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.     

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.     

The enzymatic replication of DNA.

Enzymatic mechanisms of DNA replication have been investigated using small bacteriophages as probes to illuminate the cellular systems upon which they must rely during infection. Conversion of the circular, single-stranded DNAs of phages M13, G4, and phi X174 to their duplex forms has revealed the participation of diverse ways to start a new chain and a complex DNA polymerase III holoenzyme upon which all these systems depend for chain elongation. The phi X174 system, which is the most exacting and revealing of the host chromosomal replication pattern, includes at least twenty polypeptides for making the viral DNA into a duplex and multiplying the duplex. Resolution and purification of these numerous proteins is in train and their reconstitution into a "replisome"-like structure is envisioned.     

Effect of SSB protein on cleavage of single-stranded DNA by phi X gene A protein and A* protein.

Gene A protein of bacteriophage phi X174 plays a role as a site-specific endonuclease in the initiation and termination of phi X rolling circle DNA replication. To clarify the sequence requirements of this protein we have studied the cleavage of single-stranded restriction fragments from phi X and G4 viral DNAs using purified gene A protein. The results show that in both viral DNAs cleavage occurs at the origin and at one additional site which shows striking sequence homology with the origin region. During rolling circle replication the single-stranded viral DNA tail is covered with single-stranded DNA binding (SSB) protein. Therefore, we have also studied the effect of SSB on phi X gene A protein cleavage. In these conditions only single-stranded fragments containing the complete or almost complete origin region of 30 bases are cleaved, whereas cleavage at the additional sites of phi X or G4 viral DNAs does not occur. A model for termination of rolling circle replication which is based on these findings is presented. Finally, we present evidence that the second product of gene A, the A* protein, cleaves phi X viral DNA at the additional cleavage site in the presence of SSB, not only in vitro but also in vivo. The functional significance of this cleavage in vivo is discussed.     

The nuclease specificity of the bacteriophage phi X174 A* protein.

The A* protein of bacteriophage phi X174 is a single-stranded DNA specific nuclease. It can cleave phi X viral ss DNA in many different places. The position of these sites have been determined within the known phi X174 nucleotide sequence (1). From the sequences at these sites it is clear that the A* protein recognizes and cleaves at sites that show only partial homology with the origin of RF DNA replication in the phi X DNA. Different parts of the origin sequence can be deduced that function as a signal for recognition and cleavage by the A* protein. We conclude that different parts within the DNA recognition domain of the A* protein are functional in the recognition of the origin sequence in single-stranded DNA. The existence of different DNA recognition domains in the A* protein, and therefore also in the A protein, leads to a model that can explain how the A protein performs its multiple function in the phi X174 DNA replication process (2).     

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.     

Regions of incompatibility in single-stranded DNA bacteriophages phi X174 and G4.

The intracellular presence of a recombinant plasmid containing the intercistronic region between the genes H and A of bacteriophage phi X174 strongly inhibits the conversion of infecting single-stranded phi X DNA to parental replicative-form DNA. Also, transfection with single-stranded or double-stranded phi X174 DNA of spheroplasts from a strain containing such a "reduction" plasmid shows a strong decrease in phage yield. This phenomenon, the phi X reduction effect, was studied in more detail by using the phi X174 packaging system, by which plasmid DNA strands that contain the phi X(+) origin of replication were packaged as single-stranded DNA into phi X phage coats. These "plasmid particles" can transduce phi X-sensitive host cells to the antibiotic resistance coded for by the vector part of the plasmid. The phi X reduction sequence in the resident plasmid strongly affected the efficiency of the transduction process, but only when the transducing plasmid depended on primosome-mediated initiation of DNA synthesis for its conversion to double-stranded DNA. The combination of these results led to a model for the reduction effect in which the phi X reduction sequence interacted with an intracellular component that was present in limiting amounts and that specified the site at which phi X174 replicative-form DNA replication takes place. The phi X reduction sequence functioned as a viral incompatibility element in a way similar to the membrane attachment site model for plasmid incompatibility. In the DNA of bacteriophage G4, a sequence with a similar biological effect on infecting phages was identified. This reduction sequence not only inhibited phage G4 propagation, but also phi X174 infection.     

An international collaborative study of ''genetic drift'' in Salmonella typhimurium strains used in the Ames test

The efficiency of Weigle reactivation of ultraviolet light-irradiated single and double-stranded phi X174 DNA by wild-type and excision repair-defective E. coli hosts was determined. After limited exposure to ultraviolet light, the efficiency of Weigle reactivation by an ultraviolet light-irradiated wild-type host was greater for double-stranded phi X174 DNA than for its single-stranded counterpart. However, the efficiency of inducible recovery of the double-stranded DNA molecule decreased as its exposure to ultraviolet light increased until it became constant at a value 1.5 times less than that for single-stranded form of phi X174 DNA. The efficiency of Weigle reactivation of the single-stranded DNA molecule by the same host, however, was independent of the dose to the DNA, as were the efficiencies of reactivation for both forms of phi X174 DNA by ultraviolet light-irradiated excision repair-deficient hosts. In excision repair-defective hosts the efficiency of Weigle reactivation of double-stranded phi X174 DNA was also 1.5 times less than that for the single-stranded molecule. These results suggest that the Weigle reactivation of double-stranded phi X174 DNA is mediated in part by an excision repair process, and that this component of Weigle reactivation eventually can be saturated by ultraviolet light-induced DNA damage leaving other repair processes, such as trans-damage synthesis, responsible for the remaining inducible reactivation.     

Studies of the recognition sequence of phi X174 gene A protein. Cleavage site of phi X gene A protein in St-1 RFI DNA.

It is already known that phi X gene A protein converts besides phi X RFI DNA also the RFI DNAs of the single-stranded bacteriophages G4, St-1, alpha 3 and phi K into RFII DNA. We have extended this observations for bacteriophages G14 and U3. Restriction enzyme analysis placed the phi X gene A protein cleavage site in St-1 RF DNA in the HinfI restriction DNA fragment F10 and in the overlapping HaeIII restriction DNA fragment Z7. The exact position and the nucleotide sequence at the 3'-OH end of the nick were determined by DNA sequence analysis of the single-stranded DNA subfragment of the nicked DNA fragment F10 obtained by gelelectrophoresis in denaturing conditions. A stretch of 85 nucleotides of St-1 DNA around the position of the phi X gene A protein cleavage site was established by DNA sequence analysis of the restriction DNA fragment Z7F1. Comparison of this nucleotide sequence with the previously determined nucleotide sequence around the cleavage site of phi X gene A protein in phi X174 RF DNA and G4 RF DNA revealed an identical sequence of only 10 nucleotides. The results suggest that the recognition sequence of the phi X174 gene A protein lies within these 10 nucleotides.     

Characterization of an endonuclease activity associated with Friend-murine leukemia virus

An endonuclease activity shown to be associated with Friend leukemia virus has been characterized using double-stranded phi X174 DNA as substrate. In the presence of Mg2+, the endonuclease activity was able to convert supercoiled circular DNA duplexes to the relaxed form by introducing single-stranded nicks into the DNA. Most of the nicked DNA duplexes contained only one nick per strand, since unit length DNA was the predominant species obtained when the nicked DNA was analyzed by alkaline sucrose gradient centrifugation. The regions into which the nick could be introduced were evenly distributed around the circular DNA molecule. When Mn2+ was substituted for Mg2+ in the reaction mixture, the number of nicks introduced into circular DNA duplexes by the virus associated endonuclease was greatly increased. In contrast to circular duplexes, linear duplexes and single-stranded DNA functioned poorly as substrates for the virus-associated enzyme. The Friend leukemia virus-associated endonuclease activity is with respect to these characteristics very similar to the endonuclease activity associated with the p32 protein of the avian myeloblastosis virus [1]. The molecular weight of the Friend leukemia virus endonuclease was estimated by gel filtration on a Sephacryl S-200 column to be about 45 000.     

Cleavage of single-stranded DNA by the A and A* proteins of bacteriophage phi X174.

The purified A protein and A* protein of bacteriophage phi X174 have been tested for endonuclease activity on single stranded viral phi X174 DNA. The A protein (55.000 daltons) nicks single-stranded DNA in the same way and at the same place as it does superhelical RFI DNA, at the origin of DNA replication. The A* protein (37.000 daltons) can cleave the single-stranded viral DNA at many different sites. It has however a strong preference for the origin of replication. Both proteins generate 3'OH ends and blocked 5' termini at the nick site.     

DNA synthesis in Escherichia coli cells infected with gene H mutants of bacteriophage phi X174.

Escherichia coli cells infected with gene H mutants of bacteriophage phi X174 produce two types of particles. The 110S particles contain single-stranded circular DNA; the 110S particles are not infectious, although their DNA is infectious for E. coli spheroplasts. The second type of particles, 70S particles, contain a fragment of single-stranded DNA ranging from 0.2 to 0.5 genome in length. This fragment DNA anneals only to restriction enzyme fragments of replicative-form DNA from the portion of the molecule corresponding to the origin and early region of phi X174 single-stranded synthesis, although full-round single-stranded DNA synthesis is occurring in the H mutant-infected cells. Different H mutant phages produce different proportions of 70S to 110S particles; those mutants producing the most 70S also exhibit the largest amount of degradation of intracellularly labeled DNA during infection. These results suggest that in H mutant-infected cells, full-length single-stranded DNA is synthesized; varying amounts of degradation of the single-stranded material occur, and the resulting fragment DNA is subsequently incorporated into 70S particles.     

Effects of genome size on bacteriophage phi X174 DNA packaging in vitro

Effects of the size of template DNA on the DNA packaging reaction of bacteriophage phi X174 were studied using plasmids of various sizes which contain the phi X174 origin of DNA replication and the in vitro phage synthesizing system (Aoyama, A., Hamatake, R. K., and Hayashi, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4195-4199). DNA between 78.5% and 101% of the length of phi X174 DNA produced infectious particles efficiently. Packaging of DNA smaller or larger than this range produced uninfectious defective particles. Although these particles contained circular single-stranded DNA, they suffered structural changes which altered the sedimentation properties or the ability to adsorb to the cells. Mutant phage were found from the packaging reaction of DNA larger than 101% of phi X174 DNA. These mutants deleted the termination region of DNA, suggesting that they were produced by early termination of the phage synthesizing reaction.     

Construction and characterization of recombinant plasmid DNAs containing sequences of the origin of bacteriophage phi X174 DNA replication

The synthetic DNA fragment (formula, see text) (corresponding to nucleotides 4299-4314 of the phi X DNA sequence) was cloned into either the AmpR gene or the KmR gene of plasmid pACYC 177. The DNA sequence of the KmR gene around the insertion site was determined by nucleotide sequence analysis of the pACYC 177 FnudII restriction DNA fragment N6 (345 b.p.). Of five selected plasmid DNAs, which contained inserted DNA sequences in the antibiotic resistance genes, the nucleotide sequences at and around these insertions were determined. Two recombinant plasmids (pFH 704 and pFH 614) contain the hexadecamer sequence in tandem (tail-to-tail and tail-to-head). In the recombinant plasmids pFH 812, pFH 903 and pFH 807 the DNA sequence homology with the phi X origin region was 14 (No. 4300-4313), 16 (No. 4299-4314) and 20 nucleotides (No. 4299-4318), respectively. None of the supercoiled recombinant plasmid DNAs is nicked upon incubation with phi X gene A protein. Moreover, the recombinant plasmid RFI DNAs cannot act as substitutes for phi X RFI DNA in the in vitro (+) strand synthesizing system. It has been shown earlier that single-stranded DNA, which contains the decamer sequence CAACTTGATA is efficiently nicked by the phi X gene A protein. The present results indicate that for nicking of double-stranded supercoiled DNA nucleotide sequence homology with the phi X origin region of more than 20 nucleotides is required. These results suggest a model for initiation of phi X RF DNA replication, which involves the presence of the recognition sequence CAACTTGATA of the phi X gene A protein as well as a second specific nucleotide sequence which is required for the binding of the phi X gene A protein. This binding causes local unwinding of the DNA double helix and exposure of the recognition sequence in a single-stranded form, which then can be nicked by phi X gene A protein.     

Process of attachment of phi X174 parental DNA to the host cell membrane

The phi X174-DNA membrane complex was isolated from Escherichia coli infected with phi X174 am3 by isopycnic sucrose gradient centrifugation followed by zone electrophoresis. The phi X174 DNA-membrane complex banded at two positions, intermediate density membrane fraction and cytoplasmic membrane fraction, having bouyant densities of 1.195 and 1.150 g/ml, respectively. Immediately after infection with phi X147, replicating DNA was pulse-labeled and then the incorporated label was chased. The radioactivity initially recovered in the intermediate density membrane fraction migrated to the cytoplasmic membrane fraction. The DNAs from both complexes sedimented mainly at the position of parental replicative form I (RFI). The phi X174 DNA-membrane complex contained a speficic membrane-bound protein having a molecular weigth of 80,000 which is accumulated in the host DNA-membrane complex. These results suggest that when phi X174 DNA penetrated into cells in the early phase of infection, single-stranded circular DNA was converted to parental RFI at a wall/membrane adhesion region and migrated to the cytoplasmic membrane fraction, where the parental RF could serve as a template in the replication of progeny RF.     

Uncoupling the DNA cleavage and religation activities of topoisomerase II with a single-stranded nucleic acid substrate: evidence for an active enzyme-cleaved DNA intermediate

Following its cleavage of double-stranded DNA, topoisomerase II is covalently bound to the 5'-termini of both nucleic acid strands. However, in order to isolate this enzyme-cleaved DNA complex in the presence of magnesium (the enzyme's physiological divalent cation), reactions must be terminated by the addition of a strong protein denaturant such as sodium dodecyl sulfate (SDS). Because of the requirement for a protein denaturant, it is unclear whether DNA cleavage in this in vitro system takes place prior to or is induced by the addition of SDS. To distinguish between these two possibilities, experiments were carried out to determine whether topoisomerase II bound DNA contains 3'-OH termini prior to denaturation. This was accomplished by using circular single-stranded phi X174 DNA as a model substrate for the enzyme. As found previously for topoisomerase II mediated cleavage of double-stranded DNA, the enzyme was covalently linked to the 5'-termini of cleaved phi X174 molecules. Moreover, optimal reaction pH as well as optimal salt and magnesium concentrations was similar for the two substrates. In contrast to results with double-stranded molecules, single-stranded DNA cleavage increased with time, was not salt reversible, and did not require the presence of SDS. Furthermore, cleavage products generated in the absence of protein denaturant could be labeled at their 3'-OH DNA termini by incubation with terminal deoxynucleotidyltransferase and [alpha-32P]ddATP. Finally, cleaved phi X174 molecules could be joined to a radioactively labeled double-stranded oligonucleotide by a topoisomerase II mediated intermolecular ligation reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Replication of phi X174 dna with purified enzymes. I. Conversion of viral DNA to a supercoiled, biologically active duplex

Conversion of phi X174 viral, single-stranded circular DNA to the duplex replicative form (RF), previously observed with partially purified enzymes, has now been demonstrated with the participation of 12 nearly pure Escherichia coli proteins containing approximately 30 polypeptides. To complete the synthesis of a full length complementary strand, E. coli DNA polymerase I was needed to fill the short gap left by DNA polymerase III holoenzyme, and to remove the primer and replace it with DNA. Production of supercoiled RF required the further actions of E. coli DNA ligase and gyrase. Net synthesis of viral circles was obtained by coupling the formation of RF supercoils to the actions of the phi X174-encoded gene A protein and E. coli rep protein. Viral DNA circles produced from enzymatically synthesized supercoiled RF, serving as template-substrate, were indistinguishable from those produced from RF isolated from infected cells; synthetic RF and the viral circles generated from it by replication were as biologically active in transfection of spheroplasts as the forms obtained from infected cells and virions. The conversion of single-stranded circular DNA to RF is suggested here as a model for discontinuous synthesis of the lagging strand of the E. coli chromosome. The primosome, a complex of some of the replication proteins responsible for initiations of DNA chains, will be described elsewhere. Multiplication of RF supercoils, described in the succeeding paper, proceeds by a rolling-circle mechanism in which the synthesis of viral strands may have analogies to the continuous synthesis of the leading strand of the E. coli chromosome.     

Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. I. Characterization of the termination and reinitiation reactions

The phi X174 (phi X) gene A protein-mediated termination and reinitiation of single-stranded circular (SS(c] phi X viral DNA synthesis in vitro were directly and independently analyzed. Following incubation together with purified DNA replication enzymes from Escherichia coli, ATP, [alpha-32P]dNTPs, and either the phi X A protein and phi X replicative form I (RF I) DNA, or the purified RF II X A complex, the phi X A protein was detected covalently linked to newly synthesized 32P-labeled DNA. Formation of the phi X A protein-[32P]DNA covalent complex required all the factors necessary for phi X (+) SS(c) DNA synthesis in vitro. Thus, it was a product of the reinitiation reaction and an intermediate of the replication cycle. Identification of this complex provided direct evidence that reinitiation of phi X (+) strand DNA synthesis involved regeneration of the RF II X A complex. Substitution of 2',3'-dideoxyguanosine triphosphate (ddGTP) for dGTP in reaction mixtures resulted in the formation of covalent phi X A protein 32P-oligonucleotide complexes; these complexes were trapped analogues of the regenerated RF II X A complex. They could not act catalytically due to the presence of ddGMP residues at the 3'-termini of the oligonucleotide moieties. Reaction mixtures containing ddGTP also yielded nonradioactive (+) SS(c) DNA products derived from circularization of the displaced (+) strand of the input parental template DNA. The formation of the phi X A protein-32P-oligonucleotide complexes and nonradioactive (+) SS(c) DNA were used to assay both reinitiation and termination reactions, respectively. Both reactions required DNA synthesis from the 3'-hydroxyl primer at nucleotide residue 4305 which was formed by cleavage of phi X RF I DNA by the phi X A protein. Elongation of this primer by 18, but not 11 nucleotides was sufficient to support each reaction. Reinitiation reactions proceeded rapidly and were essentially complete after 90 s. In contrast, when ddGTP was replaced with dGTP in reaction mixtures, DNA synthesis proceeded with linear kinetics for up to 10 min. These results suggested that in the presence of all four dNTPs, active templates supported more than 40 rounds of DNA synthesis.     

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.     

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.