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 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.     

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.     

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.     

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.     

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.     

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.     

Purified Escherichia coli recA protein catalyzes homologous pairing of superhelical DNA and single-stranded fragments.

Purified Escherichia coli recA protein catalyzed ATP-dependent pairing of superhelical DNA and homologous single-stranded fragments. The product of the reaction: (i) was retained by nitrocellulose filters in 1.5 M NaCl/0.15 M Na citrate at pH 7, (ii) was dissociated at pH 12.3 but was not dissociated by heating at 55 degrees C for 4 min or by treatment with 0.2% sodium dodecyl sulfate and proteinase K, (iii) contained covalently closed circular double-stranded DNA (form I DNA), (iv) contained single-stranded fragments associated with replicative form (RF) DNA, and (v) contained a significant fraction of D-loops as judged by electron microscopy. Linear and nicked circular double-stranded DNA did not substitute well for superhelical DNA; intact circular single-stranded DNA did not substitute well for single-stranded fragments. Homologous combinations of single-stranded fragments and superhelical DNA from phages phiX174 and fd reacted, whereas heterologous combinations did not. The reaction required high concentrations of protein and MgCl2. The ATPase activity of purified recA protein was more than 98% dependent on the addition of single-stranded DNA. In 1 mM MgCl2, the ability of superhelical DNA to support the ATPase activity was two-thirds as good as that of single-stranded DNA.     

DNA replication in vitro starting with an intact phiX174 phage.

Conversion of the single-stranded DNA in the intact phiX174 phage particle to the duplex replicative form (RF) has been demonstrated in lysates form phage-sensitive cells. The conversion is resistant to rifampicin and requires participation of both a "membrane" fraction of the lysate and a multienzyme replicative system. The lipopolysaccharide phage receptor, while essential, does not replace the membrane fraction. Clear, nonsedimentable extract fractions prepared with a certain nonionic detergent can replace the membrane fraction. Purification of the activity in these extracts by adsorption to polypropylene film yields a fraction with a 5-fold increase in activity relative to lipopolysaccharide and 50-fold increase relative to protein. The low buoyant density (1.03 g/cm3) suggests a high phospholipid or detergent content in this fraction.     

Uptake of homologous single-stranded fragments by superhelical DNA: a possible mechanism for initiation of genetic recombination.

Superhelical [3-H]DNA (replicative form I, RFI) of bacteriophage phiX174 slowly but spontaneously took up 32-P-labeled homologous single-stranded fragments at 4 degrees. Uptake was accelerated by heating to 75 degrees. RFI did not take up single-stranded fragments derived from DNA of Escherichia coli or from separated strands of phage lambda. Uptake was inhibited by low concentrations of ethidium bromide. Relaxed circular phiX174 DNA did not take up homologous fragments. Per molecule of RFI, the complexes contained as much as 90 nucleotide residues of homologous fragment. The 32-P-lebeled fragments were largely resistant to digestion by exonuclease I, and were not displaced by heating complexes at 60 degrees for 1 min in 16 mM or 100 mM NaCl. Under comparable conditions of temperature and salt all of the fragments were displaced from complexes in which at least one phosphodiester bond was cleaved by pancreatic DNase, but a significant fraction of the fragments was retained in complexes that were relaxed by digestion with S1 nuclease. These observations are interpreted to mean that S1 nuclease digested the plus (viral) strand of the recipient RF at the site of uptake in some instances. Transfection of E. coli by heterozygous complexes produced recombinant progeny, thereby showing that genetic information can be transferred from the fragment of plus strand to progeny plus strands. We propose that both uptake of a third strand by superhelical DNA and the action of nucleases on the resulting complex may simulate early steps in genetic recombination.     

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.     

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.     

DNA gyrase: purification and catalytic properties of a fragment of gyrase B protein.

A protein isolated from Escherichia coli complements the DNA gyrase A (NalA) protein to generate an activity that relaxes supercoiled DNA. Oxolinic acid, a known inhibitor of DNA gyrase, blocks this activity and causes double-strand cleavage of DNA at the same sites as are attacked by DNA gyrase. The protein, of molecular weight 50,000, appears to be fragment of the DNA gyrase B (Cou) protein (molecular weight, 90,000) as judged by the identical sizes of numerous peptides produced by partial proteolytic digestion. The complex of this fragment and the gyrase A protein lacks both the DNA-supercoiling and DNA-dependent ATPase activities of DNA gyrase.     

Recombination promoted by superhelical DNA and the recA gene of Escherichia coli.

When a mixture of superhelical DNA (RFI) of phage phiX174 am3 and fragments of single-stranded DNA from wild-type phiX174 was added to spheroplasts of E. coli carrying an amber suppressor, several percent of the progeny phage were recombinant. The yield of wild-type progeny was 10(3) to 10(4) times lower when the fragments came from phiX174 am3 or phage G4 am+, or when fragments were absent. Fewer recombinants were produced in proportion to the decrease in the fraction of RFI in samples treated with S1 nuclease, whereas the total yield of phage did not decrease. Transfection by fragments and superhelical DNA produced 20 to 100 times more recombinants than transfection by fragments and either nicked circular DNA or relaxed closed circular DNA. Transfection of a recA- strain by RFI DNA and fragments yielded 5-10% as many recombinants as transfection of a rec+ strain. This partial requirement for recA was bypassed by transfection with complexes of RFI AM3 DNA and am+ fragments made in vitro.     

Conversion of ?X174 and fd Single-Stranded DNA to Replicative Forms in Extracts of Escherichia coli

varphiX174 and M13 (fd) single-stranded circular DNAs are converted to their replicative forms by extracts of E. coli pol A1 cells. We find that the varphiX174 DNA-dependent reaction requires Mg(++), ATP, and all four deoxynucleoside triphosphates, but not CTP, UTP, or GTP. This reaction also involves the products of the dnaC, dnaD, dnaE (DNA polymerase III), and dnaG genes, but not that of dnaF (ribonucleotide reductase). The in vitro conversion of fd single-stranded DNA to the replicative form requires all four ribonucleoside triphosphates, Mg(++), and all four deoxynucleoside triphosphates. The reaction involves the product of gene dnaE but not those of genes dnaC, dnaD, dnaF, or dnaG. The reaction with fd DNA is inhibited by rifampicin or antibody to RNA polymerase, while the reaction with varphiX174 DNA is not affected by either. With the varphiX174 DNA-dependent reaction, activities have been detected that specifically complement extracts of dnaA, dnaB, dnaC, dnaD, or dnaG mutants.     

Mechanism of action of nalidixic acid: Purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme

A target protein for nalidixic and oxolinic acids in Escherichia coli, the nalA gene product (Pnal), was purified to homogeneity as judged by gel electrophoresis, using an in vitro complementation assay. It is a dimer of identical 110,000-dalton subunits. A polypeptide of this molecular weight is uniquely induced by a lambda nalA transducing phage, thereby showing that the purified Pnal is a product of the nalA gene. Nalidixic and oxolinic acids inhibit DNA gyrase activity and induce formation of a relaxation complex analogue. Treatment of the complex with sodium dodecyl sulfate causes a doublestrand break in the DNA substrate and the resulting linear molecule seems covalently bound to protein. Complex formation, unlike the introduction of supertwists, does not require ATP or relaxed circular DNA and is insensitive to novobiocin. DNA gyrase from a strain with a nalA mutation conferring drug resistance (nalA(r)) is 1/100 as sensitive to oxolinic and nalidixic acids with respect to inhibition of supertwisting and induction of the pre-linearization complex. Addition of Pnal restores drug sensitivity and stimulates DNA gyrase activity. DNA gyrase preparations and Pnal catalyze a third reaction sensitive to nalidixic and oxolinic acids, the ATP-independent relaxation of supertwister DNA. Relaxation by gyrase from nalA(r) cells is drug resistant. The nicking-closing activity is distinct from E. coli omega protein in several properties, including the ability to relax positively supertwisted DNA. We postulate that the nalA gene product occurs in two molecular forms, as Pnal and as a gyrase component. Both forms catalyze nicking-closing, and inhibition of this activity by nalidixic and oxolinic acids may account for the inhibition of DNA synthesis by these drugs.     

Nucleotide specificity in DNA scission by neocarzinostatin.

Using the DNA sequencing technique of Maxam and Gilbert, we show that the protein antibiotic neocarzinostatin, cleaves double-stranded phiX174 DNA restriction fragments almost exclusively at deoxythymidylic and deoxyadenylic acid residues in a reaction requiring 2-mercaptoethanol. Overall, deoxythymidylic acid residues are attacked much more frequently than are deoxyadenylic acid residues, although there is variability in the attack rate for both nucleotides at different locations in the DNA molecule. While all deoxythymidylic acid residues are sites of scission by neocarzinostatin, not all deoxyadenylic acid residues are cleavage sites. There appears to be no clear-cut nucleotide sequence specificity in determining cleavage frequency. Single-stranded DNA is a very poor substrate for neocarzinostatin-induced scission; with one single-stranded DNA fragment, cleavage occurs at a position that is not attacked in double-stranded DNA. The possible significance for its biological activity of a drug that can attack both members of a DNA base pair is discussed.     

Identification of ColE1 DNA sequences that direct single strand-to-double strand conversion by a phi X174 type mechanism.

A DNA single-strand initiation sequence, named rriA (called rri-1 previously), was detected in the origin region (Hae II fragment E) of the ColE1 plasmid [Nomura, N. & Ray, D. S. (1980) Proc. Natl. Acad. Sci. USA 77, 6566-6570]. Another site, called rriB, has been found on the opposite strand of Hae II fragment C. Both rriA and rriB (i) direct conversion of chimeric M13 phage single-stranded DNA to parental replicative form DNA in vivo by a rifampicin-resistant mechanism that is dependent on the dnaG and dnaB gene products, (ii) provide effector sites of dATP hydrolysis by primosomal protein n', and (iii) require the same primosomal proteins as phi X174 DNA for directing the in vitro conversion that rriA is the DNA sequence that determines the mechanism of lagging strand synthesis of ColE1 DNA and that the mechanism of discontinuous synthesis involves the primosomal proteins utilized in the in vitro conversion of phi X174 single strands to the double-stranded replicative form.     

Role of DNA gyrase subunits in synthesis of bacteriophage phi X174 viral DNA

The role of Escherichia coli DNA gyrase subunit A and subunit B during phi X174 viral DNA synthesis was investigated. Addition of nalidixic acid (an inhibitor of gyrase subunit A) and novobiocin (an inhibitor of gyrase subunit B) to an in vitro system capable of synthesizing phi X174 viral DNA inhibited DNA synthesis. The inhibition caused by novobiocin, however, was not due specifically to an inhibition of gyrase subunit B because DNA synthesis in an in vitro system composed of an extract containing novobiocin-resistant gyrase subunit B was also inhibited by novobiocin. The requirement for gyrase subunit A and the dispensability of gyrase subunit B during viral strand synthesis was confirmed in vivo by examining phi X174 viral DNA synthesis in host bacteria containing temperature-sensitive gyrase subunits.