Effect of phi X C protein on leading strand DNA synthesis in the phi X174 replication pathway

The influence of the bacteriophage phi X174 (phi X) C protein on the replication of bacteriophage phi X174 DNA has been examined. This small viral protein, which is required for the packaging of phi X DNA into proheads, inhibits leading strand DNA synthesis. The inhibitory effect of the phi X C protein requires a DNA template bearing an intact 30-base pair (bp) phi X origin of DNA replication that is the target site recognized by the phi X A protein. Removal of nucleotides from the 3' end of this 30-bp conserved origin sequence prevents the inhibitory effects of the phi X C protein. Leading strand replication of supercoiled DNA substrates containing the wild-type phi X replication origin results in the production of single-stranded circular DNA as well as the formation of small amounts of multimeric and sigma structures. These aberrant products are formed when the termination and reinitiation steps of the replication pathway reactions are skipped as the replication fork moves through the origin sequence. Replication carried out in the presence of the phi X C protein leads to a marked decrease in these aberrant structures. While the exact mechanism of action of the phi X C protein is not clear, the results presented here suggest that the phi X C protein slows the movement of the replication fork through the 30-bp origin sequence, thereby increasing the fidelity of the termination and reinitiation reactions. In keeping with the requirement for the phi X C protein for efficient packaging of progeny phi X DNA into proheads, the phi X C protein-mediated inhibition of leading strand synthesis is reversed by the addition of proteins essential for phi X bacteriophage formation. Incubation of plasmid DNA substrates bearing mutant 30 base pair phi X origin sequences in the complete packaging system results in the in vitro packaging and production of infectious particles in a manner consistent with the replication activity of the origin under study.     

Selective cloning of a DNA single-strand initiation determinant from phi X174 replicative-form DNA.

An M13 phage deletion mutant, M13 delta E101, developed as a vector for selecting DNA sequences that direct DNA strand initiation on a single-stranded template, has been used for cloning restriction enzyme digests of phi X174 replicative-form DNA. Initiation determinants, detected on the basis of clear-plaque formation by the chimeric phage, were found only in restriction fragments containing the unique effector site in phi X174 DNA for the Escherichia coli protein n' dATPase (ATPase). Furthermore, these sequences were functional only when cloned in the orientation in which the phi X174 viral strand was joined to the M13 viral strand. A 181-nucleotide viral strand fragment containing this initiation determinant confers a phi X174-type complementary-strand replication mechanism on M13 chimeras. The chimeric phage is converted to the parental replicative form in vivo by a mechanism resistant to rifampin, a specific inhibitor of the normal RNA polymerase-dependent mechanism of M13. In vitro, the chimeric single-stranded DNA promotes the assembly of a functional multiprotein priming complex, or primosome, identical to that utilized by intact phi X174 viral strand DNA. Chimeric phage containing the sequence complementary to the 181-nucleotide viral strand sequence shows no initiation capability, either in vivo or in vitro.     

Mechanism of replication of bacteriophage phi X174. XXII. Site-specific mutagenesis of the A* gene reveals that A* protein is not essential for phi X174 DNA replication

The A and A* proteins of phage phi X174 are encoded in the same reading frame in the viral genome; the smaller A protein is the result of a translational start signal with the A gene. To differentiate their respective functions, oligonucleotide-directed site-specific mutagenesis was used to change the ATG start codon of the phi X 174 A* gene, previously cloned into pCQV2 under lambda repressor control, into a TAG stop codon. The altered A gene was then inserted back into phi X replicative form DNA to produce an amber mutant, phi XamA*. Two different Escherichia coli amber suppressor strains infected with this mutant produced viable progeny phage with only a slight reduction in yield. In Su+ cells infected with phi XamA*, phi X gene A protein, altered at one amino acid, was synthesized at normal levels; A* protein was not detectable. These observations indicate that the A* protein increases the replicative efficiency of the phage, perhaps by shutting down host DNA replication, but is not required for replication of phi X174 DNA or the packaging of the viral strand under the conditions tested.     

DNA methylation inhibits the transfecting activity of replicative- form phi X174 DNA

Replacement of virtually all the cytosine residues with 5-methylcytosine residues in the complementary strand of the replicative form (RF) of phi X174 DNA caused a 300- to 500-fold loss in its transfecting activity. Similar results were obtained with analogously methylated M13 RF. Transfection experiments with phi X RF hemimethylated in only part of the molecule, as assessed by analysis with restriction endonucleases, indicated that gene A of phi X, which needs to be nicked at a specific site by the gene A protein for RF replication, was not the main target for this inhibition by DNA methylation. We propose that the loss of transfecting activity was due to hemimethylation of the phi X RF interfering with the processively catalyzed movement of the replication fork.     

Mechanism of replication of bacteriophage phi X174 XIX. Initiation of phi X174 viral strand DNA synthesis at internal sites on the genome.

Bacteriophage phi X174 viral strand DNA molecules shorter than genome length found late in the infectious cycle in Escherichia coli were 5' end labeled with 32P. Hybridization of the 32P-labeled molecules to restriction enzyme fragments of phi X replicative form DNA revealed an excess of phi X molecules whose 5' ends mapped in HaeIII fragments Z3 and Z4 in comparison with fragments Z1 and Z2. This suggests that initiation of phi X174 viral strand DNA synthesis may occur at internal sites on the complementary strand. There are several appropriately located sequences that might serve as n' (factor Y) recognition sequences and thereby facilitate discontinuous synthesis of the viral strand.     

Roles of phi X174 type primosome- and G4 type primase-dependent primings in initiation of lagging and leading strand syntheses of DNA replication

Eleven single strand initiation sequences (ssi) were isolated from various plasmid genomes using a plaque-morphology assay. Out of seven ssi that require dnaB and dnaC functions for replication in a crude in vitro system, six use a phi X174 type priming mechanism, and a phi X174 type primosome is assembled at these sequences from the purified proteins, n'(priA), n(priB), n"(priC), dnaT, dnaB, dnaC, and primase. The same ssi potentiate dATPase activity of n' protein, and thus represent new n' protein recognition sequences (n'-pas). Based on sequence homology, two structural groups are evident. Two sequences show a strong homology with the phi X174 site, whereas three share extensive homology with the previously characterized n'-pas of ColE1, ssiA(ColE1). All the n'-pas have a potential to form stem and loop structures, although sequence homology between the two classes is absent. In addition to the phi X174 type priming, three ssi do not require either dnaB or dnaC function for replication, and use a G4 type priming, requiring only SSB and primase. The 5' ends of primer RNA synthesized by primase are localized within the vicinity of one of the three blocks of highly conserved nucleotide sequences. Deletions of parts of these conserved sequences result in loss of priming activity, suggesting that they are important for priming on the G4 type ssi, which are termed G site. The general significance of these two types of priming in initiation of lagging or leading strand synthesis as well as various modes of initiation at origins of replication are proposed.     

Properties of the A and A proteins of bacteriophage G4. The origin of G4 replicative-form DNA replication

The A and A proteins of the bacteriophage G4 have been purified. The proteins have been analysed for their enzymatic activities on single-stranded and double-stranded DNA. The A protein introduces a single-stranded break at a specific place in the G4 replicative form I DNA. This cleavage site has been localized between nucleotides 506 and 507 in the viral (+) strand. The A protein binds covalently to the 5' end of the cleavage site. The A protein initiates the replication of the viral (+) DNA [Borrias, et al. (1979) Virology, 31, 288-298]; the cleavage site therefore identifies the origin of replication. The A protein cleaves viral (+) strand DNA at many different sites and also binds covalently to the 5' ends of the nick sites. The properties of both proteins strongly resemble the properties of the A and A proteins of the related and much butter analysed phage phi X174. These results indicate that the G4 and phi X174A and A proteins have comparable functions and also that both phages initiate the replicative form DNA in a similar way.     

Mutational analysis of the bacteriophage phi X174 replication origin

Bacteriophage phi X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type phi X174. Two phi X174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate. The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein. This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin. In a Darwinian experiment wild-type phi X174 outgrows all phi X174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage phi 174. Insertions and deletions were constructed at different positions within the phi X174 replication origin cloned in a plasmid. Small insertions and deletions in the A + T-rich spacer region do not inhibit phi X174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats.     

DNA sequences which support activities of the bacteriophage phi X174 gene A protein

The DNA sequence of 30 nucleotides which surrounds the origin of viral strand DNA replication is highly conserved amongst the icosahedral single-stranded DNA bacteriophages. The A gene of these phages encodes a protein which is required for initiation and termination of viral strand DNA synthesis and acts as a nicking-closing activity specifically within this 30-nucleotide sequence. A system of purified Escherichia coli host proteins and phi X174 gene A protein has been developed which specifically replicates in vitro the viral strand of phi X174 from RF (replicative form) I template DNA and yields single-stranded circular DNA products (RF leads to SS(c) DNA replication system). Recombinant plasmids carrying inserts derived from phage phi X174 or G4 DNA which range in length from 49 to 1175 base pairs and contain the 30-nucleotide conserved sequence have been shown to support phi X A protein-dependent DNA synthesis in vitro in this replication system. We report here that insertion of the 30-nucleotide sequence alone into pBR322 allows the resulting recombinant plasmids to support phi X A protein-dependent in vitro DNA synthesis as efficiently as phi X174 template DNA in the RF leads to SS(c) replication system. The 30-nucleotide sequence functions as a fully wild type DNA replication origin as determined by the rate of DNA synthesis and the structure of resulting DNA products. Furthermore, the DNA sequence requirements for nicking of RF I DNA by the phi X A protein and for supporting replication origin function have been partially separated. Homology to positions 1, 29, and 30 of the 30-nucleotide conserved sequence are not required for cleavage of RF I DNA by the A protein; homology to position 1 but not 29 or 30 is required for efficient DNA replication.     

Formation of rolling-circle molecules during phi X174 complementary strand DNA replication

The primosome is a mobile multiprotein priming apparatus that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC and dnaG genes; replication factor Y (protein n'); and proteins i, n, and n"). While the primosome is analagous to the phage T7 gene 4 protein and phage T4 gene 41/61 proteins in its DNA G-catalyzed priming function, its ability to act similarly also as a DNA helicase has remained equivocal. The role of the primosome in unwinding duplex DNA strands was investigated in the coliphage phi X174 SS(c)----replicative form DNA replication reaction in vitro, which requires the E. coli single-stranded DNA binding protein, the primosomal proteins, and the DNA polymerase III holoenzyme. Multigenome-length, linear, double-stranded DNA molecules were generated in this reaction, presumably via a rolling circle-type mechanism. Synthesis of these products required the presence of a helicase-catalyzed strand-displacement activity to permit multiple cycles of continuous complementary (-) strand synthesis. The participation of the primosome in this helicase activity was supported by demonstrating that other SS(c) DNA templates (G4 and alpha-3), which lack primosome assembly sites, failed to support significant linear multimer production and that replication of phi X174 with the general priming system (the DNA B and DNA G proteins and DNA polymerase III holoenzyme) resulted in a 13-fold lower rate of linear multimer synthesis.     

Gene A protein cleavage of recombinant plasmids containing the phi X174 replication origin

Synthetic oligonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p. of the origin of replication of bacteriophage phi X174 (nucleotides 4299-4328 of the phi X174 DNA sequence). The double-stranded DNA fragments were cloned into the unique SmaI or HindIII restriction sites in the kanamycin-resistance gene of pACYC177 (AmpR, KmR). Recombinant plasmids were picked up by colony hybridization. DNA sequencing showed that not only recombinant plasmids with the expected insert were formed, but also recombinant plasmids with a shorter insert. Recombinant plasmids with an insert homologous to the first 24, 25, 26, 27, 28 or all 30 b.p. of the phi X174 origin region were thus obtained. Supercoiled plasmids containing a sequence homologous to the first 27, 28 or 30 b.p. of the phi X174 origin region are nicked by the phi X174 gene A protein. However, the other supercoiled plasmids are not nicked by the phi X174 gene A protein. These results show that the first 27 b.p. of the phi X174 origin region are sufficient as well as required for the initiation step in phi X174 RF DNA replication, i.e. the cleavage by gene A protein.     

RNA priming of DNA replication by bacteriophage T4 proteins

Bacteriophage T4 DNA replication proteins have been shown previously to require ribonucleoside triphosphates to initiator new DNA chains on unprimed single-stranded DNA templates in vitro. This DNA synthesis requires a protein controlled by T4 gene 61, as well as the T4 gene 41, 43 (DNA polymerase), 44, 45, and 62 proteins, and is stimulated by the gene 32 (helix-destabilizing) protein. In this paper, the nature of the RNA primers involved in DNA synthesis by the T4 proteins has been determined, using phi X174 and f1 DNA as model templates. The T4 41 and "61" proteins synthesize pentanucleotides with the sequence pppA-C(N)3 where N in positions 3 and 4 can be G, U, C, or A. The same group of sequences is found in the RNA at the 5' terminus of the phi X174 DNA product made by the seven T4 proteins. The DNA product chains begin at multiple discrete positions on the phi X174 DNA template. The characteristics of the T4 41 and "61" protein priming reaction are thus appropriate for a reaction required to initiate the synthesis of discontinuous "Okazaki" pieces on the lagging strand during the replication of duplex DNA.     

Enzymatic properties of the bacteriophage phi X174 A protein on superhelical phi X174 DNA: a model for the termination of the rolling circle DNA replication.

Incubation of phi X174 replication form I DNA with the A* protein of phi X174 in the presence of MN2+ results in the formation of three different types of DNA molecules: open circular form DNA (RFII), linear form DNA (RFIII) and the relaxed covalently closed form DNA (RFIV). The RFII and RFIII DNAs are shown to be A* protein-DNA complexes by electron microscopy using the protein labeling technique of Wu and Davidson (1). The linear double-stranded RFIII DNA molecule carries at one end a covalently attached A* protein whereas at the other end of the molecule the single-stranded termini are covalently linked to each other. The structure of the RFIII DNA shows its way of formation. The described properties of the A* protein indicate the way the larger A protein functions in the termination step of the rolling-circle type of phi X174 DNA replication.     

Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE plasmid DNA.

The opposite strands of the ColE1 and ColE3 plasmids were isolated as circular single-stranded DNA molecules. These molecules were compared with M13 and phi X174 viral DNA with respect to their capacity to function as templates for in vitro DNA synthesis by a replication enzyme fraction from Escherichia coli. It was found for both ColE plasmids that the conversion of H as well as L strands to duplex DNA molecules closely resembles phi X174 complementary strand synthesis and occurs by a rifampicin-resistant priming mechanism involving the dnaB, dnaC, and dnaG gene products. Restriction analysis of partially double-stranded intermediates indicates that preferred start sites for DNA synthesis are present on both strands of the ColE1 HaeII-C fragment. Inspection of the nucleotide sequence of this region reveals structural similarities with the origin of phi X174 complementary strand synthesis. We propose that the rifampicin-resistant initiation site (rri) in the ColE1 L strand is required for the priming of discontinuous lagging strand synthesis during vegetative replication and that the rri site in the H strand is involved in the initiation of L strand synthesis during conjugative transfer.     

Studies on the phi X174 gene A protein-mediated termination of leading strand DNA synthesis

Recombinant RF (replicate form) I DNAs containing the bacteriophage phi X174 gene A protein-recognition sequence are cleaved by the phi X A protein yielding a phi X RF II X A protein complex (Zipursky, S.L., Reinberg, D., and Hurwitz, J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5182-5186). Such complexes support DNA synthesis in both RF I leads to SS(c) and RF I leads to RF I phi X DNA replication reactions in vitro. Two phi X A protein-recognition sequences were inserted into plasmid pBR322. Both sequences were contiguous with the same strand of the vector DNA and separated by 667 and 4275 base pairs. This recombinant plasmid (G27-4) was cleaved by the phi X A protein at either insert and both inserts support the initiation of RF leads to SS(c) DNA synthesis. This was verified by the finding that replication products were circular molecules of 667 and 4275 nucleotides. This finding is in keeping with the multifunctional activities associated with the phi X A protein; these include the site-specific nicking of RF I DNA which initiates DNA synthesis and site-specific termination resulting in the circularization of the displaced DNA strand. The phi X A protein and the Escherichia coli rep and SSb proteins catalyze the unwinding of phi X RF I DNA in vitro (Scott, J.F., Eisenberg, S., Bertsch, L.L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 193-197). Recombinant plasmid G27-4 RF I DNA was also unwound in vitro by this enzyme system; in this case, both circular and linear single-stranded DNA molecules of 667 and 4275 nucleotides, as well as full length circular single-stranded DNA were formed. Full length linear DNA was not detected. The two single-stranded circular DNA products formed as leading strands in RF leads to SS(c) reaction mixtures containing G27-4 RF I DNA differed in their ability to support lagging strand DNA synthesis. It was shown that the large single-stranded circular product included DNA sequences homologous to a replication factor Y effector sequence required for RF leads to RF and SS(c) leads to RF replication (Zipursky, S.L., and Marians, K.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6521-6525). The 4275-nucleotide, but not the 667-nucleotide, single-stranded circular DNA product was converted to a duplex structure.     

Role of polymeric forms of the bacteriophage phi X174 coded gene A protein in phi XRFI DNA cleavage.

Gene A of the phi X174 genome codes for two proteins, A and A* (Linney, E.A., and Hayashi, M.N. (1973) Nature New Biol. 245, 6-8) of molecular weights 60,000 and 35,000, respectively. The phi X A* protein is formed from a natural internal initiator site within the A gene cistron while the phi X A protein is the product of the entire A gene. These two proteins have been purified to homogeneity as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Previous studies have shown that the phi X A protein is an endonuclease which specifically introduces a discontinuity in the A cistron of the viral strand of supertwisted phi XRFI DNA. In addition to this activity, the phi X A protein also causes relaxation of supertwisted phi XRFI DNA and formation of a phi XRFH DNA . phi X A protein complex which has a discontinuity in the A cistron of the viral strand. This isolatable complex supports DNA synthesis when supplemented with extracts of uninfected Escherichia coli which lack phi X A protein and phi XRFI DNA. The phi XRFII DNA . phi X A protein complex can be attacked by exonuclease III but is not susceptible to attack by E. coli DNA polymerase I, indicating that the 5'-end of the complex is blocked. Attempts to seal the RFII structure generated from the phi XRFII DNA . phi X A protein complex with T4 DNA ligase in the presence or absence of DNA polymerase were unsuccessful. The phi X A protein does not act catalytically in the cleavage of phi XRFI DNA. Under conditions leading to the quantitative cleavage of phi XRFI DNA, the molar ratio of phi XRFI DNA to added phi X A protein was approximately 1:10. At this molar ratio, cross-linking experiments with dimethyl suberimidate yielded 10 distinct protein bands which were multiples of the monomeric phi X A protein. In the absence of DNA or in the presence of inactive DNA (phi XRFII DNA) no distinct protein bands above a trimer were detected. We found it possible in vitro to form a phi XRFII DNA . phi X A protein complex with wild-type phi XRFI DNA (phi X A gene+) and with phi XRFI DNA isolated from E. coli (su+) infected with phage phi X H90 (an am mutant in the phi X A gene). Thus, in vitro, in contrast to in vivo studies, phi X A protein is not a cis acting protein. The purified phi X A* protein does not substitute for the phi X A protein in in vitro replication of phi XRFI DNA nor does it interfere with the action of the phi X A protein which binds only to supertwisted phi XRFI DNA. In contrast, the phi X A* protein binds to all duplex DNA preparations tested. This property prevents nucleases of E. coli from hydrolyzing duplex DNAs to small molecular weight products.     

Initiation and termination of the bacteriophage phi X174 rolling circle DNA replication in vivo: packaging of plasmid single-stranded DNA into bacteriophage phi X174 coats.

The bacteriophage phi X174 viral (+) origin when inserted in a plasmid can interact in vivo with the A protein produced by infecting phi X174 phages. A consequence of this interaction is packaging of single-stranded plasmid DNA into preformed phage coats resulting in infective particles (1). This property was used to study morphogenesis and to analyse the signals for initiation and termination of the rolling circle DNA replication in vivo. It is shown that the size of the DNA had a strong effect on the encapsidation by the phage coats and the infectivity of the particle. Termination was analysed by using plasmids with two phi X (+) origins either in the same orientation or in opposite orientation. Both origins were used with equal frequency. Initiation at one origin resulted in very efficient termination (greater than 96%) at the second origin in the case of two origins in the same orientation. When the two (+) origins have opposite orientations, no correct termination was observed. The second origin in the opposite strand effectively inhibits (greater than 98%) the normal DNA synthesis; i.e. the covalently bound A protein present in the replication fork interacts with the (+) origin sequence in the opposite strand.     

The complete 30-base-pair origin region of bacteriophage phi X174 in a plasmid is both required and sufficient for in vivo rolling-circle DNA replication and packaging

The origin of replication of the isometric single-stranded DNA bacteriophages is located in a specific sequence of 30 nucleotides, the origin region, which is highly conserved in these phage genomes. Plasmids harboring this origin region are subject to rolling-circle DNA replication and packaging of single-stranded (ss) plasmid DNA into phage coats in phi X174 or G4-phage-infected cells. This system was used to study the nucleotide sequence requirements for rolling-circle DNA replication and DNA packaging employing plasmids which contain the first 24, 25, 26, 27, 28 and the complete 30-base-pair (bp) origin region of phi X174. No difference in plasmid ss DNA packaging was observed for plasmids carrying only the 30-bp origin region and plasmids carrying the 30-bp origin region plus surrounding sequences (i.e. plasmids carrying the HaeIII restriction fragment Z6B of phi X174 replicative-form DNA). This indicates that all signals for DNA replication and phage morphogenesis are contained in the 30-bp origin region and that no contribution is made by sequences which immediately surround the origin region in the phi X174 genome. The efficiency of packaging of plasmid ssDNA for plasmids containing deletions in the right part of the origin region decreases drastically when compared with the plasmid containing the complete 30-bp origin region (for a plasmid carrying the first 28 bp of the origin region to approximately 5% and 0.5% in the phi X174 and G4 systems respectively). Previous studies [Fluit, A.C., Baas, P.D., van Boom, J.H., Veeneman, G.H. and Jansz, H.S. (1984) Nucleic Acids Res. 12, 6443--6454] have shown that the presence of the first 27 bp of the origin region is necessary as well as sufficient for cleavage of the viral strand in the origin region by phi X174 gene A protein. Moreover, Brown et al. [Brown, D.R., Schmidt-Glenewinkel, T., Reinberg, D. and Hurwitz, J. (1983) J. Biol. Chem. 258, 8402--8412] have shown that omission of the last 2 bp of the origin region does not interfere with phi X174 rolling-circle DNA replication in vitro. Our results therefore suggest that for optimal phage development in vivo, signals in the origin region are utilized which have not yet been noticed by the in vitro systems for phi X174 phage DNA replication and morphogenesis.     

Rolling-circle replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro.

Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers. Thus, similarly to its activity on UV-irradiated single-stranded DNA, DNA polymerase III holenzyme can bypass pyrimidine photodimers in the more complex replicative form --->single-strand replication, which involves, in addition to the polymerizing activity, the unwinding of the duplex by the rep helicase and the participation of a more complex multiprotein replisome.     

The A* protein of phi X174 is an inhibitor of DNA replication

Extracts prepared from phi X174 infected E. coli cells inhibited in vitro RF replication The inhibition was dependent upon the presence of A* protein in the reaction and served as an assay to highly purify the A* protein. Purified A* protein bound tightly to duplex DNA as well as single-stranded DNA. The binding of the A* protein to duplex DNA inhibited (I) its single-stranded DNA specific endonucleolytic activity; (II) in vitro synthesis of viral (+) single stranded DNA on an A-RFII DNA complex template; (III) ATP hydrolysis by rep protein and unwinding of the strands of RF DNA. We propose that this inhibitory activity is responsible in vivo for the shut off of E. coli chromosome replication during phi X174 infection, and has a role in the transition from semiconservative RF DNA replication to single-stranded DNA synthesis in the life cycle of phi X174.