Bacteriophage T7: Minimal requirements for the replication of a duplex DNA molecule

DNA replication is a complex process consisting of numerous steps, each of which may require several proteins. One approach to its dissection has been to study the replication of the small, circular, single-stranded DNAs of bacteriophages such as $X174. Since their replication is dependent on host proteins, they have provided for complementation assays that have led to the identification of over 16 replication proteins encoded by E. coli (Kornberg, Supplement to DNA Replication, W. H. Freeman, 1982). Another approach has been to study the replrcation of the double-stranded DNA molecules of more complex phages such as T7 and T4. The large size of their chromosomes enable them to encode most of their own replication proteins, bypassing the host replication machinery. At the same time, their basic replication mechanisms share many similarities with those of higher organisms. Replication is initiated at multiple, site-specific origins; DNA synthesis is both bidirectional and discontinuous; and lagging strand synthesis is initiated by short RNA primers. The chromosome of phage T7 is a double-stranded linear molecule, each strand consisting of an uninterrupted sequence of 39,936 nucleotides. The nucleotide sequence of the entire molecule has been determined, and a total of 50 genes identified within that sequence (Dunn and Studier, J. Mol. Biol., in press). The growth cycle of T7 is remarkable: within 15 min after infecting an E. coli cell, the T7 DNA molecule is duplicated 200 times. To accomplish this feat, T7 has evolved an efficient and economical mechanism for the replication of its DNA, one that probably defines the minimal requirements for the rapid and accurate replication of a duplex molecule. Five proteins, all purified to homogeneity, accomplish the basic steps in initiation and elongation (Fuller et al., CSHS 47, 669-679, 1983; Richardson, in Developments /n Molecular Virology, 2, Y. Becker, ed. Martrnus Nifkoff, 1983). While more complex organisms employ more proteins, these usually represent a drvisron of labor or a refinement of the steps defined by the T7 system. initiation at the Replication Origin T7 DNA replication is initiated at a specific site, located 15% of the distance from the genetic left end of the chromosome, and proceeds bidirectionally. When this region IS deleted, initiation occurs at secondary origins, the predominant one being at the extreme left end of the molecule (Tamanoi et al., PNAS 77, 2656-2660, 1980). The primary ongrn, a 200 bp intergenic segment from 14.5% to 15.0%, contains two tandem T7 RNA polymerase promoters (41 .I A and 61 ,l B) followed by a 61 bp AT-rich (78% A + T) region (Saito et al., PNAS 77, 3917-3921, 1980). In addition, a single gene 4 protein primase site is located within the AT-nch region and an E. coli RNAase III cleavage site lies between the two promoters. Deletion of the AT-rich region or in vitro mutagenesrs of the two promoters inactivates the origin in vivo, causing initiation to occur exclusively at secondary origins. Although precise mapping of the secondary origin near the left end of the molecule has not been carried out, a T7 RNA polymerase promoter, $OL, followed by an AT-rich region, has been identified from sequence analysis (Dunn and Studier, op. cit.). T7 RNA polymerase promoters are recognized by a T7encoded RNA polymerase, the product of gene 7. Gene I is among the genes transcribed by E. coli RNA polymerase soon after infection. T7 RNA polymerase accounts for intermediate and late gene expression by initiating transcription at 17 promoters, each consisting of a conserved 23 bp sequence (Dunn and Studier, op. cit.). It has also been shown that T7 RNA polymerase is essential for T7 DNA replication in vivo (Hinkle, J. Virol. 34, 136-141, 1980). Initiation of DNA synthesis at the primary origin has been achieved using two purified phage-induced proteins, T7 RNA polymerase and T7 DNA polymerase (Roman0 et al., PNAS 78, 4107-4111, 1981; Fuller et al., op. cit.). Electron microscopic analysis of the products obtained when T7 DNA is incubated with the two purified enzymes, dNTPs, and rNTPs reveals initiation at the primary origin. Initiation has been studied to finer resolution in vitro, using plasmids into which the primary origin has been cloned. Electron microscopy and restriction enzyme analysis show that DNA synthesis initiates precisely within the primary origin and initially proceeds unidirectionally, in the same direction as transcription from the $1.1 A and 41.1 B promoters. The 5’ termini of the newly synthesized DNA molecules are covalently linked to RNA. These primer RNAs result from transcription from both the 41 .lA and the 41 .lB promoters; the transition to DNA synthesis occurs at numerous sites, many within the AT-rich region. The basic features of initiation in vitro are summarized in the scheme below. While specific initiation can be achieved in vitro using only T7 RNA polymerase and DNA polymerase, the mechanisms that pertain in vivo may be more refined. Genetic