The role of gene A protein and E. coli rep protein in the replication of phi X 174 replicative form DNA

The gene A protein of bacteriophage phi X 174 initiates replication of super-twisted RFI DNA by cleaving the viral (+) strand at the origin of replication and binding to the 5' end. Upon addition of E. coli rep protein (single-stranded DNA dependent ATPase), E. coli single-stranded DNA binding protein and ATP, complete unwinding of the two strands occurs. Electron microscopic analyses of intermediates in the reaction reveal that the unwinding occurs by movement of the 5' end into the duplex, displacing the viral strand in the form of a single-stranded loop. Since unwinding will not occur in the absence of either gene A protein or rep protein, it is presumed that the rep protein interacts to form a complex with the bound gene A protein. Single-stranded DNA binding protein facilitates the unwinding by binding to the exposed single-stranded DNA. Further addition of the four deoxyribotriphosphates and DNA polymerase III holoenzyme to the reaction results in synthesis of viral (+) single-stranded circles in amounts exceeding that of the input template. A model describing the role of gene A protein and rep protein in duplex DNA replication is presented and other properties of gene A protein discussed.     

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.     

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.     

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.     

Rep protein as a helicase in an active, isolatable replication fork of duplex phi X174 DNA

Rep protein as a helicase combines its actions with those of gene A protein and single-stranded DNA binding protein to separate the strands of phi X174 duplex DNA and thereby can generate and advance a replication fork (Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197). Tritium-labeled rep protein is bound in an active gene A protein. phi X174 closed circular duplex supercoiled DNA complex in a 1:1 ratio. Catalytic separation of the strands of the duplex by rep protein, as measured by incorporation of tritium-labeled single-stranded DNA binding protein, requires ATP at a Km value of 8 microM, and hydrolyzes two molecules of ATP for every base pair melted. When coupled to replication in the synthesis of single-strand viral circles, a "looped" rolling-circle intermediate is formed that can be isolated in an active form containing gene A protein, rep protein, single-stranded DNA binding protein, and DNA polymerase III holoenzyme. Unlike the binding of rep protein to single-stranded DNA, where its ATPase activity is distributive, binding to the replicating fork is not affected by ATP, further suggesting a processive action linked to gene A protein. Limited tryptic hydrolysis of rep protein abolishes its replicative activity without affecting significantly its binding of ATP and its ATPase action on single-stranded DNA. These results augment earlier findings by describing the larger role of rep proteins as a helicase, linked in a complex ith other proteins, at the replication fork of a duplex DNA.     

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.     

Purification of the rep protein of Escherichia coli. An ATPase which separates duplex DNA strands in advance of replication.

The product of the rep gene of Escherichia coli catalytically separates phiX174 duplex DNA strands in advance of their replication, utilizing ATP in the process (Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197). The enzyme has now been purified to near-homogeneity. Relatively large quantities were obtained from ColE1-plasmid-containing cells in which the enzyme level was 7 to 10 times above wild type. The assay for rep protein was based on its essential role, with phage-induced cistron A protein, in enzymatic synthesis of phage phiX174 (+) strands, using duplex circular DNA as template. The protein exhibits a molecular weight of 65,000 under denaturing and reducing conditions. The turnover number of the enzyme is approximately 6800 ATP molecules/min in strand separation as measured by extent of replication, or in an uncoupled reaction using single-stranded DNA effector.     

The bacteriophage lambda O and P protein initiators promote the replication of single-stranded DNA.

A soluble enzyme system that specifically initiates lambda dv plasmid DNA replication at a bacteriophage lambda replication origin [Wold et al. (1982) Proc. Natl. Acad. Sci. USA 79, 6176-6180] is also capable of replicating the single-stranded circular chromosomes of phages M13 and phi X174 to a duplex form. This chain initiation on single-stranded templates is novel in that it is absolutely dependent on the lambda O and P protein chromosomal initiators and on several Escherichia coli proteins that are known to function in the replication of the lambda chromosome in vivo, including the host dnaB, dnaG (primase), dnaJ and dnaK replication proteins. Strand initiation occurs at multiple sites following an O and P protein-dependent pre-priming step in which the DNA is converted into an activated nucleoprotein complex containing the bacterial dnaB protein. We propose a scheme for the initiation of DNA synthesis on single-stranded templates in this enzyme system that may be relevant to strand initiation events that occur during replication of phage lambda in vivo.     

Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. I. Replication of DNA containing an alteration in position 1 of the 30-nucleotide icosahedral bacteriophage origin

The influence of a C----G transversion at position 1 of the 30-base pair replication origin of bacteriophage phi X174 replicative form I DNA (phi X RFI) was examined in the RF----single-stranded circular DNA replication pathway catalyzed by the combined action of the purified phi X A protein, the Escherichia coli DNA polymerase III holoenzyme, rep helicase, and single-stranded DNA binding protein (Eisenberg, S., Scott, J.F., and Kornberg, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1594-1597; Reinberg, D., Zipursky, S.L., and Hurwitz, J. (1981) J. Biol. Chem. 256, 13143-13151). RFI DNA containing this transversion was cleaved to RFII by the phi X A protein as effectively as DNA containing the wild-type origin. The altered duplex DNA, however, supported replication at a slower rate (3- to 4-fold) than the wild-type DNA due to a defect in the termination and reinitiation reactions catalyzed by the phi X A protein. This defect resulted in the accumulation of DNA products containing long single strands covalently joined to the mutant DNA. These single strands were susceptible to nuclease S1 and exonuclease VII attack. The defect in the template DNA containing C----G transversion was not corrected when this mutant origin was placed on the same strand with a wild-type origin. This double-origin DNA was also replicated poorly and led to the accumulation of large products, in contrast to the products formed with RFI DNA containing two wild-type 30-base pair replication origins on the same strand.     

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.     

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.     

Studies on the biological role of DNA methylation: III Role in excision of one-genome long single-stranded phi X 174 DNA.

Accumulation of replicative intermediates of the bacteriophage phi X174 was observed in E. coli C infected cells when phage DNA methylation has been inhibited by nicotinamide or when cells were infected with a temperature-sensitive mutant in gene A. Analysis of the accumulating replicative intermediates by electron microscopy revealed that these molecules are composed of double-stranded DNA rings with multiple-genome length single-stranded "tails". These results suggest that the single 5-methylcytosine residue present in the phage DNA serves as a recognition site for the gene A protein mediating the excision of one-genome long phage DNA. This excision process is oligatory for the final maturation of the phage.     

Purification of a RecA protein analogue from Bacillus subtilis

We have identified in Bacillus subtilis an analogue of the Escherichia coli RecA protein. Its activities suggest that it has a corresponding role in general genetic recombination and in regulation of SOS (DNA repair) functions. The B. subtilis protein (B. subtilis Rec) has a Mr of 42,000 and cross-reacts with antisera raised against E. coli RecA protein. Its level is significantly reduced in the recombination-deficient recE4 mutant. B. subtilis Rec is induced 10- to 20-fold in rec+ strains following treatment with mitomycin C, whereas it is not induced in the recombination-deficient mutants recE4, recE45, and recA1. We have purified B. subtilis Rec about 2000-fold to near homogeneity and we describe its activities. It catalyzes DNA-dependent hydrolysis of dATP at a rate comparable to that of E. coli RecA protein. However, B. subtilis Rec has a negligible ATPase activity, although ATP effectively inhibits dATP hydrolysis. In the presence of dATP, B. subtilis Rec catalyzes DNA strand transfer, assayed by the conversion of phi X174 linear duplex DNA and homologous circular single-stranded DNA to replicative form II (circular double-stranded DNA with a discontinuity in one strand). ATP does not support strand transfer by this protein. B. subtilis Rec catalyzes proteolytic cleavage of E. coli LexA repressor in a reaction that requires single-stranded DNA and nucleoside triphosphate. This result suggests that an SOS regulatory system like the E. coli system is present in B. subtilis. The B. subtilis enzyme does not promote any detectable cleavage of the E. coli bacteriophage lambda repressor.     

Cloned bacteriophage phi X174 DNA sequence interferes with synthesis of the complementary strand of infecting bacteriophage phi X174.

The insertion of a particular phi X DNA sequence in the plasmid pACYC177 strongly decreased the capacity of Escherichia coli cells containing such a plasmid to propagate bacteriophage phi X174. The smallest DNA sequence tested that showed the effect was the HindII fragment R4. This fragment does not code for a complete protein. It contains the sequence specifying the C-terminal part of the gene H protein and the N-terminal part of the gene A protein, as well as the noncoding region between these genes. Analysis of cells that contain plasmids with the "reduction sequence" showed that (i) the adsorption of the phages to the host cells is normal, (ii) in a single infection cycle much less phage is formed, (iii) only 10% of the infecting viral single-stranded DNA is converted to double-stranded replicative-form DNA, and (iv) less progeny replicative form DNA is synthesized. The reduction process is phi X174 specific, since the growth of the related G4 and St-1 phages was not affected in these cells. The effect of the recombinant plasmids on infecting phage DNA shows similarity to the process of superinfection exclusion.     

Functional aspects of Escherichia coli rep helicase in unwinding and replication of DNA

The gene for Escherichia coli rep helicase (rep protein) was subcloned in a pBR plasmid and the protein overproduced in cells transformed with the hybrid DNA. The effect of purified enzyme on strand unwinding and DNA replication was investigated by electron microscopy. The templates used were partial duplexes of viral DNA from bacteriophage fd::Tn5 and reannealed DNA from bacteriophage Mu. The experiments with the two DNA species show DNA unwinding uncoupled from replication. The single-stranded phage fd::Tn5 DNA with the inverted repeat of transposon Tn5 could be completely replicated in the presence of the E. coli enzymes rep helicase, DNA binding protein I, RNA polymerase and DNA polymerase III holoenzyme. A block in the unwinding step increases secondary initiation events in single-stranded parts of the template, as DNA polymerase III holoenzyme cannot switch across the stem structure of the transposon.     

Characterization of a DNA binding protein of bacteriophage PRD1 involved in DNA replication.

Escherichia coli phage PRD1 protein P12, involved in PRD1 DNA replication in vivo, has been highly purified from E. coli cells harbouring a gene XII-containing plasmid. Protein P12 binds to single-stranded DNA as shown by gel retardation assays and nuclease protection experiments. Binding of protein P12 to single-stranded DNA increases about 14% the contour length of the DNA as revealed by electron microscopy. Binding to single-stranded DNA seems to be cooperative, and it is not sequence specific. Protein P12 also binds to double-stranded DNA although with an affinity 10 times lower than to single-stranded DNA. Using the in vitro phage phi 29 DNA replication system, it is shown that protein P12 stimulates the overall phi 29 DNA replication.     

Overproduction and purification of protein P6 of Bacillus subtilis phage phi 29: role in the initiation of DNA replication.

A phi 29 DNA fragment containing gene 6, required for DNA replication, has been cloned in plasmid pPLc28 under the control of the PL promoter of phage lambda. A polypeptide with an electrophoretic mobility close to that of p6 was labelled with 35S-methionine after heat induction. This protein, representing about 4% of the total E. coli protein after 1 h of induction, was obtained in a highly purified form. The protein was characterized as p6 by amino acid analysis and NH2-and COOH-terminal sequence determination. Protein p6 has an apparent molecular weight of 23,600, suggesting that the native form of the protein is a dimer. The purified protein p6 stimulated the protein-primed initiation of phi 29 DNA replication when added to purified proteins p2 (phi 29-coded DNA polymerase) and p3 (terminal protein).     

The ABC-primosome. A novel priming system employing dnaA, dnaB, dnaC, and primase on a hairpin containing a dnaA box sequence

A priming mechanism requiring dnaA, dnaB, and dnaC proteins operates on a single-stranded DNA coated with single-stranded DNA-binding protein. This novel priming, referred to as "ABC-priming," requires a specific hairpin structure whose stem carries a dnaA protein recognition sequence (dnaA box). In conjunction with primase and DNA polymerase III holoenzyme, ABC-priming can efficiently convert single-stranded DNA into the duplex replicative form. dnaA protein specifically recognizes and binds the single-stranded hairpin and permits the loading of dnaB protein to form a prepriming protein complex containing dnaA and dnaB proteins which can be physically isolated. ABC-priming can replace phi X174 type priming on the lagging strand template of pBR322 in vitro, suggesting a possible function of ABC-priming for the lagging strand synthesis and duplex unwinding. Similar to the phi X174 type priming, a mobile nature of ABC-priming was indicated by helicase activity in the presence of ATP of a prepriming protein complex formed at the hairpin. The implications of this novel priming in initiation of replication at the chromosomal origin, oriC, and in its contribution to the replication fork are discussed.     

phi X174-directed DNA and protein syntheses in infected minicells.

Phi X174-infected minicells, produced by Escherichia coli PC2251, synthesized 11 phi X174-encoded polypeptides. The infecting single-stranded viral genome was converted to a double-stranded, closed circular, replicative form (replicative form I). Little, if any, replicative form I replication took place, and synthesis of progeny single-stranded molecules could not be detected.