A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication.

The enzyme system for duplicating the duplex, circular DNA of phage phi X174 (replicative form) in stage II of the replicative life cycle was shown to proceed in two steps: synthesis of the viral (+) strand ]stage II(+)], followed by synthesis of the complementary (-) strand ]stage II(-)] [Eisenberg et al. (1976) Proc. Natl. Acad. Sci. USA 73, 3151-3155]. Novel features of the mechanism of the stage II(+) reaction have now been observed. The product, synthesized in extensive net quantities, is a covalently closed, circular, single-stranded DNA. The supercoiled replicative form I template and three of the four required proteins--the phage-induced cistron A protein (cis A), the host rep protein (rep), and the DNA polymerase III holoenzyme (holoenzyme)--act catalytically; the Escherichia coli DNA unwinding (or binding) protein binds the product stoichiometrically. In a reaction uncoupled from replication, cis A, rep, DNA binding protein, ATP, and Mg2+ separate the supercoiled replicative form I into its component single strands coated with DNA binding protein. In the presence of Mg2+, cis A, nicks the replicative form I; rep, ATP, and Mg2+ achieve strand separation with a concurrent cleavage of ATP and binding of DNA binding protein to the single strands. rep exhibits a single-stranded DNA-dependent ATPase activity. These observations suggest that the rep enzymatically melts the duplex at the replicating fork, using energy provided by ATP; this mechanism may apply to the replication of the E. coli chromosome as well.     

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.     

Replication of duplex DNA of phage phi X174 reconstituted with purified enzymes.

Replication of the covalently closed duplex replicative form (RF) of phage phi X174 DNA has been achieved by coupling two known enzyme systems: (i) synthesis of viral strand circles (SS) from RF, and (ii) conversion of SS to nearly complete RF (RF II). In this coupled system, activated RF (gene A . RF II complex) was a more efficient template and generated as many as 10 RF II molecules per RF input, at a rate commensurate with SS synthesis. The 11 proteins required for the two component systems were all needed in the coupled RF duplication system; no new factors were required. Single-stranded DNA binding protein was needed for RF duplication at only 4% the level needed in its stoichiometric participation in SS synthesis. In addition to RF II, more complex replicative forms appeared late in the reaction, and their possible origin is discussed.     

Discontinuous replication of replicative form DNA from bacteriophage phiX174.

Bacteriophage phiX174 DNA has been labeled with short pulses of [3H]thymidine during synthesis of replicative form molecules in infected Escherichia coli HF4704 cells. The replicating phiX174 DNA was isolated and analyzed by sedimentation in an alkaline sucrose gradient. During a brief pulse (5 sec at 30 degrees), the radioactivity incorporated into the complementary strand was found in chains much shorter than one genome length. Of the radioactivity incorporated into the viral strand, two-thirds was in the short pieces and the rest was in chains of one genome length or longer. RNA attachment to the 5' end of both strand components of the nascent short pieces was shown by the appearance of spleen exonuclease-digestable nascent molecules after alkali treatment. These observations suggest that the viral as well as the complementary strand is synthesized by the discontinuous mechanism with RNA primers during replication of duplex phiX174 DNA.     

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.     

RNA Synthesis Initiates In Vitro Conversion of M13 DNA to Its Replicative Form

Soluble enzyme fractions from uninfected Escherichia coli convert M13 and varphiX174 viral single strands to their double-stranded replicative forms. Rifampicin, an inhibitor of RNA polymerase, blocks conversion of M13 single strands to the replicative forms in vivo and in vitro. However, rifampicin does not block synthesis of the replicative forms of varphiX174 either in vivo or in soluble extracts. The replicative form of M13 synthesized in vitro consists of a full-length, linear, complementary strand annealed to a viral strand. The conversion of single strands of M13 to the replicative form proceeds in two separate stages. The first stage requires enzymes, ribonucleoside triphosphates, and single-stranded DNA; the reaction is inhibited by rifampicin. The macromolecular product separated at this stage supports DNA synthesis with deoxyribonucleoside triphosphates and a fresh addition of enzymes; ribonucleoside triphosphates are not required in this second stage nor does rifampicin inhibit the reaction. We presume that in the first stage there is synthesis of a short RNA chain, which then primes the synthesis of a replicative form by a DNA polymerase.     

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.     

Studies on nucleic acid reassociation kinetics: rate of hybridization of excess RNA with DNA, compared to the rate of DNA renaturation.

The rate of reaction of double-stranded replicative form (RF) [3H]DNA of bacteriophage phiX174 with excess (+)strand DNA and (+)strand RNA was measured by standard methods of hydroxyapatite chromatography. The reactions follow pseudo-first-order kinetics and the observed rate constant for the RNA-DNA reaction differs less than 25% from that of the DNA-DNA reaction. The pseudo-first-order rate constants are close to the value predicted on the basis of the second-order rate constant measured in the renaturation of the double-stranded phiX RF [3H]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.     

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.     

Isolation and characterization of the protein coded by gene A of bacteriophage phiX174 DNA.

Replication of phiX174 circular replicative form (RFI) DNA by extracts of Escherichia coli infected with bacteriophage phiX174 (amber in gene A) requires the phiX174 gene A product. This requirement has been used as an assay for the isolation of this protein. The gene A product (purified 4000-fold) caused relaxation of superhelical phiX174 RFI and formation of discontinuities in the viral strand of phiX174 RFI uniquely situated in the A region of the genome, and yielded a complex after interacting with phiX174 RFI that is active in replication of phiX RFI.     

In vitro DNA replication of recombinant plasmid DNAs containing the origin of progeny replicative form DNA synthesis of phage phi X174.

The origin of phage phi X174 progeny replicative form (RF) DNA synthesis has been inserted into the plasmid vector pBR322 and cloned. In direct contrast to pBR322, the recombinant superhelical plasmids can substitute for phi X174 RFI DNA as template in phi X174-specific reactions in vitro. We have shown that the recombinant plasmids: (i) are cleaved by the phi X174 A protein; (ii) support net synthesis of unit-length single-stranded circular DNA in the presence of the phi X174 A protein and Escherichia coli rep protein, DNA-binding protein, and DNA polymerase III elongation system; (iii) support replication of duplexes catalyzed by the phi X174 A protein and extracts of E. coli.     

Efficient correction of a mutation by use of chemically synthesized DNA.

The mutated base in the am3 lysis-defective mutant of the bacteriophage phiX174 has been corrected by a combined in vitro enzymatic DNA synthesis and in vivo replication of the heteroduplex product. Chemically synthesized oligodeoxyribonucleotides carrying the wild-type sequence have been used to prime DNA synthesis with am3 phiX174 DNA serving as a template. The resultant semisynthetic heteroduplex composed of an am3(+) strand and a wild-type (-) strand, with one mismatched base pair at position 587 on the phiX174 DNA sequence, was used to infect spheroplasts. The progeny phage were analyzed by a parallel plaque assay on wild-type host, Escherichia coli C, to screen for wild-type phenotype, and on E. coli HF4714, an amber suppressor strain, to determine the total progeny phage. When a 23-base-long synthetic primer was used, about one-third of total progeny were found to be wild type. Shorter primers yielded lower percentages of wild type; they also had poorer priming activity.     

DNA synthesis in vitro dependent upon phiX174 replicative form I DNA.

Extracts of Escherichia coli strains infected with bacteriophage phiX174 catalyze DNA synthesis dependent on double-stranded, circular phiX174 replicative form I (phiX RFI) by a semiconservative process. The reaction required Mg++, ATP, all four dNTP, and exogenous phiX RFI DNA as template and yielded phiX RFI and phiX RFII. The reaction was inhibited by nalidixic acid and novobiocin but not by rifampicin. DNA synthesis required the phiX174 gene A product and E. coli gene products dnaB, dnaC(D), dnaG, and rep.     

Migration of Escherichia coli dnaB protein on the template DNA strand as a mechanism in initiating DNA replication

The first step in conversion of varphiX174 singlestranded DNA to the duplex replicative form in vitro is the synthesis of a nucleoprotein intermediate [Weiner, J. H., McMacken, R. & Kornberg, A. (1976) Proc. Natl. Acad. Sci. USA 73, 752-756]. We now demonstrate that dnaB protein (approximately one molecule per DNA circle) is an essential component of the intermediate and retains its ATPase activity. Synthesis of RNA primers, dependent on dnaG protein (primase), occurred only on DNA that had been converted to the intermediate form. In a coupled RNA priming-DNA replication reaction the first primer synthesized was extended by DNA polymerase III holoenzyme into full-length complementary strand DNA. In RNA priming uncoupled from replication, multiple RNA primers were initiated on a varphiX174 circle. The single dnaB protein molecule present on each DNA circle participated in initiation of each of the RNA primers, which appear to be aligned at regular intervals along the template strand. We propose that dnaB protein, once bound to the template, migrates in a processive fashion along the DNA strand, perhaps utilizing energy released by hydrolysis of ATP for propulsion; in this scheme the actively moving dnaB protein acts as a "mobile promoter" signal for dnaG protein (primase) to produce many RNA primers. Schemes are proposed for participation of dnaB protein both in the initiation of replication at the origin of the Escherichia coli chromosome and in the initiation of primers for nascent (Okazaki) fragments at a replication fork.     

Association of phiX174 DNA-dependent ATPase activity with an Escherichia coli protein, replication factor Y, required for in vitro synthesis of phiX174 DNA.

phiX174 DNA-dependent DNA synthesis is catalyzed in vitro by the combination of at least 11 purified protein fractions: dnaB, dnaC(D), and dnaG gene products, DNA polymerase III, DNA elongation factors I and II, DNA binding protein, and replication factors W, X, Y, and Z. The reaction requires ATP, 4 dNTPs, and Mg+2 and is specific for phiX174 (or phiXahb) DNA. Purified replication factor Y contains phiX174 (or phiXahb) DNA-dependent ATPase (or dATPase) activity. The ATPase activity is poorly stimulated by other single-stranded DNA, by double-stranded DNA, or by RNA. The products of the phiX174 DNA-dependent ATPase activity of factor Y are Pi and ADP (or dADP). The association of phiX174 DNA-dependent ATPase activity with factor Y was shown in the following ways: (a) the two activities copurified with a constant ratio; (b) they comigrated on native polyacrylamide gel electrophoresis; (c) both activities were heat-inactivated at the same rate; and (d) both showed identical patterns of N-ethylmaleimide sensitivity.     

Nucleotide sequences of the separate origins of synthesis of bacteriophage G4 viral and complementary DNA strands.

Bacteriophage G4 has physically separated origins of synthesis of its viral and complementary DNA strands. Chain termination and "plus and minus" DNA sequencing methods have been used to obtain the nucleotide sequence of these two origins. The unique origin at which the complementary DNA strand is initiated has located in the untranslated region between genes F and G. This sequence, which has considerable secondary structure, contains a stretch which is complementary to the RNA primer that is observed during synthesis in vitro of the G4 complementary DNA strand [Bouché, J.P., Rowen, L. & Kornberg, A. (1978) J. Biol. Chem., in press]. This G4 origin shows extensive sequence homology with the bacteriophage lambda origin of DNA replication [Denniston-Thompson, K., Moore, D. D., Kruger, D. E., Furth, M. E. & Blattner, F. R. (1977) Science 198, 1051-1056]. The sequence around the site in gene A at which G4 viral DNA strand synthesis is initiated by the nicking action of the cistron A protein is very similar to that of bacteriophage phiX174. An (A + T)-rich stretch flanked by (G + C)-rich sequences may be involved in the interaction between the DNA and protein.     

THE PROCESS OF INFECTION WITH BACTERIOPHAGE ϕX174, XXX. REPLICATION OF DOUBLE-STRANDED ϕX DNA

Intermediates involved in the replication of double-stranded varphiX174 RF DNA have been identified and partially characterized. Analysis of pulselabeled RF DNA suggests that the synthesis of progeny RF molecules involves, in part, the addition of nucleotides to linear complementary strands on a circular parental strand as template, so as to produce intermediate DNA strands of greater than viral length. Electron microscopy reveals DNA rings with "tails" and "double rings," which could be the intermediate structures. A model is postulated for the replication process.     

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.     

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.