Enzymatic replication of viral and complementary strands of duplex DNA of phage phiX174 proceeds by seprate mechanisms.

Multiplication of the duplex, circular, phage phiX174DNA (replicative form, RF) in stage II of the replicative life cycle has been observed with a crude enzyme preparation [Eisenberg et al. (1976) Proc, Natl. Acad, Sci. USA 73, 1594-1597]. This stage has now been partially reconstituted with purified proteins and subdivided into two stages: II(+) and II(-). In stage II(+), viral (+) strand synthesis is carried out by four proteins: the phage-induced, cistron A-dependent protein, rep-dependent protein, DNA unwinding protein, and DNA polymerase III holenzyme. In stage II(-), complementary (-) strand synthesis utilizes the product of stage II(+) as template and the multiprotein system previously identified in the stage I synthesis of a complementary strand on the viral DNA template to produce RF. The multiprotein system includes DNA unwinding protein, proteins i and n, dnaB protein, dnaC protein, dnaG protein, and DNA polymerase III holoenzyme. A discussion of these two separate mechanism for synthesis of (+) and (-) strands suggests that they may account for essentially all the replicative stages in the life cycle of phiX174.     

Enzyme-catalyzed DNA unwinding: Studies on Escherichia coli rep protein

Replication in vitro of the replicative form (RF) I DNA of bacteriophage varphiX174 requires the phage-induced cistron A (cisA) protein, the host rep protein, DNA-binding protein, ATP, and DNA polymerase III plus replication factors. The rep protein is a single-stranded DNA-dependent ATPase. In this paper we show that varphiX174 RF I DNA cut by the cisA protein acts as a duplex DNA cofactor for the rep protein ATPase activity, provided that DNA-binding protein is present. In this latter reaction the duplex DNA is unwound by the rep protein with concomitant hydrolysis of ATP. The extents of ATP hydrolysis, DNA unwinding, and, where appropriate, DNA synthesis are proportional to the amounts of DNA-binding protein present. Two ATP molecules are hydrolyzed per base pair unwound. We propose that the obligatory requirement for the cisA protein in the unwinding of varphiX174 RF I DNA is not simply due to its endonuclease activity but rather is due to its provision of a site for the binding of the rep protein. The rep protein in the presence of DNA-binding protein, but in the absence of cisA protein, unwinds duplex DNA when one strand extends to generate a single-stranded leader region preceding the duplex. We show that rep protein translocates along the leader single strand in a 5'-to-3' direction only and then invades the duplex DNA. The rep protein shows a directional specificity for translocation and unwinding. A model is presented to explain the mechanism of DNA unwinding catalyzed by the rep protein.     

Conservation of the primosome in successive stages of phi X174 DNA replication.

Synthesis of a complementary strand to match the single-stranded, circular, viral (+) DNA strand of phage phi X174 creates a parental duplex circle (replicative form, RF). This synthesis is initiated by the assembly and action of a priming system, called the primosome [Arai, K. & Kornberg, A (1981) Proc. Natl. Acad. Sci. USA 78, 69-73; Arai, K., Low, R. L. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 707-711]. Of the seven proteins that participate in the assembly and function of the primosome, most all of the components remain even after the DNA duplex is completed and covalently sealed. Remarkably, the primosome in the isolated RF obviates the need for supercoiling of RF by DNA gyrase, an action previously considered essential for the site-specific cleavage by gene A protein that starts viral strand synthesis in the second stage of phi X174 DNA replication. Finally, priming of the synthesis of complementary strands on the nascent viral strands to produce many copies of progeny RF utilizes the same primosome, requiring the addition only of prepriming protein i. thus a single primosome, which becomes associated with the incoming viral DNA in the initial stage of replication, may function repeatedly in the initiation of complementary strands at the subsequent stage of RF multiplication. These patterns of phi X174 DNA replication suggest that a conserved primosome also functions in the progress of the replicating fork of the Escherichia coli chromosome, particularly in initiating the synthesis of nascent (Okazaki) fragments.     

recA protein of Escherichia coli promotes branch migration, a kinetically distinct phase of DNA strand exchange.

The recA protein of Escherichia coli promotes the complete exchange of strands between full-length linear duplex and single-stranded circular DNA molecules of bacteriophage phi X-174, converting more than 50% of the single-stranded DNA into heteroduplex replicative form II-like structures. Kinetically, the reaction can be divided into two phases, formation of short heteroduplex regions (D loops) and extension of the D loops via branch migration. recA protein participates directly in both phases. D loops are formed efficiently in the presence of ATP or the nonhydrolyzable ATP analog adenosine 5'-[gamma-thio]triphosphate, whereas D-loop extension requires continuous ATP hydrolysis. Complete strand exchange requires a stoichiometric amount of recA protein and is strongly stimulated by the single-stranded-DNA-binding protein of E. coli.     

ϕX174 cistron A protein is a multifunctional enzyme in DNA replication

The cistron A protein induced by phage varphiX174 nicks (produces a single-strand break in) the viral strand of the superhelical varphiX duplex DNA, thereby forming a complex with the DNA. The protein, seen bound to the DNA in the electron microscope, was located in the restriction endonuclease fragment between nucleotides 4290 and 4330 on the varphiX map [Sanger, F., Air, G. M., Barrel, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison, C. A., III, Slocomb, P. M. Y. & Smith, M. (1977) Nature 265, 687-695]. Replication also was initiated at this point, thus identifying the site of cistron A protein nicking and binding as the origin of replication.The cisA-DNA complex (separated from free cistron A protein), upon the addition of Escherichia coli rep protein, ATP, and DNA binding protein, is unwound to generate a single-stranded linear [presumably the nicked (+) strand] and a circular [presumably the (-) strand] molecule. The cisA-DNA complex, upon the further addition of DNA polymerase III holoenzyme and deoxynucleoside triphosphates, supports replication to generate viral, single-stranded circles, as many as 15 circles per cisA-DNA complex.The replicating intermediates seen in the electron microscope are a novel form of "rolling circle" [Gilbert, W. & Dressler, D. H. (1969) Cold Spring Harbor Symp. Quant. Biol. 33, 473-485]. The 5' end (presumably with the cistron A protein bound to it) is locked in the replication fork and loops back to accompany the strand-separation and replication fork around the template [(-) strand] circle. Thus, the multiple functions of cistron A protein include: (i) nicking the viral strand at the origin of replication to initiate a round of replication, (ii) participating in a complex which supports fork movement in strand separation and replication, (iii) nicking again at the regenerated origin to produce a unit-length DNA, and (iv) ligating the newly generated 3'-OH end to the 5'-phosphate-complexed end to form a circular viral molecule.     

Initiation of general recombination catalyzed in vitro by the recA protein of Escherichia coli.

Homogeneous recA protein catalyzes the hybridization of single-stranded DNA to homologous regions in duplex DNA. The products are D-loops, which are formed with equal efficiency in linear and supercoiled molecules. This assimilation reaction can be separated into two partial reactions. In the first, recA protein binds to duplex DNA and produces a reA protein-DNA complex. The binding shows a sigmoidal dependence on recA protein concentration, requires ATP, GTP or the gamma-thio analog of ATP, and Mg2+, but does not require hydrolysis of the nucleoside triphosphate. In the second reaction, single-stranded regions of the recA protein-ATP-duplex DNA intermediate hybridize with free complementary single strands to produce D-loop structures. This reaction is coupled to ATP hydrolysis and is analogous to the renaturation of single-stranded DNA catalyzed by the recA protein [Weinstrock, G.M., McEntee, K. & Lehman, I.R. (1979) Proc. Natl. Acad. Sci. USA 76, 126-130]. Hydrolysis of ATP appears to be required in these reactions for dissociation of recA protein from the DNA.     

Purification and characterization of an activity from Saccharomyces cerevisiae that catalyzes homologous pairing and strand exchange.

An activity that catalyzes the formation of joint molecules from linear M13mp19 replicative form DNA and circular M13mp19 viral DNA was purified 1000- to 2000-fold from mitotic Saccharomyces cerevisiae cells. The activity appeared to reside in a Mr 132,000 polypeptide. The reaction required that the substrates be homologous and also required Mg2+. There was no requirement for ATP. The reaction required stoichiometric amounts of protein and showed a cooperative dependence on protein concentration. Electron microscopic analysis of the joint molecules indicated they were formed by displacement of one strand of the linear duplex by the single-stranded circular molecule. This analysis also showed that heteroduplex formation started at the 3'-homologous end of the linear duplex strand followed by extension of the hybrid region toward the 5'-homologous end of the linear duplex strand (3'-to-5' direction).     

In vitro synthesis of bacteriophage phi X174 by purified components.

An in vitro system capable of synthesizing infectious phi X174 phage particles was reconstituted from purified components. The synthesis required phi X174 supercoiled replicative form DNA, phi X174-encoded proteins A, C, J, and prohead, Escherichia coli DNA polymerase III holoenzyme, rep protein, and deoxyuridinetriphosphatase (dUTPase, dUTP nucleotidohydrolase, EC 3.6.1.23) as well as MgCl2, four deoxyribonucleoside triphosphates, and ATP. Phage production was coupled to the synthesis of viral single-stranded DNA. More than 70% of the synthesized particles sedimented at the position of mature phage in a sucrose gradient and associated with the infectivity. The simple requirement of the host proteins suggests that the mechanism of viral strand synthesis in the phage-synthesizing reaction resembles that of viral strand synthesis during the replication of replicative form DNA.     

Synthesis of phiX174 viral DNA in vitro depends on phiX replicative form DNA.

A cell-free system that catalyzes phiX174 replicative form I (supercoiled circular duplex, RFI)-dependent phiX174 DNA synthesis has been isolated from Escherichia coli infected with phiX174 phage. The products formed with such preparations are viral strands as judged by hybridization to poly(U,G) followed by equilibrium centrifugation in CsCl. This phiX174 DNA-synthesizing involves formation of DNA-protein complexes that sediment in neutral sucrose with S values of 50, 60-70, and higher. The 50S complex contained a rolling-circle replicative intermediate DNA with an extended tail of single-stranded viral DNA. The DNA contained in the 60-70S region was a mixture of circular and linear single-stranded DNA, RFI, and RFII with an extended single-stranded tail. Such complexes have been isolated during in vivo progeny phiX174 DNA synthesis [Fujisawa, H. & Hayashi, M. (1976) J. Vriol. 19,409]. In vitro, maximal phiX174 DNA synthesis was shown to require the genetically defined proteins E. coli dna B, dna C, dna G, dna Z, rep. phiX174 gene A product, and other phiX174 coded proteins. The synthesis of phiX174 DNA is ATP-dependent and is inhibited by nalidixic acid and novobiocin but is resistant to rifampicin.     

Escherichia coli host factor required specifically for the phi X174 stage III reaction: in vitro identification and partial purification.

A cell-free extract prepared from phi X174-infected Escherichia coli cells sustained in vitro synthesis of viral DNA (stage III reaction) when supplemented with fraction II from uninfected cells. The reaction was dependent upon deoxyribonucleoside triphosphate, ATP, added phi X174 replicative form I DNA template, and the fraction II from uninfected cells. This reaction differed from the stage II reaction (semiconservative replication of duplex replicative form DNA) by the production of stable viral protein-DNA complexes sensitive to anti-phi X174 antiserum. Three types of protein-DNA complexes were identified, 50S, 92S, and a 114S complex that cobanded in CsCl and cosedimented in neutral sucrose gradients with a phi X174 phage marker. The sensitivity of these complexes to anti-phi X174 antiserum and Staphylococcus aureus provided a relatively rapid biochemical assay for direct measurement of the amount of DNA synthesized by the stage III reaction. With this assay, an E. coli factor (SIII) required specifically for the synthesis of viral protein-DNA complexes was identified and purified 200-fold from uninfected E. coli cells. The partially purified SIII factor was required for the synthesis of DNA and viral protein-DNA complexes in the phi X174-infected cell extracts and could not be replaced by rep protein, single-strand binding protein, or DNA polymerase III holoenzyme.     

Recombinational bypass of pyrimidine dimers promoted by the recA protein of Escherichia coli.

recA protein, in the presence of single-stranded DNA binding protein and ATP, promotes the complete exchange of strands between circular single-stranded DNA containing pyrimidine dimers and a homologous linear duplex, converting the pyrimidine dimer-containing single-stranded DNA to a circular duplex. Bypass of a pyrimidine dimer during the branch-migration phase of the reaction requires approximately 20 seconds, a rate 1/50th of that in the absence of the dimer. The circular duplex product is specifically incised by the pyrimidine dimer-specific T4 endonuclease V, and the resulting 3' hydroxyl termini can serve as primers for deoxynucleotide polymerization by DNA polymerase I. These findings indicate that recA protein serves a direct role in recombinational repair and demonstrate that the pyrimidine dimers that have been bypassed can be processed by enzymes of the excision-repair pathway.     

Role of DNA gyrase in phiX replicative-form replication in vitro.

Preparations containing DNA gyrase activity Gellert, M., Mizuchi, K., O'Dea, M.H. & Nash, H.A. (1976) Proc. Natl. Acad. Sci. USA 73, 3872-3876] have been extensively purified from Escherichia coli. Such fractions, in the presence of ATP and Mg2+, catalyze supertwisting of relaxed circular double-stranded DNA replicative forms of a number of DNAs that results in the formation of superhelical replicative forms. Relaxed phiX174 replicative form (phiX RFIV) is not attacked by the A protein endonuclease coded for by the phiX DNA genome. After exposure to preparations of DNA gyrase, the relaxed phiX174 replicative form is converted to phiX RFI which can then be attacked by the phiX gene A protein and participate in replication of duplex phiX DNA.     

3'' homologous free ends are required for stable joint molecule formation by the RecA and single-stranded binding proteins of Escherichia coli.

The RecA protein of Escherichia coli is important for genetic recombination in vivo and can promote synapsis and strand exchange in vitro. The DNA pairing and strand exchange reactions have been well characterized in reactions with circular single strands and linear duplexes, but little is known about these two processes using substrates more characteristic of those likely to exist in the cell. Single-stranded linear DNAs were prepared by separating strands of duplex molecules or by cleaving single-stranded circles at a unique restriction site created by annealing a short defined oligonucleotide to the circle. Analysis by gel electrophoresis and electron microscopy revealed that, in the presence of RecA and single-stranded binding proteins, a free 3' homologous end is essential for stable joint molecule formation between linear single-stranded and circular duplex DNA.     

Stable three-stranded DNA made by RecA protein.

When RecA protein, in the form of a nucleoprotein filament containing circular single-stranded DNA (plus strand only), reacts with homologous linear duplex DNA, a directional transfer ensues of a strand from the duplex DNA to the nucleoprotein filament, resulting in the displacement of the linear plus strand in the 5' to 3' direction. The initial homologous synapsis, however, can occur at either end of the duplex DNA, or anywhere in between, and when homology is restricted to different regions of the duplex DNA, the joint molecules that form in each region show striking differences in stability upon deproteinization: distal joints greater than proximal joints much greater than medial joints. In the deproteinized distal joints, which are thermostable, 2000 nucleotide residues of the circular plus strand are resistant to P1 nuclease; both strands of the original duplex DNA remain resistant to P1 nuclease, and the potentially displaceable linear plus strand, which has a 3' homologous end, remains resistant to Escherichia coli exonuclease I. These observations suggest that RecA protein promotes homologous pairing and strand exchange via long three-stranded DNA intermediates and, moreover, that, once formed, such triplex structures in natural DNA are stable even when RecA protein has been removed.     

recA protein-catalyzed strand assimilation: stimulation by Escherichia coli single-stranded DNA-binding protein.

The single-stranded DNA-binding protein of Escherichia coli significantly alters the strand assimilation reaction catalyzed by recA protein [McEntee, K., Weinstock, G. M. & Lehman, I. R. (1979) Proc. Natl. Acad. Sci. USA 76, 2615--2619]. The binding protein (i) increases the rate and extent of strand assimilation into homologous duplex DNA, (ii) enhances the formation of a complex between recA protein and duplex DNA in the presence of homologous or heterologous single-stranded DNA, (iii) reduces the rate and extent of ATP hydrolysis catalyzed by recA protein in the presence of single-stranded DNA, (iv) reduces the high concentration of recA protein required for strand assimilation, and (v) permits detection of strand assimilation in the presence of the ATP analog, adenosine 5'-O-(O-thiotriphosphate). Single-stranded DNA-binding protein purified from a binding protein mutant (lexC) is considerably less effective than wild-type binding protein in stimulating strand assimilation, a result which suggests that single-stranded DNA-binding protein participates in general recombination in vivo.     

Unwinding associated with synapsis of DNA molecules by recA protein.

In the presence of adenosine 5'-[gamma-thio]triphosphate, a nonhydrolyzable analog of ATP, Escherichia coli recA protein extensively unwinds duplex DNA in a reaction that is strongly stimulated by either homologous or heterologous single-stranded DNA [Cunningham, R.P., Shibata, T., DasGupta, C. & Radding, C.M. (1979) Nature (London) 281, 191-195]. In the presence of ATP and homologous circular single-stranded DNA, recA protein also unwinds circular duplex DNA that is nicked at a heterologous site. When DNA ligase seals this nick, the product is a highly negatively superhelical molecule that can be relaxed by E. coli topoisomerase I. This unwinding requires a high degree of homology since phi X174 single-stranded DNA does not serve as a cofactor in the unwinding of G4 DNA, even though these molecules are 70% homologous. Like synapsis itself, and unlike strand exchange which follows synapsis, unwinding is sensitive to inhibition by ADP. Because recA protein unwinds duplex DNA when neither the single-stranded DNA nor the duplex DNA has a free end in the region of homology, unwinding can be initiated or mediated by a synaptic structure that differs from that of a simple D loop. The paired circular single strand in the synaptic structure behaves like one strand of an under-wound helix because E. coli topoisomerase I can interwind it with its complement.     

Escherichia coli replication factor Y, a component of the primosome, can act as a DNA helicase.

The primosome is a mobile multienzyme DNA replication-priming complex that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC, dnaG, and dnaT genes as well as proteins n and n" and replication factor Y). It has been shown previously that the primosome, in combination with the E. coli DNA polymerase III holoenzyme, can form replication forks in vitro that move at rates similar to those measured in vivo and that the primosome and one of the components of the primosome, the DNA B protein, have DNA helicase activity. Evidence is presented here that another component of the primosome, replication factor Y, possesses DNA helicase activity as well. Factor Y helicase activity requires the presence of E. coli single-stranded DNA binding protein, Mg2+, and hydrolyzable ATP or dATP. Helicase activity is stimulated 15-fold when the enzyme is actively loaded onto single-stranded DNA through a primosome assembly site, and duplex DNA is unwound unidirectionally, 3'----5', along the DNA strand to which the protein is bound.     

Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication.

The simian virus 40 (SV40) large T antigen (large tumor antigen), in conjunction with a topoisomerase, a DNA binding protein, and ATP, catalyzed the conversion of a circular duplex DNA molecule containing the SV40 origin of replication to a form with unusual electrophoretic mobility that we have named form U. Analysis of this molecule revealed it to be a highly underwound covalently closed circle. DNA unwinding was not detected with DNA containing a SV40 T-antigen binding site II mutation that renders the DNA inactive in replication. The unwinding reaction requires the action of a helicase, and SV40 T-antigen preparations contain such an activity. The T-antigen-associated ability to unwind DNA copurified with other activities intrinsic to T antigen [ability to support replication of SV40 DNA containing the SV40 origin, poly(dT)-stimulated ATPase activity, and DNA helicase]. However, in contrast to the unwinding activity, the SV40 T-antigen-associated helicase activity was not sequence-specific. A variety of labeled oligonucleotides hybridized with circular single-stranded DNA were displaced by T antigen in the presence of ATP.     

Polarity of heteroduplex formation promoted by Escherichia coli recA protein.

When recA protein pairs circular single strands with linear duplex DNA, the circular strand displaces its homolog from only one end of the duplex molecule and rapidly creates heteroduplex joints that are thousands of base pairs long [DasGupta, C., Shibata, T., Cunningham, R. P. & Radding, C. M. (1980) Cell 22, 437-446]. To examine this apparently polar reaction, we prepared chimeric duplex fragments of DNA that had M13 nucleotide sequences at one end and G4 sequences at the other. Circular single strands homologous to M13 DNA paired with a chimeric fragment when M13 sequences were located at the 3' end of the complementary strand but did not pair when the M13 sequences were located at the 5' end. Likewise circular single-stranded G4 DNA paired with chimeric fragments only when G4 sequences were located at the 3' end of the complementary strand. To confirm these observations, we prepared fd DNA labeled only at the 5' or 3' end of the plus strand, and we examined the susceptibility of these labeled ends to digestion by exonucleases when joint molecules were formed. Eighty percent of the 5' label in joint molecules became sensitive to exonuclease VII. Displacement of that 5' end by recA protein was concerted because it did not occur in the absence of single-stranded DNA or in the presence of heterologous single strands. By contrast, only a small fraction of the 3' label became sensitive to exonuclease VII or exonuclease I. These observations show that recA protein forms heteroduplex joints in a concerted and polarized way.     

Isolation of an intermediate which precedes dnaG RNA polymerase participation in enzymatic replication of bacteriophage phi X174 DNA.

Conversion of phi X174 single-stranded DNA to the duplex replicative form (RF) in vitro requires at least 10 purified proteins. Three stages - strand initiation, elongation, and termination - comprise this conversion. We now identify a separate stage in strand initiation which precedes dnaG RNA polymerase participation. Incubation of five proteins - protein i, protein n, DNA unwinding protein, dnaB protein, and dnaC protein - with ATP and phi X174 DNA forms an intermediate which enables subsequent stages measured by DNA synthesis to proceed 20 times faster. The intermediate can be isolated in quantitative yield by gel filtration or by ultracentrifugation. Protein i and protein n are required in less than stoichiometric amounts and appear to be absent from the isolated intermediate. Whereas formation of the intermediate is sensitive to antibody to protein i and to N-ethylmaleimide (an inhibitor of protein n and dnaC protein), the intermediate itself is resistant to these reagents. DNA unwinding protein complexes the DNA in a ratio of 60 molecules per circle. Synthesis of the intermediate appears to require stoichiometric quantities of dnaB protein and dnaC PROTEin but their presence in the intermediate has not been established as yet.