# Patent

## Title
Recombinant type II restriction endonuclease, NmeAIII, and a process for producing the same

## Abstract

A protein is described that has an amino acid sequence characterized by at least 90% sequence identity with SEQ ID NO: 24, the protein being capable of recognizing a sequence consisting of 5′-GCCGAG-3′ within the double-stranded DNA and cleaving the substrate predominantly at 21/19 nucleotides from the recognition site. A method is also described that utilizes the protein for creating a DNA tag for use as a unique identifier for paired end sequencing of DNA or serial analysis of gene expression.

## Background
CROSS REFERENCE
This application is a §371 application of international application number PCT/US2007/088522 filed on 21 Dec. 2007, which claims priority from U.S. provisional application No. 60/877,265 filed on 27 Dec. 2006, herein incorporated by reference.


BACKGROUND
Restriction endonucleases are enzymes that occur naturally in certain unicellular microbes—mainly bacteria and archaea—and that function to protect these organisms from infections by viruses and other parasitic DNA elements. Restriction endonucleases bind to specific sequences of nucleotides (‘recognition sequence’) in double-stranded DNA molecules (dsDNA) and cleave the DNA, usually within or close to the sequence, disrupting the DNA and triggering its destruction. Restriction endonucleases commonly occur with one or more companion enzymes termed modification methyltransferases. Methyltransferases bind to the same sequences in dsDNA as the restriction endonucleases they accompany, but instead of cleaving the DNA, they alter it by the addition of a methyl group to one of the bases within the sequence. This methylation (‘modification’) prevents the restriction endonuclease from binding to that site thereafter, rendering the site resistant to cleavage. Methyltransferases function as cellular antidotes to the restriction endonucleases they accompany, protecting the cell's own DNA from destruction by its restriction endonucleases. Together, a restriction endonuclease and its companion modification methyltransferase(s) form a restriction-modification (R-M) system, an enzymatic partnership that accomplishes for microbes what the immune system accomplishes, in some respects, for multicellular organisms.
A large and varied class of restriction endonucleases has been classified as ‘Type II’ restriction endonucleases. These enzymes cleave DNA at defined positions, and in purified form can be used to cut DNA molecules into precise fragments for gene cloning and analysis. The biochemical precision of Type II restriction endonucleases far exceeds anything achievable by chemical methods, making these enzymes the reagents sine qua non of molecular biology laboratories. In this capacity, as molecular tools for gene dissection, Type II restriction endonucleases have had a profound impact on the life sciences in the past 35 years, transforming the academic and commercial arenas, alike. Their utility has spurred a continuous search for new restriction endonucleases, and a large number have been found (Roberts and Macelis, Nucl. Acids Res. 29:268-269 (2001)). (REBASE®, http://rebase.neb.com/rebase). Today, Type II endonucleases, recognizing more than 250 unique DNA recognition sequences, are known among the several thousand enzymes that have been characterized. Concomittantly, the production and purification of these enzymes has been improved by the cloning and over-expression of the genes that encode them in non-natural production strain host cells such as E. coli. 
Since the various restriction enzymes appear to perform similar biological roles, in much the same ways, it might be thought that they would resemble one another closely in amino acid sequence and behavior. Experience shows this not to be true, however. Surprisingly, far from resembling one another, most Type II restriction enzymes appear unique, resembling neither other restriction enzymes nor any other known kind of protein. Type II restriction endonucleases seem to have arisen independently of one another for the most part during evolution, and to have done so hundreds of times, so that today's enzymes represent a heterogeneous collection rather than a discrete family. Some restriction endonucleases act as homodimers, some as monomers, and others as heterodimers. Some bind symmetric sequences, others asymmetric sequences; some bind continuous sequences, others discontinuous sequences; some bind unique sequences, others multiple sequences. Some are accompanied by a single methyltransferase, others by two, and yet others by none at all. When two methyltransferases are present, sometimes they are separate proteins, and at other times they are fused. The orders and orientations of restriction and modification genes vary, with all possible organizations occurring. Several kinds of methyltransferases exist, some methylating adenines (m6A-MTases), others methylating cytosines at the N-4 position (m4C-MTases), or at the 5 position (m5C-MTases). Usually there is no way of predicting, a priori, which modifications will block a particular restriction endonuclease, which kind(s) of methyltransferases(s) will accompany that restriction endonuclease in any specific instance, nor what their gene orders or orientations will be.
A hallmark of restriction endonucleases is their great variability in amino acid sequence and catalytic behavior; each occurs in unique enzymatic association, adapted to unique microbial circumstances; and each presents the experimenter with a unique challenge. Sometimes a restriction endonuclease can be cloned and over-expressed in a straightforward manner but more often than not it cannot, and what works well for one enzyme can work not at all for the next. Success with one is no guarantee of success with another.
SUMMARY
In an embodiment of the invention, a protein is provided that has an amino acid sequence characterized by at least 50% sequence identity, more particularly at least 80% sequence identity, more particularly at least 90% sequence identity, with SEQ ID NO:24, the protein having restriction endonuclease activity. The protein is further characterized as being capable of recognizing a sequence consisting of 5′-GCCGAG-3′ within the DNA and cleaving the double-stranded DNA substrate predominantly at 19 nucleotides on one strand and 21 nucleotides on the complementary strand from the recognition site (21/19). In an additional embodiment, a DNA fragment encoding the protein is provided and also a vector that includes the DNA fragment, and a host cell containing the vector for expressing the protein.
In an embodiment of the invention, a method is provided that includes the steps of: creating a DNA tag by cleaving a target DNA with the protein described above; for use in sequencing applications such as for a unique identifier for paired end sequencing of DNA or for serial analysis of gene expression.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows a region of Neisseria meningitidis DNA sequence (SEQ ID NO:1) which contains a stop codon at the 13th amino acid position of the sequence fragment shown.
FIG. 1B shows a pAII17 plasmid containing the N. meningitidis 1,733,738 to 1,736,684 DNA fragment in which the stop codon is replaced with tryptophan by changing “tag” nucleotides to “tgg”. The primers (SEQ ID NOS:3 and 4) used to amplify the pAII17 plasmid have a phosphate (P) at the 5′ end. The product of amplification is a linear molecule with phosphate groups at the 5′ ends of each strand so that ligation can occur to generate a DNA encoding an NmeAIII carrying a point mutation. This was achieved using a Phusion site directed mutagenesis kit (New England Biolabs, Inc. (NEB), Ipswich, Mass.).
FIG. 2 shows NmeAIII endonuclease activity of a protein encoded by a DNA sequence corresponding to the modified extended NmeAIII 1791 reading frame, in which the stop codon at position 32 is replaced by a tryptophan.

    Lanes 1 and 16 contain size standards: lambda-HindIII and PhiX174-HaeIII.
    Lanes 2 to 7 contain serial dilution of NmeAIII crude extract with lambda DNA, from 8 μl per 50 μl reaction (lane 2) to 0.25 μl crude extract per 50 μl reaction (lane 7).
    Lanes 8 and 9: size standards—lambda BstEII and pBR322-MspI.


FIG. 3A shows the mapping of NmeAIII recognition sites.

    Lanes 2-5 contain pBR322 DNA cleaved by NmeAIII and each of the following restriction enzymes: ClaI (lane 2), NruI (lane 3), NdeI (lane 4) and PstI (lane 5).
    Lane 8-11: PhiX174 cleaved by NmeAIII and each of the following restriction enzymes: PstI (lane 8), SspI (lane 9), NciI (lane 10) and StuI (lane 11).
    Lanes 1 and 12: lambda-HindIII, PhiX-HaeIII size standards.
    Lanes 6 and 7: lambda BstEII, pBR322-MspI size standards.


FIG. 3B shows the mapping of NmeAIII recognition sites.

    Lanes 2-4 and 6-8 contain pBC4 DNA cleaved by NmeAIII and each of the following restriction enzymes: NdeI (lane 2), AvrII (lane 3), PmeI (lane 4), AscI (lane 6), SpeI (lane 7) and EcoRV (lane 8).
    Lanes 1 and 9 contain lambda-HindIII and PhiX-HaeIII DNA size standards.
    Lane 5 contains lambda-BstEII and pBR322-MspI DNA size standards.


FIG. 4A shows how the location of the NmeAIII cleavage site is determined on the top 5′-GCCGAG-3′ strand.
DNA (pBC4) having an NmeAIII recognition site at position 3741 and a convenient primer (3475-3497) (SEQ ID NO:5) was cut with NmeAIII, yielding ends indicated by the arrows (1). The bottom strand (SEQ ID NO:6) served as the template for dideoxy DNA sequencing extension (2). The primer was annealed and extended through the NmeAIII site. When the reaction reached the end of the molecule opposite the NmeAIII cleavage site, the Taq polymerase added an extra A base (3) (SEQ ID NO:7). SEQ ID NO:8 includes in parentheses an additional sequence that is generally cleaved and therefore not reflected in the extended primer sequence. A sequencing profile of NmeAIII was generated for NmeAIII cleaved pBC4 DNA (ABI377 Sequencer) showing the cleavage site (4) (SEQ ID NO:9). The height of the peaks denotes the frequency of occurrence of a base in the fragment population. At position “N” the height of the peaks is dramatically reduced because of the enzyme cleavage site. Low amounts of single bases can be detected past the cleavage site because the enzyme cleavage is not 100% effective.
FIG. 4B shows how the NmeAIII cleavage site was determined on the bottom 5′-CTCGGC-3′ strand.
A DNA (PhiX174) having an NmeAIII site at 1022 and a convenient primer (713-734) (SEQ ID NO:10) was cut with NmeAIII, yielding ends indicated by the arrows (1). The bottom strand (SEQ ID NO:11) serves as the template for dideoxy DNA sequencing extension (2). The primer was annealed and extended through to the NmeAIII cleavage site. When the reaction reaches the end of the molecule the Taq polymerase added an extra A base (3) (SEQ ID NOS:12 and 13). Sequencing Profile of NmeAIII cut PhiX174 DNA (ABI377 Sequencer, Applied Biosystems, Inc., Foster City, Calif.) is provided in (4) (SEQ ID NO:14) as described above.
FIGS. 4C-1 and 4C-2 show a comparison of NmeAIII and MmeI cleavage positions for a DNA substrate having the identical sequence of nucleotides in the DNA sequence after the recognition site up to the putative cut site. The results are consistent with FIGS. 4A and 4B. This shows that the difference in cleavage specificity and cleavage distance from the recognition site is independent of the character of the intervening sequence.
An NmeAIII and an MmeI recognition site was cloned into the polylinker of pUC19 DNA adjacent to the same intervening sequence between the position of cutting and recognition. The intervening sequence chosen is the sequence flanking the NmeAIII site at 3426 in PhiX174. The DNA was then cleaved with NmeAIII or MmeI (position of cleavage indicated by the arrows) in (1) (SEQ ID NO:15) and (2)(SEQ ID NO:16).
The primer (NEB1224) was annealed and extended through the NmeAIII site or MmeI site. When the reaction reached the end of the molecule (SEQ ID NOS:17-20), the Taq polymerase added an extra A base. An extra A base was added at the two positions for MmeI, indicating variability in the cleavage position (3) and (4). Sequencing profiles for NmeAIII and MmeI (SEQ ID NOS:21 and 22) were generated showing the cleavage sites for these enzymes.
FIG. 5 shows a restriction map for the pAII17-NmeAIII plasmid.
FIGS. 6-1 to 6-2 shows a DNA fragment encoding NmeAIII (2814 bp) (SEQ ID NO:23).
FIG. 7 shows an NmeAIII amino acid sequence (937 amino acids) (SEQ ID NO:24).
FIGS. 8-1 to 8-6 show an NmeAIII-pAII17 construct (SEQ ID NO:25).



DETAILED DESCRIPTION OF THE EMBODIMENTS
Present embodiments of the invention relate to a DNA fragment that codes for a polypeptide possessing site-specific restriction endonuclease and methyltransferase activities. An amino acid sequence of interest from Neisseria meningitidis Z2491 was identified because it has significant similarity to the amino acid sequence of MmeI (see for example, U.S. Pat. No. 7,115,407). Genomic DNA was obtained from the N. meningitidis strain and the DNA fragment for the putative gene was amplified by PCR and cloned into an expression vector, pRRS, and introduced into E. coli. The transformed cells were grown, induced and assayed for restriction endonuclease activity. However, the hypothetical enzyme did not appear to have any detectable endonuclease activity.
It was decided to try to convert the hypothetical enzyme into an active restriction endonuclease.
Further examination of the protein sequence revealed that the NMA1791 hypothetical protein sequence was shorter than the MmeI protein sequence but nevertheless shared significant similarity in the BLAST search starting from near the beginning of the NMA1791 sequence, with residue 13 corresponding to MmeI residue 144, and with further matches throughout the rest of the sequences. It was hypothesized that the observed truncation of the NMA1791 hypothetical protein sequence was the reason for the lack of functional endonuclease activity.
Consequently, the sequence was extended. The reading frame (306 nucleotides) was extended in a 5′ direction to the predicted TTG start codon before encountering a stop codon. Even this longer potential reading frame did not extend similarity to the beginning of MmeI sequence, nor was there a plausible putative start codon (ATG, GTG or TTG).
After additional experimentation, it was determined that the extension of the putative open reading frame to a plausible start codon, ATG, located at position 1,733, 738 in the N. meningitidis DNA sequence (Genbank Accession No. NC—003116.1) yielded an amino acid sequence with significant similarity to the MmeI amino acid sequence from very near the start of the both sequences. This potential extension of the reading frame for NMA1791, however, contained a stop codon, 5′-TAG-3′, at amino acid position 32 of the new reading frame. We cloned and expressed the N. meningitidis DNA from this alternative start position to the end of the NMA1791 reading frame (positions 1,733,738 to 1,736,551) and assayed for restriction endonuclease activity. No activity was observed.
The cloned DNA was then sequenced and it was confirmed that the cloned sequence matched the sequence in the database sequence with a TAG stop codon located at position 32 of this identified potential open reading frame.
It was again concluded that this naturally occurring sequence does not express a restriction endonuclease in N. meningitidis. 
Construction of a Novel Active Endonuclease that Recognizes GCCGAG and a Clone for Expressing the Endonuclease
The amino acid sequence from the identified extended potential open reading frame was aligned with the amino acid sequence of MmeI and 19 other sequences that all have highly significant similarity to MmeI (and each other). At the position where the extended potential sequence had a stop codon, TAG, we observed that the homologous sequences encoded a hydrophobic residue: most often a tryptophan, but sometimes a phenylalanine or a leucine or isoleucine. We reasoned that changing the stop codon to a codon for any of phenylalanine, leucine, isoleucine or tryptophan might introduce activity into the inactive enzyme. In Examples 2 and 3, we showed that this approach was effective. The nucleotide codon for tryptophan is TGG, while the observed stop codon at this position in N. meningitidis DNA was TAG. By changing the TAG to TGG and expressing the DNA from the ATG codon at position 1,733,738 (GenBank coordinate for N. meningitidis Z2491), a polypeptide was obtained that had restriction endonuclease function.
The creation of an active restriction endouclease was practically achieved as follows:

    
    
        (a) Site-directed mutagenesis was performed using an NEB (Ipswich, Mass.) kit on the expression vector that had the N. meningitidis DNA from 1,733,738 to 1,736,551 to change the A of the TAG stop codon to G, to code for the tryptophan TGG codon.
        (b) The altered DNA was then expressed in E. coli and a protein extract from the E. coli cells assayed for endonuclease activity (Example 1).
        (c) From these manipulations, a site-specific endonuclease was obtained (FIG. 2).
        (d) We purified this endonuclease and characterized the recognition sequence and position of cleavage relative to the recognition sequence (Examples 2 and 3).
    
    


We found that the altered DNA so expressed produced a restriction endonuclease, which we named NmeAIII, which recognizes 5′-GCCGAG-3′ in double-stranded DNA and cleaves either 21 and 19 nucleotides, or 20 and 18 nucleotides away from the recognition sequence as indicated: GCCGAGN21/N19 or GCCGAGN20/N18 (see Examples 2 and 3).
In summary, a novel DNA fragment (FIGS. 6-1 and 6-2) or variants is described that encode a novel restriction endonuclease, having the following properties:

    
    
        (a) recognizes the nucleotide sequence 5′-GCCGAG-3′ in a double-stranded DNA molecule as shown below,
    
    











5′-GCCGAG-3′



 



3′-CGGCTC-5′






    
    
        (wherein G represents guanine, C represents cytosine, A represents adenine and T represents thymine);
        (b) cleaves DNA in the phosphodiester bond following the 21st or 20th nucleotide 3′ to the recognition sequence 5′-GCCGAG-3 and preceding the 19th or 18th nucleotide 5′ to the complement strand of the recognition sequence 5′-CTCGGC-3′ to produce a 2 base 3′ extension:
    
    











5′-GCCGAG(N21)/-3′ or 5′-GCCGAG(N20)/-3′



 



3′-CGGCTC(N19)/-5′ or 3′-CGGCTC(N18)/-5′;



and






    
    
        (c) methylates the recognition sequence specified in (a) in vivo to protect the host DNA from cleavage by the NmeAIII endonuclease activity.
    
    


Although a full length sequence for DNA encoding the restriction endonuclease is provided, present embodiments additionally encompass fragments of varying lengths that encode the NmeAIII endonuclease. The amino acid at position 32 of the NmeAIII endonuclease may be preferentially selected from tryptophan, phenylalanine, leucine or isoleucine.
NmeAIII endonuclease fits the category of Type IIE restriction endonucleases in that it seems to require two or more recognition sequences for efficient cleavage, with one site being cleaved and one site acting as an effector (Roberts et al. Nucl. Acids Res. 31: 1805-1812 (2005)). The cleavage patterns observed are stable but partial digest DNA fragment banding patterns were observed even with excess enzyme, indicating that the enzyme does not cleave the substrate DNA completely. Addition of excess short oligonucleotides containing the specific DNA recognition sequence can drive the endonuclease cleavage of the DNA to near completion, which is consistent with the Type IIE type of endonuclease.
The ability of NmeAIII to cleave at a predominantly single site at a long reach position from the recognition site has a variety of uses in molecular biology. For example, the 19-21 base pair reach from the recognition site offers a means for making DNA tags that can be used for “Paired Ends” DNA sequencing where sequences from both ends of random clones are determined and assembled into sequence contigs. The sequence data and their linking information can then be used to construct clone maps in the form of scaffolds. This can be effectively performed using paired end ditagging and multiplex sequencing techniques (Ng et al. Nucleic Acids Res. 34(12):e84 (2006)). DNA tags can also be used for generating duel end sequence data (sequences generated from each end of a template) in shotgun sequence assembly (Dempsey et al. J. Bacteriol. 188(16): 5904-5914 (2006)). In such strategies, a recognition site is embedded in a cloning vector or in an adapter oligonucleotide ligated to the nucleic acid to be interrogated. The longer the distance between the cleavage site and the fixed recognition sequence, the more novel sequence is obtained and thus the more useful the enzyme for such purposes.
All references cited above and below, as well as U.S. provisional application Ser. No. 60/877,265 filed Dec. 27, 2006, are incorporated by reference.
EXAMPLES
Example I
Creating the NmeAIII DNA Fragment and Cloning that Fragment into E. Coli 
The NMA1791 reading frame, labeled as a hypothetical protein in Genbank database (NP—284504), was analyzed and found to be incomplete. A potential start codon, ATG, was identified at position 1,733,738 in the N. meningitidis genomic sequence. The identified reading frame starting at this position gave an amino acid sequence that has significant similarity to the known endonucleases throughout the entire sequence. This identified potential start codon was in frame with the NMA1791 incomplete reading frame; however, there was a stop codon, TAG, at amino acid position 32, nucleotide position 1,733,831 to 1,733,833, in N. meningitidis genome sequence that disrupted this reading frame.
The identified, extended reading frame from 1,733,738 to 1,736,551 was cloned and expressed in E. coli to see if this DNA fragment from N. meningitidis would express an endonuclease activity.
Oligonucleotide primers were synthesized to specifically amplify the identified DNA fragment from N. meningitidis Z2491 genomic DNA for expression in a cloning vector such as pRRS (Skoglund, Gene 88: 1-5 (1990)) or pAII17 (U.S. Pat. No. 5,371,006). The forward primer contained a PstI site for cloning, a stop codon in frame with the lacZ gene of the pRRS vector, a consensus E. coli ribosome-binding site, 5′-AGGAGGT-3, an NdeI site at the start of the reading frame for cloning, the ATG start codon, and 21 nucleotides matching the N. meningitidis DNA sequence from position 1,733,738 to 1,733,758:







(SEQ ID NO: 26)







5′- GTT CTG GAG TTA AGG AGG TAA CAT ATG AAA ACC


 


CTG CTC CAA CTC -3′






The reverse primer introduced BamHI and SalI sites for cloning and had 23 nucleotides that matched the N. meningitidis Z2491 DNA sequence 3′ to the end of the NmeAIII open reading frame at position 1,736,684 to 1,736,662:







(SEQ ID NO: 27)







5′-GTT GGA TCC GTC GAG CCT ACG CAA TTG CTT TTG


 


CGG -3′






The NmeAIII gene was amplified in a PCR reaction by combining:

    
    
        40 μl 10× Thermopol buffer (NEB, Ipswich, Mass.)
        24 μl 4 mM dNTP solution
        20 μl forward primer (10 mM stock) (SEQ ID NO:26)
        20 μl reverse primer (10 mM stock) (SEQ ID NO:27)
        1 μl N. meningitidis Z2491 genomic DNA (200 mg/ml stock)
        295 μl dH2O
        3 μl (6 units) Vent® DNA polymerase (NEB, Ipswich, Mass.)
    
    


The reaction was mixed and aliquoted into 5 tubes of 80 μl each. MgSO4 was added (100 mM stock) to bring the final concentration of Mg++ ions to 2 mM, 3 mM, 4 mM, 5 mM and 6 mM, respectively. The cycling parameters were 95° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 3 minutes, for 25 cycles. The reactions were analyzed by gel electrophoresis. The 3 mM through 6 mM Mg++ reactions were found to contain a DNA band of the desired size of approximately 2.9 kb. These reactions were pooled and the 2.9 kb band was gel purified. The 2.9 kb amplified N. meningitidis DNA fragment was digested with NdeI and BamHI endonucleases (NEB, Ipswich, Mass.) in NEBuffer BamHI (NEB, Ipswich, Mass.) according to the manufacturer's instructions. The small fragments cleaved off the ends of the 2.9 kb DNA fragment were removed, along with the endonucleases, by purification on a Zymo Research “DNA Clean and Concentrate 5” spin column according to the manufacturer's instructions (Zymo Research, Orange, Calif.).
The amplified, cleaved N. meningitidis DNA fragment was ligated to pAII17 vector, previously cleaved with NdeI and BamHI and dephosphorylated, in a 20 ml reaction using NEB T4 DNA ligase according to the manufacturer's instructions (NEB, Ipswich, Mass.). 5 μl of the ligation reaction was transformed into 50 μl chemical competent E. coli ER2566 cells (NEB, Ipswich, Mass.) and the cells were plated on L-broth plates containing 100 μg/ml ampicillin and incubated at 37° C. overnight. Six representatives were analyzed as follows: plasmid from each colony was isolated by miniprep procedures and digested with BamHI and NdeI endonucleases to determine if they contained the correct size insert. 5 of the 6 transformants had the correct size insert of approximately 2900 bp. The five clones were tested to see if they produced endonuclease activity. The clones were grown to mid log phase (klett of 120 to 150) at 37° C. in 30 mL L-broth containing 100 μg/ml ampicillin. The culture was then induced by the addition of IPTG to 0.4 mM and grown for an additional 90 minutes. The cells were harvested by centrifugation, suspended in 1.5 mL sonication buffer (20 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, pH7.5) and broken by sonication. The crude lysate was cleared by centrifugation and the supernatant recovered. The lysate was assayed for endonuclease activity by serial dilution of the lysate in 1× reaction buffer NEBuffer 4 (NEB, Ipswich, Mass.) containing 20 μg/ml lambda DNA substrate, or 20 μg/ml pBC4 DNA substrate linearized with NdeI, and supplemented with SAM (S-adenosyl-L-methionine) at 80 μM final concentration. The reactions were incubated for 30 minutes at 37° C. The reaction products were analyzed by agarose gel electrophoresis on a 1% agarose gel in 1×TBE buffer.
None of the clones produced endonuclease activity. The clones were sequenced and the TAG stop codon at position 1,733,831 to 1,733,833 of the N. meningitidis genomic sequence was present, indicating that the sequence in the database was correct; this stop codon was not an artificial sequencing error but was the accurate sequence of the N. meningitidis DNA. The DNA fragment encoding the open reading frame from N. meningitidis DNA, even when extended from the shortened version found in the database (NMA1791) to include all the sequence having similarity to MmeI, did not encode an active endonuclease.
We reasoned that we might be able to create a DNA fragment encoding an endonuclease by changing the TAG stop codon described above to code for an amino acid. The choice of the amino acid to insert at this position was guided by comparison with amino acid sequences of MmeI, CstMI and other sequences identified from sequence databases that have highly significant scores (Expectation value, E, of E less than e-20) in a BLAST search of the database using the extended N. meningitidis open reading frame amino acid sequence. For the comparison twenty sequences were aligned with the extended N. meningitidis sequence using ClustalW, (www.ebi.ac.uk/clustalw) of which 65% of the sequences examined had a tryptophan at the position of the N. meningitidis stop codon, 15% had a phenylalanine, 10% had a leucine, 5% an isoleucine, and 5% had a cysteine. All except the cysteine are hydrophobic residues. We chose to replace the stop codon with sequence coding for tryptophan, although it is likely we could use phenylalanine, leucine or isoleucine at this position, and possibly cysteine as well. To create the changed DNA sequence we employed the Phusion Site-Directed Mutagenesis Kit (NEB, Ipswich, Mass.). Two primers were synthesized, one of which had TGG in place of TAG at the position of the N. meningitidis stop codon, and used the primers according to the manufacturer's directions for the mutagenesis (FIG. 1). The sequence of the forward primer was: 5′-pTGGAACGAATTTTTCGCCATTTTC-3′ (SEQ ID NO:28) where p indicates a phosphate group. The second base, “G” (underlined) differs from the N. meningitidis sequence “A” at this position. The reverse primer was: 5′-pGAAGGTGTCTTTCTCGCG-3′ (SEQ ID NO:29). One of the pAII17 clones carrying the N. meningitidis DNA fragment from 1,733,738 to 1,736,684 was employed as the template for PCR amplification with these primers in a reaction consisting of:

    
    
        0 μl 5× Phusion HF buffer
        1 μl pAII17-N. meningitidis plasmid
        15 μl forward mutagenic primer (SEQ ID NO:28)
        15 μl reverse primer (SEQ ID NO:29)
        7.5 μl dNTPs (10 mM stock)
        202 μl dH2O
        3 μl (6 units) Phusion DNA polymerase
    
    


Amplification Conditions Were:
98° C. for 1 minute for one cycle, then 25 cycles of 98° C. for 10 seconds, 63° C. for 20 seconds, and 72° C. for 4 minutes. The amplified DNA was purified on a Zymo Research (Orange, Calif.) Spin column according to the manufacturer's instructions and eluted in 20 μl TE. 3 μl of the DNA was ligated in a 20 μl reaction with T4 DNA ligase (NEB, Ipswich, Mass.). 4 μl of the ligation reaction was transformed into 50 μl chemical competent ER2566 cells (NEB, Ipswich, Mass.) and plated on LB plates containing 100 micrograms/mL ampicillin.
Two individual transformants were grown to express the altered DNA fragment to test for endonuclease activity. A colony was inoculated into 60 mL LB containing 100 micrograms per ml ampicillin in a 250 ml flask and grown to mid log phase at 37° C. with shaking. At mid log, the cells were induced to express protein from the DNA fragment by addition of IPTG to 0.4 mM final concentration and grown for an additional 2 hours. The cells were then harvested by centrifugation, resuspended in 3 ml sonication buffer and lysed by sonication. The crude cell extract was clarified by centrifugation and tested for endonuclease activity by serial dilution of the lysate in 1× reaction buffer NEBuffer 4 (NEB, Ipswich, Mass.) supplemented with SAM (S-adenosyl-L-methionine) at 80 μM final concentration and either 20 μg/ml lambda DNA substrate, or 20 μg/ml pBC4 DNA substrate linearized with NdeI. The reactions were incubated for 30 minutes at 37° C. The reaction products were analyzed by agarose gel electrophoresis on a 1% agarose gel in 1×TBE buffer. Both transformants tested expressed a specific endonuclease (FIG. 1).
Example II
Determination of the NmeAIII Specific DNA Recognition Sequence
The NmeAIII endonuclease was purified from the crude cell extract on a 1 ml Heparin HiTrap column (Pharmacia, Piscataway, N.J.). The crude extract was diluted to 10 ml in buffer A (20 mM Tris pH7.5, 1 mM DTT, 0.1 mM EDTA) containing 50 mM NaCl and applied to the column previously washed and equilibrated in buffer A containing 50 mM NaCl. A 40-milliliter linear gradient from 50 mM to 1M NaCl in buffer A was applied to the column and 1 ml fractions were collected. The fractions were assayed for endonuclease activity. The NmeAIII endonuclease activity eluted at approximately 0.35M NaCl.
The purified NmeAIII from the heparin column was used to digest pUC19, pBR322, PhiX174 and pBC4 DNAs in order to map the locations of NmeAIII cutting in these DNAs. NmeAIII cutting was mapped to approximate positions of 70, 1125 and 3530 in pBR322 DNA, to approximate positions of 1050, 3440 and 4740 in PhiX174 DNA, and to approximate positions of 1900, 3715, 4990, 5385, 6995 and 9750 in pBC4 (FIG. 3). The distance between positions of cutting was entered into the REBPredictor program as fragment sizes for pBR322 DNA (http://tools.neb.com/REBpredictor/index.php). The fragment sizes entered were 2405, with a 4% error margin, 1050 with 6% margin and 900 with 10% error margin. A number of possible recognition sequences were returned, and these were examined to see where they occur in pBR322 and the other DNAs mapped for NmeAIII. One of the potential recognition sequences predicted by REBPredictor, 5′-GCCGAG-3′, correlated with the position of NmeAIII cutting.
We found that the sequence 5′-GCCGAG-3′ occurs in pBR322 at positions 116, 1168 and 3489, with the 116 and 1168 positions oriented to cleave the DNA 5′ to the site (at a smaller position number than the recognition sequence by approximately 20 nucleotides, i.e. at approximately positions 96 and 1148), while the 3489 site is oriented to cleave the DNA 3′ to the recognition site (i.e., at a position approximately 25 nucleotides greater than the position number of the recognition site, or 3514 in this case). These 5′-GCCGAG-3′ positions agree with the observed positions cut by the endonuclease. The 5′-GCCGAG-3′ sequence positions in PhiX174 and pBC4 agree with the experimentally observed positions of cleavage, from which we determine that NmeAIII endonuclease recognizes the sequence 5′-GCCGAG-3′ in double stranded DNA.
Example III
Determination of the NmeAIII Cleavage Site
The position of the NmeAIII DNA cleavage site relative to the recognition sequence was determined by cleaving a suitable DNA molecule and then performing DNA sequencing from a suitable primer to the end of the cleaved DNA template. Multiple NmeAIII cut sites were tested because we observed some small variation in the exact distance from the recognition sequence to the position of cleavage, which we believe is due to the nature of the DNA sequence occurring between any given recognition site and cutting positions. Several DNAs (PhiX174, pBC4, pUC19 and pBR322) were employed as the templates. These templates were chosen because they have more than three NmeAIII recognition sites to test and primers available to sequence through these sites. The templates were cleaved with NmeAIII by combining:

    
    
        25 μl 10× NEBuffer #4 (NEB, Ipswich, Mass.)
        5 μl PhiX174 DNA (5 μg)
        2.5 μl BSA
        218 μl dH2O
        0.6 μl SAM (32 mM stock)
        2 μl NmeAIII (20 units)

and incubating for 15 minutes at 25° C. The cleaved DNA was purified and concentrated using a Qiagen QiaPrep DNA spin column according to the manufacturer's instructions (Qiagen, Valencia, Calif.). The DNA was eluted in a volume of 50 μl (FIG. 3)

Sequencing Reactions

    
    


The sequencing reactions were performed using an ABI377 DNA sequencer according to the manufacturer's instructions, using cleaved template DNAs (PhiX174 or pBC4) and one of several primers:








P344-350:



5′-TGATAGGGTCTTTTACCAGCACT-3′
(SEQ ID NO: 30)


 


P344-351:


5′-TAATGGCGTCGAGCGTCCGGTT-3′
(SEQ ID NO: 31)


 


NEB1224:


5′-CGCCAGGGTTTTCCCAGTCACGAC-3′
(SEQ ID NO: 32)






The results indicate NmeAIII cleaves DNA at a significant distance from the recognition sequence, either between the 21st and the 22nd nucleotides 3′ to the recognition sequence in the 5′-GCCGAG-3 strand of the DNA, and between the 19th and 20th nucleotides 5′ to the recognition sequence in the complement stand, 5′-CTCGGC-3′, or one base closer to the recognition sequence, to produce a 2 base 3′ extension (FIGS. 4A and 4B). The position of cleavage observed varied somewhat between two positions, largely between the 21st and 22nd nucleotides 3′ to the recognition sequence in the top strand, 5′-GCCGAG-3′, but sometimes between the 20th and 21st nucleotides. Likewise, there was some variation in the bottom strand cutting position, largely between the 19th and 20th nucleotides 5′ to the recognition sequence in the complement stand, 5′-CTCGGC-3′, or one base closer to the recognition sequence between the 18th and 19th nucleotides 5′ to the recognition sequence in the complement stand, 5′-CTCGGC-3′.
Because NmeAIII cut at a large distance from the DNA sequence that is recognized and specifically bound by the enzyme, it may be that the particular DNA sequence between the recognition sequence and the position of cutting affects whether the cut position is 21/19 or 20/18 nucleotides away from the recognition sequence. This theory was tested and compared with results for MmeI, which previous to NmeAIII had the longest distance of cutting away from the recognition site of any known type II endonuclease.
A DNA was constructed having either the MmeI recognition site, 5′-TCCRAC-3′, or the NmeAIII recognition site, 5′-GCCGAG-3′, followed by the same DNA sequence, from Phi174 in which we observed NmeAIII cutting at 21/19 nucleotides from the recognition site at 3426. The MmeI construct was cut and sequenced with MmeI as above, and the NmeAIII construct was similarly tested. The results showed that NmeAIII cuts this construct predominantly at 21/19 bases from the recognition site, while MmeI produces a mixture of 21/19 and 20/18 away from its recognition site (FIGS. 4C-1 and 4C-2). These results showed that NmeAIII has a slightly longer reach than MmeI, though not by a whole nucleotide. NmeAIII will thus be more likely to cut one base farther from its recognition sequence than will MmeI. However, for some intervening sequences between the recognition sites and cut positions, these enzymes will cleave at the same distance, and for others NmeAIII will cleave one base farther than MmeI.
NmeAIII Characteristics:
The NmeAIII endonuclease was assayed in NEBuffer 1, NEBuffer 2, NEBuffer 3 and NEBuffer 4, all supplemented with BSA to 100 micrograms/ml and SAM at 80 micromolar final concentration. NmeAIII was most active in NEBuffer 4, with NEBuffer 1 yielding approximately 10% of the activity observed in NEBuffer 4, and NEBuffer 2 and NEBuffer 3 yielding less than 5% activity (NEB, Ipswich, Mass.).

## Claims

1. An isolated protein, comprising: an amino acid sequence which is at least 90% sequence identical to SEQ ID NO: 24, the amino acid sequence comprising, at a position corresponding to position 32 of SEQ ID NO: 24, an amino acid residue selected from the group consisting of tryptophan, phenylalanine, leucine, and cysteine, wherein said isolated protein (i) has endonuclease activity, (ii) recognizes a sequence consisting of 5′-GCCGAG-3′ within a double-stranded DNA substrate, and (iii) cleaves said double-stranded DNA substrate predominantly at 19 nucleotides on one strand and 21 nucleotides on the complementary strand from the recognition site.

2. The protein of claim 1, comprising the amino acid sequence of SEQ ID NO:24.

3. The protein of claim 1, comprising the amino acid sequence of SEQ ID NO:24, except that at a position corresponding to position 32 of SEQ ID NO:24, the amino acid residue is selected from the group consisting of tryptophan, phenylalanine, leucine, and cysteine.

4. A kit comprising an isolated protein that has an amino acid sequence which is at least 90% sequence identical to SEQ ID NO: 24, the amino acid sequence comprising, at a position corresponding to position 32 of SEQ ID NO: 24, an amino acid residue selected from the group consisting of tryptophan, phenylalanine, leucine, and cysteine, wherein said isolated protein (i) has endonuclease activity, (ii) recognizes a sequence consisting of 5′-GCCGAG-3′ within a double-stranded DNA substrate, and (iii) cleaves said double-stranded DNA substrate predominantly at 19 nucleotides on one strand and 21 nucleotides on the complementary strand from the recognition site.

5. A method comprising the steps of:
(a) creating a DNA tag by cleaving a target DNA with an isolated protein comprising an amino acid sequence which is at least 90% sequence identical to SEQ ID NO: 24, the amino acid sequence comprising, at a position corresponding to position 32 of SEQ ID NO: 24, an amino acid residue selected from the group consisting of tryptophan, phenylalanine, leucine, and cysteine, wherein said isolated protein (i) has endonuclease activity, (ii) recognizes a sequence consisting of 5′-GCCGAG-3′ within a double-stranded DNA substrate, and (iii) cleaves said double-stranded DNA substrate predominantly at 19 nucleotides on one strand and 21 nucleotides on the complementary strand from the recognition site; and
(b) using the DNA tag as a unique identifier for paired end sequencing of DNA or serial analysis of gene expression.