) equilibrated in binding buffer. The column was washed with binding buffer, then eluted with a 100 ml linear gradient of 60 mM imidazole to 1 M imidazole in binding buffer. Fractions of 1.35 ml were collected. Fractions were analyzed for the presence of beta in an SDS polyacrylamide gel stained with Coomassie Blue. Fractions 28-52, containing most of the beta subunit, were pooled (35 ml, 82 mg). Remaining contaminating protein was removed by chromatography on MonoQ. The S. aureus beta becomes insoluble as the ionic strength is lowered and, thus, the pool of beta was dialyzed overnight against Buffer A containing 400 mM NaCl. The dialyzed pool became slightly turbid indicating it was at its solubility limit at these concentrations of protein and NaCl. The insoluble material was removed by centrifugation (64 mg remaining) and, then, diluted 2-fold with Buffer A to bring the conductivity to 256. The protein was then applied to an 8 ml MonoQ column equilibrated in Buffer A plus 250 mM NaCl and then eluted with a 100 ml linear gradient of Buffer A from 0.25 M NaCl to 0.75 M NaCl; fractions of 1.25 ml were collected (FIG. 4C). Under these conditions, approximately 27 mg of the beta flowed through the column and the remainder eluted in fractions 1-18 (24 mg).
Example 9
The S. aureus Beta Subunit Protein Stimulates S. aureus Pol III-L and E. coli Core
The experiment of FIG. 5A, tests the ability of S. aureus beta to stimulate S. aureus Pol III-L on a linear polydA-oligodT template. Reactions are also performed with E. coli beta and Pol III core. The linear template was polydA of average length of 4500 nucleotides primed with a 30mer oligonucleotide of T residues. The first two lanes show the activity of Pol III-L either without (lane 1) or with S. aureus beta (lane 2). The result shows that the S. aureus beta stimulates Pol III-L approximately 5-6 fold. Lanes 5 and 6 show the corresponding experiment using E. coli core with (lane 6) or without (lane 5) E. coli beta. The core is stimulated over 10-fold by the E. coli beta subunit under the conditions used.
Although Gram positive and Gram negative cells diverged from one another long ago and components of one polymerase machinery would not be expected to be interchangable, it was decided to test the activity of the S. aureus beta with E. coli Pol III core. Lanes 3 and 4 shows that the S. aureus beta also stimulates E. coli core about 5-fold. This result can be explained by an interaction between the clamp and the polymerase that has been conserved during the evolutionary divergence of gram positive and gram negative cells. A chemical inhibitor that would disrupt this interaction would be predicted to have a broad spectrum of antibiotic activity, shutting down replication in Gram negative and Gram positive cells alike. This assay, and others based on this interaction, can be devised to screen chemicals for such inhibition. Further, since all the proteins in this assay are highly overexpressed through recombinant techniques, sufficient quantities of the protein reagents can be obtained for screening hundreds of thousands of compounds.
In summary, the results show that S. aureus beta, produced in E. coli, is indeed an active protein (i.e., it stimulates polymerase activity). Furthermore, the results shows that Pol III-L functions with a second protein (i.e., S. aureus beta). Before this experiment, there was no assurance that Pol III-L, which is significantly different in structure from E. coli alpha, would function with another protein. For example, unlike E. coli alpha, which copurifies with several accessory proteins, Pol III-L purified from B. subtilis as a single protein with no other subunits attached (Barnes et al., “Purification of DNA Polymerase III of Gram-positive Bacteria,” Methods in Enzy., 262:35-42 (1995), which is hereby incorporated by reference). Finally, if one were to assume that S. aureus beta would function with a polymerase, the logical candidate would have been the product of the dnaE gene (alpha-small) instead of polC (Pol III-L) since the dnaE product is more homologous to E. coli alpha subunit than Pol III-L.
Example 10
The S. aureus Beta Subunit Behaves as a Circular Sliding Clamp
The ability of S. aureus beta to stimulate Pol I11-L could be explained by formation of a 2-protein complex between Pol III-L and beta to form a processive replicase similar to the Type II class (e.g., T7 type). Alternatively, the S. aureus replicase is organized as the Type III replicase which operates with a circular sliding clamp and a clamp loader. In this case, the S. aureus beta would be a circular protein and would require a clamp loading apparatus to load it onto DNA. The ability of the beta subunit to stimulate Pol III-L in FIG. 5A could be explained by the fact that the polydA-oligodT template is a linear DNA and a circular protein could thread itself onto the DNA over an end. Such “end threading” has been observed with PCNA and explains its ability to stimulate DNA polymerase delta in the absence of the RFC clamp loader (Burgers et al., “ATP-Independent Loading of the Proliferating Cell Nuclear Antigen Requires DNA Ends,” J. Biol. Chem., 268:19923-19926 (1993), which is hereby incorporated by reference).
To distinguish between these possibilities, S. aureus beta was examined for ability to stimulate Pol III-L on a circular primed template. In FIG. 5B, assays were performed using circular M13 mp18 ssDNA coated with E. coli SSB and primed with a single oligonucleotide to test the activity of beta on circular DNA. Lane 1 shows the extent of DNA synthesis using Pol III-L alone. In lane 2, Pol III-L was supplemented with S. aureus beta. The S. aureus beta did not stimulate the activity of Pol III-L on this circular DNA (nor in the absence of SSB). Inability of S. aureus beta to stimulate Pol III-L is supported by the results of FIG. 6, lane 1 that analyzes the product of Pol III-L action on the circular DNA in an agarose gel in the presence of S. aureus beta. In summary, these results show that S. aureus beta only stimulates Pol III-L on linear DNA, not circular DNA. Hence, the S. aureus beta subunit behaves as a circular protein.
Lane 3 shows the result of adding both S. aureus beta and E. coli gamma complex to Pol III-L. Again, no stimulation was observed (compare with lane 1). This result indicates that the functional contacts between the clamp and clamp loader were not conserved during evolution of Gram positive and Gram negative cells.
Controls for these reactions on circular DNA are shown for the E. coli system in Lanes 4-6. Addition of only beta to E. coli Pol III core did not result in stimulating the polymerase (compare lanes 4 and 5). However, when clamp loader complex was included with beta and core, a large stimulation of synthesis was observed (lane 6). In summary, stimulation of synthesis is only observed when both beta and clamp loader complex were present, consistent with inability of the circular beta ring to assemble onto circular DNA by itself.
Example 11
Pol III-L Functions as a Pol III-Type Replicase with Beta and a Clamp Loader Complex to Become Processive
Next, it was determined whether S. aureus Pol III-L requires two components (a beta clamp and a clamp loader) to extend a primer full length around a circular primed template. In FIG. 6, a template circular M13 mp18 ssDNA primed with a single DNA oligonucleotide was used. DNA products were analyzed in a neutral agarose gel which resolves starting materials (labeled ssDNA in FIG. 6) from completed duplex circles (labelled RFII for replicative form II). The first two lanes show, as demonstrated in other examples, that Pol III-L is incapable of extending the primer around the circular DNA in the presence of only S. aureus beta. In lane 4 of FIG. 6, E. coli clamp loader complex (also known as gamma complex) and beta subunit were mixed with S. aureus Pol III-L in the assay containing singly primed M13mp18 ssDNA coated with SSB. If the beta clamp, assembled on DNA by clamp loader complex, provides processivity to S. aureus Pol III-L, the ssDNA circle should be converted into a fully duplex circle (RFII) which would be visible in an agarose gel analysis. The results of the experiment showed that the E. coli beta and clamp loader complex did indeed provide Pol III-L with ability to fully extend the primer around the circular DNA to form the RFII (lane 4). The negative control using only E. coli clamp loader complex and beta is shown in lane 3. For comparison, lane 6 shows the result of mixing the three components of the E. coli system (Pol III core, beta, and clamp loader complex). This reaction gives almost exclusively full length RFII product. The qualitatively different product profile that Pol III-L gives in the agarose gel analysis compared to E. coli Pol III core with beta and clamp loader complex shows that the products observed using Pol III-L is not due to a contaminant of E. coli Pol III core in the S. aureus Pol III-L preparation (compare lanes 4 and 6).
It is generally thought that the polymerase of one system is specific for its SSB. However, these reactions are performed on ssDNA coated with the E. coli SSB protein. Hence, the S. aureus Pol III-L appears capable of utilizing E. coli SSB and the E. coli beta. It would appear that the only component that is not interchangeable between the Gram positive and Gram negative systems is the clamp loader complex.
Thus, the S. aureus Pol III-L functions as a Pol III type replicase with the E. coli beta clamp assembled onto DNA by a clamp loader complex.
Example 12
Purification of Two DNA Polymerase III-Type Enzymes from S. aureus Cells
The MonoQ resin by Pharmacia has very high resolution which would resolve the three DNA polymerases of S. aureus. Hence, S. aureus cells were lysed, DNA was removed from the lysate, and the clarified lysate was applied onto a MonoQ column. The details of this procedure are: 300 L of S. aureus (strain 4220, a gift of Dr. Pat Schlievert, University of Minnisota) was grown in 2×LB media at 37° C. to an O.D. of approximately 1.5 and then were collected by centrifugation. Approximately 2 kg of wet cell paste was obtained and stored at −70° C. 122 g of cell paste was thawed and resuspended in 192 ml of cell lysis buffer followed by passage through a French Press cell (Aminco) at 40,000 psi. The resultant lysate was clarified by high speed centrifugation (1.3 g protein in 120 ml). A 20 ml aliquot of the supernatant was dialyzed 2 h against 2 L of buffer A containing 50 mM NaCl. The dialyzed material (148 mg, conductivity =101 mM NaCl) was diluted 2-fold with Buffer A containing 50 mM NaCl and then loaded onto an 8 ml MonoQ column equilibrated in Buffer A containing 50 mM NaCl. The column was washed with Buffer A containing 50 mM NaCl, and then eluted with a 160 ml linear gradient of 0.05 M NaCl to 0.5 M NaCl in Buffer A. Fractions of 2.5 ml (64 total) were collected, followed by analysis in an SDS polyacrylamide gel for their replication activity in assays using calf thymus DNA.
Three peaks of DNA polymerase activity were identified (FIG. 7). Previous studies of cell extracts prepared from the Gram positive organism Bacillus subtilis identified only two peaks of activity off a DEAE column (similar charged resin to MonoQ). The first peak was Pol II, and the second peak was a combination of DNA polymerases I and III. The DNA polymerases I and III were then separated on a subsequent phosphocellulose column. The middle peak in FIG. 7 is much larger than the other two peaks and, thus, it was decided to chromatograph this peak on a phosphocellulose column. The second peak of DNA synthetic activity was pooled (fractions 37-43; 28 mg in 14 ml) and dialyzed against 1.5 L P-cell buffer for 2.5 h. Then, the sample (ionic strength equal to 99 mM NaCl) was applied to a 5 ml phosphocellulose column equilibrated in P-cell buffer. After washing the column in 10 ml P-cell buffer, the column was eluted with a 60 ml gradient of 0-0.5 M NaCl in P-cell buffer. Seventy fractions were collected and then analyzed for DNA synthesis using calf thymus DNA as template. This column resolved the polymerase activity into two distinct peaks (FIG. 7B).
Hence, there appear to be four DNA polymerases in Staphylococcus aureus. They were designated here as peak 1 (first peak off MonoQ), peak 2 (first peak off phosphocellulose), peak 3 (second peak of phosphocellulose), and peak 4 (last peak off Mono Q) (see FIG. 7). Peak 4 was presumably Pol III-L, as it elutes from MonoQ in a similar position as the Pol III-L expressed in E. coli (compare FIG. 7A with FIG. 2).
Example 13
Demonstration That Peak 1 (Pol III-2) Functions as a Pol III-Type Replicase with E. coli Beta Assembled on DNA by E. coli Clamp Loader Complex
To test which peak contained a Pol III-type of polymerase, an assay was used in which the E. coli clamp loader complex and beta support formation of full length RFII product starting from E. coli SSB coated circular M13mp18 ssDNA primed with a single oligonucleotide. In FIG. 8, both Peaks 1 and 2 are stimulated by the E. coli clamp loader complex and beta subunit and, in fact, Peaks 2 and 3 are inhibited by these proteins (the quantitation is shown below the gel in the figure). Further, the product analysis in the agarose gel shows full length RFII duplex DNA circles only for peaks 1 and 4. These results, combined with the NEM, pCMB, and KC1 characteristics in Tables 2 and 3 below, suggest that there are two Pol III-type DNA polymerases in S. aureus and that these are partially purified in peaks 1 and 4.
Next, it was determined which of these peaks of DNA polymerase activity correspond to DNA polymerases 1, I, and III, and which peak is the unidentified DNA polymerase. In the Gram positive bacterium B. subtilis, Pol III is inhibited by pCMB, NEM, and 0.15 M NaCl, Pol II is inhibited by KCl, but not NEM or 0.15 M KCL, and Pol I is not inhibited by any of these treatments (Gass et al., “Further Genetic and Enzymological Characterization of the Three Bacillus subtilis Deoxyribonucleic Acid Polymerases,” J. Biol. Chem., 248:7688-7700 (1973), which is hereby incorporated by reference). Hence, assays were performed in the presence or absence of pCMB, NEM, and 0.15 M KCl (see Tables 2 and 3 below). Peak 3 clearly corresponded to Pol I, because it was not inhibited by NEM, pCMB, or 0.15 M NaCl. Peak 2 correspond to Pol II, because it was not inhibited by NEM, but was inhibited by pCMB and 0.15 M NaCl. Peaks 1 and 4 both had characteristics that mimic Pol III; however, peak 4 elutes on MonoQ at a similar position as Pol III-L expressed in E. coli (see FIG. 2B). Hence, peak 4 is likely Pol III-L, and peak 1 is likely the unknown polymerase.







TABLE 2







Expected Characteristics of Polymerases












Polymerase
pCMB
NEM
0.15 M KCl







Pol I
not inhibited*
not inhibited
not inhibited



Pol II
inhibited**
not inhibited
not inhibited



Pol III-L
inhibited
inhibited
not inhibited







*Not inhibited is defined as greater than 75% remaining activity



**Inhibited is defined as less than 40% remaining activity













TABLE 3







Observed Characteristics










Peak
pCMB
NEM
0.15 M KCL assignment





Peak1
inhibited
inhibited
new polymerase


Peak2
inhibited
not inhibited
Pol II


Peak3
not inhibited
not inhibited
Pol I


Peak4
inhibited
inhibited
Pol III-L









Example 14
Identification and Cloning of S. aureus dnaE
This invention describes the finding of two DNA polymerases that function with a sliding clamp assembled onto DNA by a clamp loader. One of these DNA polymerases is likely Pol III-L, but the other has not been identified previously. Presumably, the chromatographic resins used in earlier studies did not have the resolving power to separate the enzyme from other polymerases. This would be compounded by the low activity of Pol III-2. To identify a gene encoding the second Pol III, the amino acid sequences of the Pol III alpha subunit of Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Haemophilis influenzae, and Helicobacter pylori were aligned using Clustal W (1.5). Two regions about 400 residues apart were conserved and primers were designed for the following amino acid sequences:
Upstream, corresponding in E. coli to residues 385-399 (SEQ. ID. No. 43)







Leu Leu Phe Glu Arg Phe Leu Asn Pro Glu Arg Val


  1               5                  10


 


Ser Met Pro


         15






Downstream, corresponding in E. coli to residues 750-764 (SEQ. ID. No. 44)







Lys Phe Ala Gly Tyr Gly Phe Asn Lys Ser His Ser


  1               5                  10


 


Ala Ala Tyr


         15






The following primers were designed to these two peptide regions using codon preferences for S. aureus: 

Upstream (SEQ. ID. No. 45)

cttctttttg aaagatttct aaataaagaa cgttattcaa tgcc  44

Downstream (SEQ. ID. No. 46)

ataagctgca gcatgacttt tattaaaacc ataacctgca aattt  45

Amplification was performed using 2.5 units of Taq DNA Polymerase (Gibco, BRL), 100 ng S. aureus genomic DNA, 1 mM of each of the four dNTPs, 1 μM of each primer, and 3 mM MgCl2 in 100 μl of Taq buffer. Thirty-five cycles of the following scheme were repeated: 94° C., 1 min; 55° C., 1 min; 72° C., 90 sec. The PCR product (approximately 1.1 kb) was electrophoresed in a 0.8% agarose gel and purified using a Geneclean III kit (Bio 101). The product was then divided equally into ten separate aliquots and used as a template for PCR reactions, according to the above protocol, to reamplify the fragment for sequencing. The final PCR product was purified using a Quiagen Quiaquick PCR Purification kit, quantitated via optical density at 260 nM, and sequenced by the Protein/DNA Technology Center at Rockefeller University. The same primers used for PCR were used to prime the sequencing reactions.

Next, the following additional PCR primers were designed to obtain more sequence information 3′ to the first amplified section.
Upstream (SEQ. ID. No. 47)

agttaaaaat gccatatttt gacgtgtttt agttctaat  39

Downstream (SEQ. ID. No. 48)

cttgcaaaag cggttgctaa agatgttgga cgaattatgg gg  42

These primers were used in a PCR reaction using 2.5 units of Taq DNA Polymerase (Gibco, BRL) with 100 ng S. aureus genomic DNA as a template, 1 mM dNTP's, 1 μM of each primer, and 3 mM MgCl2 in 100 1 of Taq buffer. Thirty-five cycles of the following scheme were repeated: 94° C., 1 min; 55° C., 1 min; 72° C., 2 min 30 seconds. The 1.6 Kb product was then divided into 5 aliquots, and used as a template in a set of 5 PCR reactions, as described above, to amplify the product for sequencing. The products of these reactions were purified using a Qiagen Qiaquick PCR Purification kit, quantitated via optical density at 260 nm, and sequenced by the Protein/DNA Technology Center at Rockefeller University. The sequence of this product yielded about 740 bp of new sequence 3′ of the first sequence.

As this gene shows better homology to the Gram negative Pol III α subunit compared to Gram positive Pol III-L, it will be designated the dnaE gene.
Example 15
Identification and Cloning of S. aureus dnaX
The fact that the S. aureus beta stimulates Pol III-L and has a ring shape suggests that the Gram positive replication machinery is of the three component type. This implies the presence of a clamp loader complex. This is not a simple determination to make as the B. subtilis genome shows homologs to only two of the five subunits of the E. coli clamp loader (dnaX encoding gamma, and holB encoding delta prime). On the basis of the experiments in this application, which suggests that there is a clamp loader, it was believed that these two subunit homologues are part of the clamp loader for the S. aureus beta.
As a start in obtaining the clamp loading apparatus, a strategy was devised to obtain the gene encoding the tau subunit of S. aureus. In E. coli, the tau and gamma subunits are derived from the same gene. Tau is the full length product, and gamma is about 2⁄3 the length of tau. Gamma is derived from the dnaX gene by what was originally believed to be an efficient translational frameshift mechanism that, after it occurs, incorporates only one unique C-terminal residue before encountering a stop codon. To identify the dnaX gene of S. aureus by PCR analysis, the dnaX genes of B. subtilis, E. coli, and H. influenzae were aligned. Upon comparison of the amino acid sequence encoded by these dnaX genes, two areas of high homology were used to predict the amino acid sequence of the S. aureus dnaX gene product. PCR primers were designed to these sequences, and a PCR product of the expected size was indeed produced. DNA primers were designed to two regions of high similarity for use in PCR that were about 100 residues apart. The amino acid sequences of these regions were:
Upstream, corresponding to residues 39-48 of E. coli (SEQ. ID. No. 49)









His Ala Tyr Leu Phe Ser Gly Pro Arg Gly



  1               5                  10






Downstream, corresponding to residues 138-148 of E. coli (SEQ. ID. No. 50)










His Ala Tyr Leu Phe Ser Gly Pro Arg Gly



  1               5                  10






The DNA sequence of the PCR primers was based upon the codon usage of S. aureus. The primers are as follows: 

Upstream (SEQ. ID. No. 51)

cgcggatccc atgcatattt attttcaggt ccaagagg  38

Downstream (SEQ. ID. No. 52)

ccggaattct ggtggttctt ctaatgtttt taataatgc  39

The first 9 nucleotides of the upstream primer (SEQ. ID. No. 51) contain a BamHI site, which is underlined, and do not correspond to amino acid codons; the 3′ 29 nucleotides correspond to the amino acid sequence of SEQ. ID. No. 49. The EcoRI site of the downstream primer (SEQ. ID. No. 52) is underlined and the 3′ 33 nucleotides correspond to the amino acid sequence of SEQ. ID. No. 50.

The expected PCR product, based on the alignment, is approximately 268 bp between the primer sequences. Amplification was performed using 500 ng genomic DNA, 0.5 mM dNTPs, 1 μM of each primer, 1 mM MgSO4, 2 units vent DNA polymerase in 100 μl of vent buffer. Forty cycles were performed using the following cycling scheme: 94° C., 1 min; 60° C., 1 min.; 72° C., 30s. The approximately 300 bp product was digested with EcoRI and BamHI and purified in a 0.7% agarose gel. The pure digested fragment was ligated into pUC18 which had been digested with EcoRI and BamHI and gel purified in a 0.7% agarose gel. Ligated products were transformed into E. coli competent DH5a cells (Stratagene), and colonies were screened for the correct chimera by examining minipreps for proper length and correct digestion products using EcoRI and BamHI. The sequence of the insert was determined and was found to have high homology to the dnaX genes of several bacteria. This sequence was used to design circular PCR primers. Two new primers were designed for circular PCR based on this sequence.
A circular PCR product of approximately 1.6 kb was obtained from a HincII digest of chromosomal DNA that was recircularized with ligase. This first circular PCR yielded most of the remaining dnaX gene. The two primers were as follows:
Rightward (SEQ. ID. No. 53)

tttgtaaagg cattacgcag gggactaatt cagatgtg  38

Leftward (SEQ. ID. No. 54)

tatgacattc attacaaggt tctccatcag tgc  33

Genomic DNA (3 μg) was digested with HincII, purified with phenol/chloroform extraction, ethanol precipitated and redissolved in 70 μl T.E. buffer. The genomic DNA was recircularized upon adding 4000 units T4 ligase (New England Biolabs) in a final volume of 100 μl T4 ligase buffer (New England Biolabs) at 16° C. overnight. The PCR reaction consisted of 90 ng recircularized genomic DNA, 0.5 mM each dNTP, 100 μmol of each primer, 1.4 mM magnesium sulfate, and 1 unit of elongase (GIBCO) in a final volume of 100 μl elongase buffer (GIBCO). 40 cycles were performed using the following scheme: 94° C., 1 min.; 55° C., 1 min.; and 68° C., 2 min. The resulting PCR product was approximately 1.6 kb. The PCR product was purified from a 0.7% agarose gel and sequenced directly. A stretch of approximately 750 nucleotides was obtained using the rightward primer used in the circular PCR reaction. To obtain the rest of the sequence, other sequencing primers were designed in succession based on the information of each new sequencing run.

This sequence, when spliced together with the previous 300 bp PCR sequence, contained the complete N-terminus of the gene product (stop codons are present upstream) and possibly lacked only about 50 residues of the C-terminus. The amino terminal region of E. coli tau shares what appears to be the most conserved region of the gene as this area shares homology with RFC subunit of the human clamp loader and with the gene 44 protein of the phage T4 clamp loader. An alignment of the N-terminal region of the S. aureus tau protein with that of B. subtilis and E. coli is shown in FIG. 10. Among the highly conserved residues are the ATP binding site consensus sequence and the four cystine residues that form a Zn2+ finger.
After obtaining 1 kb of sequence in the 5′ region of dnaX, it was sought to determine the remaining 3′ end of the gene. Circular PCR products of approximately 800 bps, 600 bps, and 1600 bps were obtained from Apo I, or Nsi I or Ssp I digest of chromosomal DNA that were recircularized with ligase.
Rightward (SEQ. ID). No. 55)

gagcactgat gaacttagaa ttagatatg  29

Leftward (SEQ. ID. No. 56)

gatactcagt atctttctca gatgttttat tc  32

Genomic DNA (3 g) was digested with, Apo I, or Nsi I or Ssp I, purified with phenol/chloroform extraction, ethanol precipitated, and redissolved in 70 1 T.E. buffer. The genomic DNA was recircularized upon adding 4000 units of T4 ligase (New England Biolabs) in a final volume of 100 1 T4 ligase buffer (New England Biolabs) at 16° C. overnight. The PCR reaction consisted of 90 ng recircularized genomic DNA, 0.5 mM each dNTP, 100 μmol of each primer, 1.4 mM magnesium sulfate, and I unit of elongase (GIBCO) in a final volume of 100 1 elongase buffer (GIBCO). 40 cycles were performed using the following scheme: 94° C., 1 min.; 55° C., 1 min.; 68° C., 2 min. The PCR products were directly cloned into pCR II TOPO vector using the TOPO TA cloning kit (Invitrogen Corporation) for obtaining the rest of the C terminal sequence of S. aureus dnaX. DNA sequencing was performed by the Rockefeller University sequencing facility.

Example 16
Identification and Cloning of S. aureus dnaB
In E. coli, the DnaB helicase assembles with the DNA polymerase III holoenzyme to form a replisome assembly. The DnaB helicase also interacts directly with the primase to complete the machinery needed to duplicate a double helix. As a first step in studying how the S. aureus helicase acts with the replicase and primase, S. aureus was examined for presence of a dnaB gene.
The amino acid sequences of the DnaB helicase of Escherichia coli, Salmonella typhimurium, Haemophilis influenzae, and Helicobacter pylori were aligned using Clustal W (1.5). Two regions about 200 residues apart showed good homology. These peptide sequences were:
Upstream, corresponding to residues 225-238 of E. coli DnaB (SEQ. ID. No. 57)







Asp Leu Ile Ile Val Ala Ala Arg Pro Ser Met Gly


  1               5                  10


 


Lys Thr






Downstream, corresponding to residues 435-449 of E. coli DnaB (SEQ. ID. No. 58)








Glu Ile Ile Ile Gly Lys Gln Arg Asn Gly Pro Ile


  1               5                  10


 


Gly Thr Val


         15






The following primers were designed from regions which contained conserved sequences using codon preferences for S. aureus: 

Upstream (SEQ. ID. No. 59)

gaccttataa ttgtagctgc acgtccttct atgggaaaaa c  41

Downstream (SEQ. ID. No. 60)

aacattatta agtcagcatc ttgttctatt gatccagatt caacgaag  48

A PCR reaction was carried out using 2.5 units of Taq DNA Polymerase (Gibco, BRL) with 100 ng. S. aureus genomic DNA as template, 1 mM dNTP's, 1 μM of each primer, 3 mM MgCl2 in 100 μl of Taq buffer. Thirty-five cycles of the following scheme were repeated: 94° C., 1 min.; 55° C., 1 min.; and 72° C., 1 min. Two PCR products were produced, one was about 1.1 kb, and another was 0.6 kb. The smaller one was the size expected. The 0.6 kb product was gel purified and used as a template for a second round of PCR as follows. The 0.6 kb PCR product was purified from a 0.8% agarose gel using a Geneclean III kit (Bio 101) and then divided equally into five separate aliquots, as a template for PCR reactions. The final PCR product was purified using a Quiagen Quiaquick PCR Purification kit, quantitated via optical density at 260 nM, and sequenced by the Protein/DNA Technology Center at Rockefeller University. The same primers used for PCR were used to prime the sequencing reaction. The amino acid sequence was determined by translation of the DNA sequence in all three reading frames, and selecting the longest open reading frame. The PCR product contained an open reading frame over its entire length. The predicted amino acid sequence shares homology to the amino acid sequences encoded by dnaB gene of other organisms.

Additional sequence information was determined using the circular PCR technique. Briefly, S. aureus genomic DNA was digested with various endonucleases, then religated with T4 DNA ligase to form circular templates. To perform PCR, two primers were designed from the initial sequence.
First primer (SEQ. ID. No. 61)

gatttgtagt tctggtaatg ttgactcaaa ccgcttaaga accgg  45

Second primer (SEQ. ID. No. 62)

atacgtgtgg ttaactgatc agcaacccat ctctagtgag aaaatacc  48

The first primer matches the sequence of the coding strand and the second primer matches the sequence of the complementary strand. These two primers are directed outwards from a central point, and allow determination of new sequence information up to the ligated endonuclease site. A PCR product of approximately 900 bases in length was produced using the above primers and template derived from the ligation of S. aureus genomic DNA which had been cut with the restriction endonuclease Apo I. This PCR product was electrophoresed in a 0.8% agarose gel, eluted with a Qiagen gel elution kit, divided into five separate aliquots, and used as a template for reamplification by PCR using the same primers as described above. The final product was electrophoresed in an 0.8% agarose gel, visualized via staining with ethidium bromide under ultraviolet light, and excised from the gel. The excised gel slice was frozen, and centrifuged at 12,000 rpm for 15 minutes. The supernatant was extracted with phenol/chloroform to remove ethidium bromide, and was then cleaned using a Qiagen PCR purification kit. The material was then quantitated from its optical density at 260 nm and sequenced by the Protein/DNA Technology Center at the Rockefeller University.

The nucleotide sequence contained an open reading frame over its length, up to a sequence which corresponded to the consensus sequence of a cleavage site of the enzyme Apo I. Following this point, a second open reading frame encoded a different reading frame up to the end of the product. The initial sequence information was found to match the initial sequence and to extend it yet further towards the C-terminus of the protein. The second reading frame was found to end in a sequence which matched the 5′-terminus of the previously determined sequence and, thus, represents an extension of the sequence towards the N-terminus of the protein.
Additional sequence information was obtained using the above primers and a template generated using S. aureus genomic DNA circularized via ligation with T4 ligase following digestion with Cla I. The PCR product was generated using 35 cycles of the following program: denaturation at 94° C. for 1 min.; annealing at 55° C. for 1 min.; and extension at 68° C. for 3 minutes and 30 s. The PCR products were electrophoresed in a 0.8% agarose gel, eluted with a Qiagen gel elution kit, divided into five separate aliquots, and used as a template reamplification via PCR with the same primers described above. The final product was electrophoresed in an 0.8% agarose gel, visualized via staining with ethidium bromide under ultraviolet light, and excised from the gel. The excised gel slice was frozen, and centrifuged at 12,000 rpm for 15 min. The supernatant was cleaned using a Qiagen PCR purification kit. The material was then quantitated via optical density at 260 nm and sequenced by the Protein/DNA Technology Center at Rockefeller University. The open reading frames continued past 500 bases. Therefore, the following additional sequencing primers were designed from the sequence to obtain further information:
First primer (SEQ. ID. No. 63)

cgttttaatg catgcttaga aacgatatca g  31

Second primer (SEQ. ID. No. 64)

cattgctaag caacgttacg gtccaacagg c  31

The N-terminal and C-terminal nucleotide sequence extensions generated using this circular PCR product completed the 5′ region of the gene (encoding the N-terminus of DnaB); however, a stop codon was not reached in the 3′ region and, thus, a small amount of sequence is still needed to complete this gene.
The alignment of the S. aureus dnaB with E. coli dnaB and the dnaB genes of B. subtilis and S. typhimurium is shown in FIG. 11.
Example 17
Identification and Cloning of S. aureus holB
The S. aureus holB was identified by searching the S. aureus database with the sequences of S. pyogenes δ′ subunit. The S. aureus holB encodes a 253 residue protein of about 28 kDa. The holB gene was amplified by PCR using an upstream 69-mer primer as follows:
Upstream Primer (SEQ. ID. No. 65):

ggataacaat tccccgctag caataatttt gtttaacttt aagaaggaga tatacccatg  60

gatgaacag  69

which contains an NcoI site (underlined), and a downstream 39-mer primer as follows:

Downstream Primer (SEQ. ID. No. 66):

aattttaaag gatccgtgta taatattcta attttcccg  39

which contains a BamHI site (underlined). The PCR product was digested with NcoI and BamHI, purified, and ligated into the NcoI and BamHI sites of pET11a to produce plasmid pETSaholB.

Example 18
Purification of S. aureus δ′
The pETSaholB plasmid of Example 17 was transformed into E. coli BL21(DE3)recA. A single colony was used to innoculate 2 L of LB media supplemented with 200 μg/ml ampicillin. Cells (2 L) were grown at 37° C. to OD600=0.5 at which point the temperature was lowered to 15° C. and 0.5 mM IPTG was added. After 16 hr of induction, cells were collected by centrifugation, resuspended in 50 mM Tris-HCl (pH 7.5), 10% sucrose, 1 M NaCl, 30 mM spermidine, 5 mM DTT, and 2 mM EDTA. Cells were lysed by two passages through a French press (15,000 psi), followed by centrifugation at 13,000 rpm for 30 min at 4° C. Ammonium sulfate (0.3 g/ml) was added to the clarified lysate. The pellet was backwashed in 30 ml buffer A containing 0.1 M NaCl and 0.24 g/ml ammonium sulfate using a Dounce homogenizer, then the pellet was recovered by centrifugation. The resulting pellet was resuspended in 20 ml of buffer A and dialyzed against buffer A. The dialyzed protein was applied to a 20 ml FFQ Sepharose column equilibrated in buffer A and eluted with a 200 ml linear gradient of 0-500 mM NaCl in buffer A; 80 fractions were collected. Peak fractions (54-75) were combined (72 mg) and dialyzed against buffer A. The 6′ preparation was aliquoted and stored frozen at −80° C.
Example 19
Identification and Cloning of S. aureus holA
The S. aureus holA gene was identified by searching the S. aureus database with the sequences of E. coli and S. pyogenes 6 subunits. The S. aureus holA gene encodes a 288 residue protein of about 32 kDa. The holA gene was amplified by PCR using an upstream 28-mer primer as follows:
Upstream Primer (SEQ. ID. No. 67):

gggagtttgt aatccatgga tgaacagc  28

which contains a NcoI site (underlined), and a downstream 37-mer primer as follows:

Downstream Primer (SEQ. ID. No. 68):

ctgaacacct attaccctag gcatctaact cacaccc  37

which contains a BamHI site (underlined). The PCR product was digested with NcoI and BamHI, purified, and ligated into the NcoI and BamHI sites of pET11a to produce plasmid pETSaholA.

Example 20
Purification of S. aureus δ
The pETSaholA plasmid of Example 19 was transformed into E. coli NovaBlue (recA1 lac[F′proA+B+lacqZΔJM15::Tn10(TcR)) (Novagen). A single colony was used to innoculate 12 L of LB media supplemented with 200 μg/ml ampicillin. Cells (12 L) were grown at 37° C. to OD600=0.5 at which point the temperature was lowered to 15° C. and 0.5 mM IPTG was added. After 16 hr of induction, cells were collected by centrifugation, resuspended in 50 mM Tris-HCl (pH 7.5), 10% sucrose, 1 M NaCl, 30 mM spermidine, 5 mM DTT, and 2 mM EDTA. Cells were lysed by two passages through a French press (15,000 psi), followed by centrifugation at 13,000 rpm for 30 min at 4° C. Ammonium sulfate (0.3 g/ml) was added to the clarified lysate. The resulting pellet was resuspended in 250 ml of buffer A. The dialyzed protein was applied to a 100 ml FFQ Sepharose column equilibrated in buffer A and eluted with a 1000 ml linear gradient of 0-500 mM NaCl in buffer A; 80 fractions were collected. Peak fractions (40-49) were combined (65 mg) and dialyzed against buffer A. The dialyzed protein was applied to a 8 ml MonoQ Sepharose column equilibrated in buffer A and eluted with a 80 ml linear gradient of 0-500 mM NaCl in buffer A; 80 fractions were collected. Peak fractions of the 6 preparation were stored frozen at −80° C.
Example 21
Constitution of a Processive S. aureus DNA Polymerase III Enzyme from Three Components
The PolC (alpha-large) requires the β clamp for processivity, which in turn requires the clamp loader (τδδ′) for assembly onto DNA. The S. aureus clamp loader, τδδ′ complex, was assembled by mixing the three proteins as follows: 400 μg of r and 80 μg each of 6 and 6′ were mixed in buffer A containing no NaCl and preincubated at 15° C. for 10 min. The mixture was injected onto a 1 ml MonoQ column equilibrated in buffer A, and then eluted with a 30 ml linear gradient of 0-500 mM NaCl in buffer A; 60 fractions were collected. Fractions were analyzed in a 10% SDS-polyacrylamide gel stained with Coomassie Blue. Peak fractions (40-50) were combined and concentrated using a Centricon 30 concentrator.
The ability of the three components to work together to form the processive Pol III was tested by determining whether τδδ′ and β clamp could confer the ability of PolC to completely extend a single primer full circle around a large 7.2 kb circular M13mp18 ssDNA genome. Replication reaction contained 70 ng (25 fmol) on singly primed M13 mp18 ssDNA, 20 ng S. aureusβ, 50 ng S. aureus PolC, either 30 ng or 90 ng of S. aureus τδδ′ (when indicated), and 0.82 μg of S. pyogenes SSB in 24 μl of 20 mM Tris-HCl (pH 7.5), 4% glycerol, 0.1 mM EDTA, 5 mM DTT, 2 mM ATP, 8 mM MgCl2, 40 μg/ml BSA, and 60 mM each of dGTP and dCTP. Reactions were pre-incubated for 2 min at 37° C. to assemble protein complexes on the primer terminus. DNA synthesis was initiated upon addition of 1.5 μl dATP and 32P-TTP (specific activity 2,000-4,000 cpm/μmol) and synthesis was allowed to proceed for 1 min before being quenched with an equal volume (25 μl) of a solution of 1% SDS and 40 mM EDTA. One-half of the quenched reaction was analyzed for total DNA synthesis using DE81 paper as described, and the other half was analyzed by agarose gel phoresis. An autoradiogram of the agarose gel analysis of the replication products is depicted in FIG. 13, which shows that the presence of PolC and β, but absence of τδδ′ (lane 1) gives no full length circular duplex (RFII). However, in the presence of τδδ′ (lanes 2 and 3), full length circular duplex DNA (RFII) is produced, as expected for the action of a processive Pol III holozyme.
Example 22
General Induction/Purification Conditions for S. pyogenes 
The purification protocols for S. pyogenes proteins were performed using following standardized conditions. Cells were grown from a single colony, freshly transformed overnight. Cells were grown in 200 μg/ml Ampicillin to OD600=0.3-0.4, at which point cultures were chilled prior to addition of IPTG (to a final concentration of 0.5 mM) and were allowed to incubate for 16 hrs at 15° C. Following this, all procedures were performed at 4° C. Cell paste (1-2 g/liter of culture) was resuspended (10 ml/g cell paste) in 50 mM Tris-HCl (pH 7.5)/10% Sucrose/1 M NaCl/5 mM DTT/30 mM Spermidine/1×Heat lysis buffer (50 mM Tris-HCl (pH 7.5), 1% Sucrose, 100 mM NaCl, 2 mM EDTA). Cells were lysed by two passages through the French Press (15,000 psi) followed by centrifugation at 14,000 rpm at 4° C. Ammonium sulfate, when added to the cleared lysate, was added gradually. Precipitate was allowed to settle on ice for a minimum of 30 min prior to collection by centrifugation. Protein pellets were resuspended in buffer A (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 5 mM DTT, 10% glycerol) and dialyzed for over 3 hours in the same buffer. Column design is based on the manufacturer's suggested capacities: Fast Flow Q (FFQ) and MonoQ are 20 mg protein/ml resin, Heparin-Affigel agarose is 1.2 mg protein/ml resin. Elution was performed using 10 column volume (c.v.) gradients, and the entire gradient elution profile was collected in 80 fractions. Unless mentioned otherwise all columns were equilibrated and eluted with buffer A.
Example 23
Identification of a S. pyogenes holA gene Encoding a Functional Delta Subunit and Purification of the Delta Subunit
Alignment of E. coli delta subunit with 10 other putative holA products from unfinished genome databases of Gram negative bacteria indicates a region of conserved amino acid sequence. Amino acids Q140 to L230 of E. coli delta were used to search the B. subtilis genome database for a Gram positive delta homolog. This search revealed yqeN, a potential reading frame of unknown function, as the highest scoring sequence. Although the score was low, it was treated as a candidate for Gram positive delta. The alignment with E. coli delta is shown in FIG. 12A. A Streptococcus pyogenes genome database was searched with yqeN. Two contigs which represent N- (contig 206) and C- (contig 264) termini of S. pyogenes delta subunit were identified. The alignment of the putative S. pyogenes holA with B. subtilis yqeN is shown in FIG. 12B. The following primers were used to obtain PCR products for delta subunit:
holA Upstream (SEQ. ID No. 69)

ggagcagatt gcttttgata catatgattg gcctattc  38

holA Downstream (SEQ. ID No. 70)

ttgtctccgc atcaaactgg gatccaagag catcatacgc gtatgg  46

These primers were used to amplify the holA gene from S. pyogenes genomic DNA. The PCR product was digested with NdeI and BamHI, purified and ligated into the pET11a vector to produce pET11a.S.p. holA.

The pET11a.S.p.holA plasmid was transformed into the BL21(DE3)RccA-strain of E. coli. A single colony from an overnight transformation was used to innoculate 12 L LB broth supplemented with 200 μg/ml Ampicillin. Cells were grown at 37° C. to OD600=0.5, at which point the temperature was lowered to 15° C. and 0.5 mM IPTG was added. Induction proceeded for 16 hrs. In the morning, cells were collected by centrifugation and resuspended in 50 mM Tris-HCl (pH 7.5)/10% Sucrose/1×Heat Lysis Buffer/1 M NaCl/30 mM Spermidine/5 mM DTT. Cells were lysed by two passages through the French press (15,000 psi), followed by centrifugation at 13,000 rpm for 30 min. The supernatant was decanted and ammonium sulfate was added to a final concentration of 0.226 g/ml. The resulting pellet was collected by centrifugation and resuspended in 20 ml of buffer A. The resuspended pellet was dialyzed against buffer A containing no salt. The dialyzed protein (500 mg) was loaded onto a FFQ- Sepharose (35 ml) column and eluted with a linear gradient from 0-500 mM NaCl (10 c.v.). The peak fractions (21-45) were combined and dialyzed against buffer A (0 NaCl) for 3 hrs, then diluted to a conductivity of 50 mM NaCl and loaded (160 mg) onto a 120 ml Heparin-Affigel column. Protein was eluted with a linear gradient of 0-500 mM NaCl (10 c.v.). The fractions containing the least contaminants (39-51) were precipitated with ammonium sulfate (0.226 g), collected by centrifugation, resuspended 5 ml of buffer A, and dialyzed in buffer A containing 200 mM NaCl. The delta subunit was stored at −80° C. The final delta subunit preparation is shown in the lane marked δ of the Coomassie Blue stained SDS-polyacrylamide gel of FIG. 14. Yield=65 mg.
Example 24
Identification of S. pyogenes holB Encoding Delta Prime and Purification of the Delta Prime Subunit
A search of the S. pyogenes genome database with the predicted B. subtilis delta prime amino acid sequence revealed a DNA sequence in contig #209 (previously known as contig #210) that predicted a high scoring match for a gene encoding a delta prime protein. The following primers were used to obtain PCR products for holB:
holB Upstream (SEQ. ID. No. 71)

gcctaggata agggagggta catatggatt tagcgc  36

holB Downstream (SEQ. ID. No. 72)

cgggcaagtc ttttgacaag cttcggatcc ccataacgaa ttcc  44

The PCR product obtained from these primers was digested with NdeI and BamHI, purified and ligated into the pET11a vector to produce pET11a.S.p. holB.

The pET11a.S.p.holB plasmid was transformed into the BL21 (DE3)RecA-strain of E. coli. A single colony from an overnight transformation was used to innoculate 12L LB broth supplemented with 200 μg/ml Ampicillin. Cells were grown at 37° C. to O.D. 600=0.4, at which point the temperature was lowered to 15° C. and 0.5 mM IPTG was added. Induction proceeded for 16 hrs. In the morning, cells were collected by centrifugation and resuspended in 100 ml 50 mM Tris-HCl (pH 7.5)/10% Sucrose/1×Heat Lysis Buffer. Lysis was initiated upon addition of 0.4 mg/ml lysozyme followed by a 1 hr incubation on ice. Lysate was clarified by centrifugation at 13,000 rpm for 30 min. Ammonium sulfate was added to the supernatant to a final concentration of 0.3 g/ml. The protein pellet was resuspended in buffer A(0.1 M NaCl)+0.24 g/ml ammonium sulfate and clarified by centrifugation. The resulting protein pellet was resuspended in 20 ml of buffer A and dialyzed against buffer A. The dialyzed protein (450 mg) was loaded onto a 30 ml FFQ- Sepharose column and eluted with a linear gradient from 0-500 mM NaCl. The peak fractions were combined (fr#20-30 containing 130 mg) and dialyzed against buffer A and loaded (70 mg) onto a 50 ml Heparin-Affigel column. Protein was eluted with a linear gradient of 0-500 mM NaCl. Delta prime binds weakly to both resins and elutes in the beginning of the gradient. This delta prime subunit was stored frozen at −80° C. The final delta prime subunit preparation is shown in lane marked δ′ of the Coomassie Blue stained SDS-polyacrylamide gel of FIG. 14. Yield=40 mg.
Example 25
Identification of the S. pyogenes dnaX Gene and Purification of the Tau Subunit
A search of the S. pyogenes genome database with the putative B. subtilis tau amino acid sequence revealed a DNA sequence in contig #284 (previously known as contig #289) with a high scoring match which predicted a gene encoding for a tau subunit protein. A set of PCR primers to 5′- and 3′-termini of the putative gene sequence were designed to include restriction enzyme recognition sequences for NdeI and BamHI sites, respectively. These primers are:
dnaX Upstream (SEQ. ID. No. 73)

ggagttaaaa acatatgtat caagctcttt atc  33

dnaX Downstream (SEQ. ID. No. 74)

cgtgggtaag ggcaaaacgg atcccttatg tatttcag  38

A PCR product obtained with the above primers was digested with NdeI and BamHI, purified and ligated into pET11a vector to produce pET11a.S.p.dnaX.

The pET11a.S.p.dnaX plasmid was transformed into the BL21(DE3)RecA-strain of E. coli. A single colony from an overnight transformation was used to innoculate 24L LB broth supplemented with 200 μg/ml Ampicillin. Cells were grown at 37° C. to O.D.600=0.5, at which point the temperature was lowered to 15° C. and 0.5 mM IPTG was added. Induction proceeded for 16 hrs. In the morning, cells were collected by centrifugation and resuspended in 200 mls of 50 mM Tris-HCl (pH 7.5)/10% Sucrose/1×Heat Lysis Buffer/1 M NaCl/30 mM Spermidine/5 mM DTT/5 mM EDTA. Cells were lysed by two passages through the French press (15,000 psi), followed by centrifugation at 13,000 rpm for 30 min. The supernatant (2.4 gm) was dialyzed against buffer A containing 50 mM NaCl, loaded onto a 120 ml FFQ column (without ammonium sulfate precipitation) and eluted with a linear gradient of 100-700 mM NaCl. The peak fractions (fr#41-55) were combined, diluted with buffer A containing no salt (a dilution of 1/5) to a conductivity of 100 mM NaCl, loaded (310 mg) onto a 300 ml Heparin-Affigel column, and eluted with a linear gradient of 100-500 mM NaCl. The peak fractions (fr#21-36) were combined, dialyzed against buffer A, loaded (87 mg) onto 10 ml FFQ column, and eluted as described for the first FFQ column. The peak fractions (fr#27-41) were concentrated by centrifugation in Centriprep 30 filtration unit and frozen at −80° C. The final tau subunit preparation is shown in the lane marked T of the Coomassie Blue stained SDS-polyacrylamide gel of FIG. 14. Yield=103 mg.
Example 26
Identification of the S. pyogenes dnaN Gene and Purification of the Beta Subunit
A search of the S. pyogenes genome database with the putative B. subtilis beta subunit amino acid sequence revealed a DNA sequence (contig #266) with a high scoring match which predicted a gene encoding for a beta subunit protein. A set of PCR primers to 5′- and 3′-termini of the putative gene sequence were designed to include restriction enzyme recognition sequences for NdeI and BamHI, respectively. The primers were:
dnaN Upstream (SEQ. ID. No. 75)

ggagttcata tgattcaatt ttcaaattaa tcgc  34

dnaN Downstream (SEQ. ID. No. 76)

tatcagctcc tggatccagt accttccatt gattagcc  38

A PCR product obtained with these primers was digested with NdeI and BamHI, purified and ligated into pET16b vector to produce pET16b.S.p.dnaN.

The pET16b.S.p.dnaN plasmid was transformed into the BL21(DE3)RecA- strain of E. coli. A single colony from an overnight transformation was used to innoculate 15L LB broth supplemented with 200 μg/ml Ampicillin. Cells were grown at 37° C. to O.D.600=0.4, at which the point temperature was lowered to 15° C. and 0.5 mM IPTG was added. Induction proceeded for 16 hrs. In the morning, cells were collected by centrifugation and resuspended in 100 ml 50 mM Tris-HCl (pH 7.5)/10% Sucrose/1×Heat Lysis Buffer/1 M NaCl/5 mM DTT/30 mM Spermidine/5 mM EDTA. Cells were lysed by two passages through the French press (15,000 psi), followed by centrifugation at 13,000 rpm for 30 min. Ammonium sulfate was added to the supernatant to a final concentration of 0.3 g/ml. The resulting protein pellet was resuspended and dialyzed against buffer A containing 50 mM NaCl. The dialyzed protein (300 mg) was loaded onto a 45 ml FFQ- Sepharose column and eluted with a linear gradient from 50-500 mM NaCl. The peak fractions (16-30) were combined, dialyzed against buffer A containing 50 mM NaCl, loaded onto a 25 ml EAH-Sepharose column, and eluted with a linear gradient of 50-500 mM NaCl. The fractions containing the least contaminants were combined into two pools (pool 110-17, pool II 19-27). Each pool was further purified on a 8 ml MonoQ column (performed under conditions described for the FFQ column above). The final beta subunit preparation is shown in the lane marked β of the Coomassie Blue stained SDS-polyacrylamide gel of FIG. 14. Yield=48 mg.
Example 27
Identification of the S. pyogenes polC Gene and Purification of the Alpha-Large Polymerase Subunit
A search of the B. subtilis genome database with the E. coli alpha subunit amino acid sequence revealed two DNA sequences with a high scoring match which predicted two genes encoding alpha-like polymerase subunits. The DNA sequence with the second highest scoring match which encoded the largest of the two polymerase subunits also appeared to encode for the epsilon exonuclease domain at the N-terminus of the putative alpha subunit. A search of the B. subtilis genome database with S. pyogenes DNA sequence confirmed this nucleotide sequence to encode the Gram positive homolog of the E. coli replicative polymerase subunit (alpha). This Gram negative alpha-like subunit lacked homology to epsilon. The gene encoding the large alpha polypeptide sequence (alpha-large) will be referred to as the product of the polC gene and the gene encoding the smaller Gram-negative alpha-like polymerase (alpha-small) will be referred to as the product of the polE or dnaE gene (see Example 28).
The alpha-large polymerase polypeptide is a product of two overlapping contigs; contig #197 (renamed #193) encodes the N-terminal 630 amino acids, and contig #278 (renamed #273) encodes the C-terminal 1392 amino acids. The putative Open Reading Frame generates a 1464 amino acid polypeptide (SEQ. ID. No. 18). Since the polC nucleotide sequence contained several NdeI sites, a primer was designed to mutate two restriction endonuclease sites in the pET11a nucleotide sequence upstream of the N-terminus of the gene; an XbaI restriction site was mutated to an NheI restriction site and an NdeI restriction site at the starting ATG was removed. A 74mer primer which spans from mutated XbaI site upstream of T7 promoter includes NheI site, rbs site (ribosome binding site), mutated NdeI site and first 10 amino acid codons of polC gene sequence. The following primers were used in a PCR reaction to amplify polC gene from S. pyogenes genomic DNA:
polC Upstream (SEQ. ID. No. 77)

ggataacaat tccccgctag caataatttt gtttaacttt aagaaggaga tatacccatg  60

tcagatttat tcgc  74

polC Downstream (SEQ. ID. No. 78)

cggtgtctct atctaaatga ctcatttggg atcctcgctt tatacggtat gtcacag  57

Elongase (BRL) produced the best amplification results. PCR reaction conditions were: 5 pg genomic DNA, 20 ng of each primer, 1 ml Elongase, 60 μM each dNTP, in 100 ml Elongase reaction buffer for 1 min at 94° C., 1 min at 55° C., and 6 min at 60° C. repeated for 40 cycles. The resulting 4000 bp PCR fragment was digested with NheI and BamHI, purified and ligated into the pET11a vector (digested with XbaI and BamHI) to produce pET11a.S.p.polC.

The pET11a.S.p.polC plasmid was transformed into the BL21(DE3)RecA-strain of E. coli. A single colony from an overnight transformation was used to innoculate 24L LB broth supplemented with 200 μg/ml Ampicillin. Cells were grown at 37° C. to OD600=0.4 at which point temperature was lowered to 15° C. and 0.5 mM IPTG was added. Induction proceeded for 16 hrs. In the morning, cells (12g) were collected by centrifugation and resuspended in 100 ml 50 mM Tris-HCl (pH 7.5)/10% Sucrose/1×Heat Lysis Buffer/1 M NaCl/5 mM DTT/30 mM Spermidine/5 mM EDTA. Cells were lysed by two passages through the French press (15,000 psi), followed by centrifugation at 13,000 rpm for 30 min. Ammonium sulfate was added to the supernatant to a final concentration of 0.226 g/ml. The precipitate was collected by centrifugation. The protein pellet (220 mg resuspended in buffer A) was dialyzed against buffer A containing 150 mM NaCl, loaded onto an 8 ml FFQ column equilibrated with buffer A containing 150 mM NaCl, and eluted with a linear gradient of buffer A containing 150-600 mM NaCl. The fractions containing the least contaminants (fr#42-64) were combined and precipitated with ammonium sulfate (0.226 g/ml). The precipitate was collected by centrifugation and resuspended in buffer A (10 mg/ml in 5 ml). A fraction (1 ml=10 mgs) of the concentrated protein was dialyzed, loaded onto 10 ml ssDNA-agarose column, and eluted with a linear gradient of 50-500 mM NaCl. The peak fractions (fr#30-50) were combined and concentrated with ammonium sulfate (as above). The final alpha-large subunit preparation is shown in lane marked αL of the Coomassie Blue stained SDS-polyacrylamide gel of FIG. 14. Yield=4 mgs.
Example 28
Identification of the S. pyogenes dnaE Gene and Purification of the Alpha-Small Polymerase
A search of the B. subtilis genome database using the E. coli alpha subunit amino acid sequence revealed two DNA sequences with a high scoring match which predicted two genes encoding for alpha-like polymerase subunits. The DNA sequence with the highest scoring match encodes a smaller alpha polymerase which does not contain an exonuclease domain. The putative short alpha DNA sequence is a product of the open reading frame in contig #253 of the S. pyogenes genome database. A set of PCR primers to 5′- and 3′-termini of the putative gene sequence were designed to include restriction enzyme recognition sequences for NdeI and BamHI, respectively. The primers were:
α-Short Upstream (SEQ. ID. No. 79)

gggaacaaga taaccaagga ggaacccatg gttgctcaac ttg  43

α-Short Downstream (SEQ. ID. No. 80)

cgaatagcag cgttcatacc aggatcctcg ccgccactgg  40

A PCR product obtained with these primers was digested with NdeI and BamHI, purified and ligated into pET11a vector to produce pET11a.S.p.dnaE.

The pET 11a.S.p.dnaE plasmid was transformed into the BL21 (DE3)RecA- strain of E. coli. A single colony from an overnight transformation was used to innoculate 12L LB broth supplimented with 200 μg/ml Ampicillin. Cells were grown at 37° C. to OD600=0.4, at which point temperature was lowered to 15° C. and 0.5 mM IPTG was added. Induction proceeded for 16 hrs. In the morning, cells were collected by centrifugation and resuspended in 100 mls 50 mM Tris-HCl (pH 7.5)/10% Sucrose/1×Heat Lysis Buffer/5 mM DTT/30 mM Spermidine/1 M NaCl/5 mM EDTA. Cells were lysed by two passages through the French press (15,000 psi), followed by centrifugation at 13,000 rpm for 30 min. Ammonium sulfate was added to the supernatant to a final concentration of 0.226 g/ml. The precipitate was collected by centrifugation. The protein pellet (resuspended in buffer A) was then dialyzed against buffer A. The dialyzed protein (600 mg) was loaded onto a 30 ml FFQ and eluted with a linear gradient of buffer A containing 50-500 mM NaCl. The peak fractions (200 mg in fr#70-79) were dialyzed and loaded onto a 100 ml Heparin-Affigel column. The fractions containing the least contaminants (100 mg from fr#18-30) were pooled and dialyzed against buffer A containing 300 mM NaCl. The dialysate (50 mg) was loaded onto a 50 ml ssDNA-agarose column and eluted with a linear gradient of 300 mM-1 M NaCl. The final alpha-small subunit preparation is shown in lane marked as of the Coomassie Blue stained SDS-polyacrylamide gel of FIG. 14. Yield=25 mg.
Example 29
Identification of the S. pyogenes ssb Gene and Purification of the Single Strand DNA-Binding Protein
Search of the S. pyogenes genome using the B. subtilis SSB amino acid sequence identified a polypeptide in contig #230(212) as having highest homology to single strand binding protein of several Gram negative bacteria. This contig lacked the first 26 amino acids at the N-terminus. Circular PCR was employed to identify the DNA encoding the N-terminus of the putative SSB protein. S. pyogenes genomic DNA was digested overnight with ApoI (5 μg chromosomal DNA in a 501l reaction). The DNA was extracted with phenol and precipitated with ethanol. The ApoI digested chromosomal DNA was self-ligated to generate circular template for future use in the circular PCR. A circular PCR was performed with primers designed to anneal back-to-back to amplify circularized ApoI reaction fragments. The primers were:
ssb.circ Upstream (SEQ. ID. No. 81)

accattttgg cttttaaagg tacggttaac agcaagtgtg aaggtagcc  49

ssb.circ Downstream (SEQ. ID. No. 82)

gaacgcgagg cagatttcat taactgtgtg atctggcg  38

The PCR reaction conditions were as follows: 100 ng circularized S. pyogenes genomic DNA, 20 ng each primer, 1 ml Elongase, 60 μM each dNTP, 100 1 Elongase reaction buffer. Amplification was performed for 40 cycles as follows: denature, 1 min at 94° C.; anneal, 1 min at 55° C.; and extend, 5 min at 68° C. PCR products were cloned into the Topo TA vector following instructions of the manufacturer (Promega). Several positive clones were sequenced to obtain N-terminal nucleotide sequence. This information lead to design of the following primers with which the use of a standard PCR reaction generated whole ssb gene products. The primers were:

ssb Upstream (SEQ. ID. No. 83)

tttaaaagag ggtagcatat gattaataat gtagtactag ttggtcgc  48

ssb Downstream (SEQ. ID. No. 84)

tttaaattta aacctaggtt caatccattc tgactagaat ggaagatcgt c  51

The resulting PCR product was digested with NdeI and BamHI, purified and ligated into pET11a vector to produce pET11a.S.p. ssb.

The pET11a.S.p.ssb plasmid was transformed into the BL21 (DE3)RecA- strain of E. coli. A single colony from an overnight transformation was used to innoculate 12L LB broth supplemented with 200 μg/ml Ampicillin. Cells were grown at 37° C. to OD600=0.5, at which point 0.5 mM IPTG was added. At the end of the 3 hr induction, cells were collected by centrifugation and resuspended in 100 ml of 50 mM Tris-HCl (pH 7.5)/10% Sucrose/1×Heat Lysis Buffer/5 mM DTT/5 mM EDTA. The cell lysis was initiated upon addition of 0.4 mg/ml lysozyme followed by a 1 hr incubation on ice. The lysate was clarified by centrifugation at 13,000 rpm for 30 min. The SSB protein was significantly purified by sequential fractionation with ammonium sulfate in the following manner. Solid ammonium sulfate was added to the clarified lysate to a final concentration of 0.24 g/ml and the precipitated protein was collected by centrifugation at 13,000 rpm for 30 min. The resulting pellet was homogenized in buffer A(0.1 M NaCl)+0.24 g/ml ammonium sulfate and the precipitate was collected by centrifugation. This procedure was repeated with buffer A(0.1 M NaCl)+0.2 g/ml ammonium sulfate, buffer A(0.1 M NaCl+0.15 g/ml ammonium sulfate, and buffer A(0.1 M NaCl)+0.13 g/ml ammonium sulfate. The final pellet was resuspended in buffer A +0.15 M NaCl and dialyzed against the same buffer. The resulting pellet was resuspended in buffer A and dialyzed against buffer A containing 500 mM NaCl. The dialysate (300 mg) was diluted to 0.15 M NaCl before it was loaded onto a 20 ml MonoQ column and eluted with a linear gradient of 0.15 M-0.5 M NaCl in buffer A. The SSB protein elutes in the very beginning of the gradient. The peak fractions were combined (150 mg in fractions 16-30), diluted to 0.05 M NaCl, loaded onto a 10 ml ssDNA-agarose column, and eluted with 0.5 M NaCl. The peak fractions (32-62) were combined and frozen. The SSB was further purified over a MonoQ column to remove contaminating polymerase activity. The final single strand DNA binding protein preparation is shown in lane marked ssb of the Coomassie Blue stained SDS-polyacrylamide gel of FIG. 14. Yield=120 mg.
Example 30
First Demonstration that S. pyogene holA Encodes a Delta Subunit Involved In Replication: Assembly of τδδ′ Complex
Gel filtration is a standard analytical technique to demonstrate direct protein-protein interaction. Purified τ, δ, δ′ proteins were used to examine whether they form a protein complex assembly. Gel filtration of t mixed with either δ, δ′, or both δ and δ′ was performed using an HR 10/30 Superose 6 column equilibrated with buffer A containing 100 mM NaCl. Either δ (200 μg), δ′ (200 μg), or a mixture of δ and δ′ (200 μg each) was incubated for 30 min at 15° C. in 100 μl of buffer A containing 100 mM NaCl, and the entire mixture was injected onto the column. The mixture was resolved on the column by collection of 170 μl fractions after the initial void (6.6 μl) volume was collected. Fractions were analyzed by 10% SDS-polyacrylamide gels (301/lane) stained with Coomassie Blue.
The results, in FIG. 15, demonstrate that under these conditions the τ protein exhibits no (weak) interaction with the delta (FIG. 15B) and the delta prime subunits (FIG. 15C) individually, and yet assembles readily into a complex when all the subunits are mixed in the reaction (FIG. 15A). The τ protein was mixed with a 2-fold molar excess of each δ and δ′, then gel filtered. A complex of τδδ′ was formed as demonstrated by coellution of δ and δ′ with τ (fr#22-30) whereas excess δδ′ complex elutes in later fractions (fr#38-46). To determine whether individual δ or δ′ subunits interact with t, the τ subunit was mixed with either δ or δ′ and then gel filtered. The results demonstrate that a gel filterable complex does not form when τ is mixed with δ (FIG. 15B) or δ′ (FIG. 15C) subunits individually, as indicated by the absence of these subunits in the T containing fractions (fr#20-26). Therefore, it appears that the presence of both δ and δ′ subunits is essential for the formation of the τδδ′ complex.
Example 31
Second Demonstration that S. pyogenes holA Encodes Delta: Functional Assembly of δ on DNA
Gel filtration was used to demonstrate that the τ, δ, δ′ proteins form a functional clamp loading complex which is able to load the β clamp onto a circular DNA molecule The reaction contained 0.5 μmol of gp2 nicked pBluescript plasmid (a circular double strand plasmid with a single nick produced by M13 gp2 protein), 1 pmol [32P]β, 0.5 pmol τδδ′ complex, 0.25 pmol of either δ, δ′, τ were used in individual experiments when a subassembly of the complex was tested (τδ, τδ′, δδ′) in 75 μl buffer B (20 mM Tris-HCl (pH 7.5), 20% glycerol, 0.1 mM EDTA, 5 mM DTT, 2 mM ATP, 8 mM MgCl2). β was incubated with nicked DNA for 10 min at 37° C. either alone, or in combination with various assemblies of the τ complex. All gel filtration experiments were performed at 4° C. The reaction mixtures were applied to a 5 ml column of Bio-Gel 15 M (Bio-Rad) equilibrated in buffer B containing 100 mM NaCl. Fractions of 170 μl were collected and quantitated in the Scintillation counter.
The results, in FIG. 16, demonstrate that the assembly of the ring onto a circular DNA molecule requires the presence of τ, δ, and δ′ proteins (FIG. 16A). In absence of any one of the subunits, loading onto DNA does not occur (FIGS. 16B-E). The clamp loader complex (τδδ′) can be supplied as a mixture of τ, δ, δ′ subunits or as an assembled complex (purified from unassembled subunits by gel filtration, or by ion exchange chromatography on MonoQ). Proteins bound to the large DNA molecule elute in the early fractions (void fr#10-17) and resolve from free proteins that elute in later fractions (fr#18-35).
Example 32
The τ Subunit Product of the dnaX Gene Binds α-Large
The interaction of S. pyogenes α and τ proteins was examined by analyzing a mixture of the proteins by gel filtration. Gel filtration of τ, α-large or a mixture of α-large and τ was performed using an HR 10/30 Superose 6 column equilibrated with buffer A containing 100 mM NaCl. Either α-large (400 μg) (200 μM) or a mixture of α-large and t was incubated for 30 min at 15° C. in 100 μl of buffer A containing 100 mM NaCl, and the entire mixture was injected onto the column. The mixture was resolved on the column by collection of 170 μl fractions after the initial void (6.6 ml) volume was collected. Fractions were analyzed by 10% SDS-polyacrylamide gels (30 μl/lane) stained with Coomassie Blue.
The results show a complex of αLτ was formed as demonstrated by coellution of α-large and T (fr#30-38) proteins (FIG. 17A) compared to the elution profile of individual proteins (FIGS. 17B-C). Also, the migration of the τ in the αLτ complex changes significantly to a larger complex (4 fractions, from fr#37 to fr#33).
Example 33
Formation of αLτδδ′ Complex
To determine whether a αLτδδ′ complex could form, the following components were mixed: α-large (400 μg, 2.5 nmol), τ (200 μg, 1.3 nmol), δ (200 μg, 4.8 nmol), δ′ (200 μg, 5.75 μmol) in a final volume of 150 μl. The mixture was diluted to 300 ml with buffer A to lower conductivity of the sample to that equivalent of 100 mM NaCl and incubated for 30 min at 15° C. The mixture was injected onto a Superose 6 column (equilibrated with buffer A containing 100 mM NaCl) and fractions (170 μl) were collected after an initial 6.6 ml of void volume was collected. Fractions were analyzed by 10% SDS-polyacrylamide gels (30 μl/lane) stained with Coomassie Blue.
A gel filterable complex (FIG. 18) of αLτδδ′ was formed as demonstrated by coellution of τ, δ and δ′ with α-large (fr#14-26), whereas excess δδ′ complex elutes in later fractions (fr#30-38). The migration of the τδδ′ protein complex in the αLτδδ′ complex does not change significantly. The complex might dissociate under the nonequilibrium conditions of gel filtration due to low concentration of proteins, salt concentration and speed of resolution.
Next, ion exchange chromatography was used to analyze the protein mixture to prepare the reconstituted αLτδδ′ complex of S. pyogenes. The αLτδδ′ complex was reconstituted upon mixing α-large (10 mg, 62 nmol), T (6 mg, 72 nmol), δ (3.3 mg, 80 nmol), δ′ (1.6 mg, 90 nmol). The α, τ, δ, δ′ protein mixture was dialyzed for 2 hrs against buffer A containing 50 mM NaCl. The entire mixture was loaded onto a 1 ml MonoQ column equilibrated in buffer A containing 50 mM NaCl. Proteins were eluted with a 20 column volume linear gradient of 50-500 mM NaCl in buffer A and 0.25 ml fractions were collected. Fractions were analyzed by 10% SDS-polyacrylamide gels (20 μl/lane) stained with Coomassie Blue.
Generally, the reconstitution of the αLτδδ′ complex on a MonoQ column results in a tight salt resistant complex which elutes at 500 mM NaCl. The high concentration of the proteins in the eluted fractions contributes to stability of the complex.
Example 34
The S. pyogenes Three Component Pol III-L Polymerase is Rapid and Processive In DNA Synthesis
It was previously demonstrated (i.e., in Examples 29 and 30) that the putative delta subunit plays an integral part in the assembly of the τδδ′ complex (FIG. 15) and that this complex is sufficient to assemble β clamps onto circular primed DNA (FIG. 16). It was also shown that the strong interaction between the α-large and τsubunits (FIG. 17) results in an isolatable αLτδδ′ complex (FIG. 18), similar to that of the E. coli DNA polymerase III*.
The MonoQ fractions containing αLτδδ′ complex were then used to assemble β onto primed DNA and determine whether this now resulted in rapid and processive DNA synthesis. Replication reactions contained 70 ng of singly primed M13mp18 ssDNA and 0.82 μg of S. pyogenes SSB in 25 μl buffer C (20 mM Tris-HCl (pH 7.5), 4% glycerol, 0.1 mM EDTA, 5 mM DTT, 2 mM ATP, 8 mM MgCl2) with 60 μM each of dGTP, dCTP, and dATP, 30 μM cold TTP and 20 μM [α-32P] TTP (specific activity of 2,000-4,000 cpm/pmol). The complex is assembled onto DNA in the following manner: 40 ng (3:1) or 140 ng (10:1) of the αLτδδ′ complex and 60 ng of β protein were preincubated for 2 min at 30° C. in presence of SSB coated primed M13 DNA and two nucleotides (dCTP and dGTP). Reactions were initiated by addition of the two remaining nucleotides dATP and TTP and quenched with an equal volume of 1% SDS/40 mM EDTA. Each time point is a separate reaction.
A time course of replication on singly primed circular M13mp18 ssDNA is shown in FIG. 19. The agarose gel analysis shows conversion of the oligonucleotide primed single stranded DNA to the slower migrating replicative form II. The fact that the speed of synthesis is independent of the concentration of polymerase in the reaction indicates that the αLτδδ′ complex synthesizes DNA in a rapid and a highly processive manner. The S. pyogenes αLτδδ′ complex in presence of the β clamp, completely replicates (is able to complete replication of) 7250 nt of M13 mp18 ssDNA in 8-9 sec.
Example 35
The S. pyogenes DnaE (α-small) Forms a Three-Component Polymerase with τδδ′ and β
The S. pyogenes DnaE (α-small) polymerase is more homologous to E. coli a than S. pyogenes PolC. Thus, it seems reasonable to expect that the DnaE polymerase may also function with the β clamp (FIGS. 21A-B). To test DnaE for function with τδδ′ and β, replication reactions contained 70 ng (25 fmol) of 30-mer singly primed M13 mp18 ssDNA, 0.82 μg of S. pyogenes SSB, and 3.3 ng-300 ng of DnaE (25 fmol −2.3 pmol) in 23.5 μl of 20 mM Tris-HCl (pH 7.5), 4% glycerol, 0.1 mM EDTA, 5 mM dithiothreitol (DTT), 40 μg/ml BSA, 2 mM ATP, 8 mM MgCl2, and 60 μM each of dGTP and dCTP. When present, reactions included 43.3 ng of β and 10 ng of τδδ′. Reactions were preincubated for 3 min at 37° C., and then NaCl was added to 40 mM followed by another 2 min at 37° C. DNA synthesis was initiated upon addition of 1.5 μl of 1.5 mM dATP, 0.5 mM [α32P]-dTTP (specific activity 2,000-4,000 cpm/pmol). Aliquots of 25 μl were removed at the indicated times and quenched with an equal volume (25 μl) of 1% SDS, 40 mM EDTA. One-half of the quenched reaction was analyzed for total deoxynucleotide incorporation using DE81 filter paper and the other half was analyzed on a 0.8% neutral agarose gel. The effect of TMAU was also examined, in which 100 μM TMAU in DMSO (2% DMSO final concentration) was present. In this case, replication was allowed to proceed for 1 min before being quenched with 25 μM of 1% SDS, 40 mM EDTA.
At a saturating concentration of DnaE polymerase, the time course of primer extension shows that it completes an M13mp18 primed ssDNA template within 2 minutes for a speed of at least 60 nucleotides/s (FIG. 21C). This rate of synthesis holds true for the highest amount of DnaE in the rightmost panel of the figure. As the DnaE concentration is decreased, a longer time is required to complete the circular template, indicating that the DnaE polymerase is not processive over the entire length of the M13mp18 template. If the DnaE polymerase were fully processive during synthesis of the 7.2 kb ssDNA circle, the product profile over time would be qualitatively similar at all concentrations of enzyme, but the overall intensity of the profile would be diminished. This particular experiment was performed in the absence of β, but presence of τδδ′. When repeated in the presence of β but without τδδ′, and in the absence of both β and τδδ′, results similar to those shown in FIG. 21C were observed.
In the presence of β and τδδ′, DnaE polymerase is stimulated in synthesis at low concentration, indicating that β increases the processivity and/or speed of DnaE (FIGS. 21C-D). At higher concentrations of DnaE, the presence of P/τδδ′ has no effect on the rate of synthesis, and thus β does not increase the intrinsic speed of the enzyme (i.e., panels 3 and 4 of FIG. 21D). Hence, the effect of the β clamp on DnaE is primarily due to an increase in processivity. The profile of product length over time remains essentially unchanged at the different DnaE concentrations, and therefore the processivity of DnaE, with β is at least equal to the 7.2 kb length of the M13mp18 substrate.
The DnaE sequence does not show homology to an exonuclease, implying that it may have no associated nuclease activity. The DnaE preparation was examined for the presence of a 3′-5′ exonuclease (FIG. 21E). The DnaE and PolC polymerases were each incubated with a 5′ 32P-labeled oligonucleotide, followed by analysis in a sequencing gel. The result showed no degradation of the oligonucleotide by DnaE. PolC is a known 3′-5′ exonuclease and it digests the end-labeled oligonucleotide as expected.
Gram positive PolC is known to be inhibited by the antibiotic hydroxyphenylaza-uracil (“HPUra”) and its derivatives. In FIG. 21F, the PolC·τδδ′, β and DnaE were tested for inhibition of synthesis on SSB coated primed M13mp18 ssDNA by an HPUra derivative, trimethylanilino-uracil (“TMAU”). The PolC·τδδ′ β enzyme was prevented from forming the RFII product by TMAU. In contrast, the DnaE polymerase was not affected by TMAU in the presence of τδδ′/β (nor in the absence of τδδ′/β, not shown).
Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

## Claims

1. An isolated DNA molecule that encodes the amino acid sequence comprising SEQ ID NO:28.

2. The isolated DNA molecule according to claim 1, wherein the DNA molecule comprises the nucleotide sequence of SEQ ID NO:27.

3. An expression system comprising an expression vector into which is inserted a heterologous DNA molecule according to claim 1.

4. The expression system according to claim 3, wherein the heterologous DNA molecule is in sense orientation and correct reading frame relative to a promoter.

5. A host cell comprising a heterologous DNA molecule according to claim 1.