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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

A DNA primase specified by I-like plasmids.

An enzyme has been isolated from Escherichia coli strains harboring the I-like plasmid R64drd11, which is capable of initiating DNA synthesis on the circular, single-stranded DNA of phages phi X174, fd, and G4. In the conversion of these templates to duplex forms in vitro, the enzyme can substitute for the functions of E. coli dna B-dnaB-dnaC-dnaG proteins, E. coli RNA polymerase, and E. coli dnaG protein, respectively. The enzyme requires all four ribonucleoside triphosphates for optimal activity, although a combination of ATP, CTP, and GTP can almost completely satisfy the rNTP requirement. The enzyme appears to cooperate specifically with DNA polymerases III because single-stranded DNA-dependent synthesis takes place in extracts deficient in DNA polymerases I and II but not in extracts from a dnaZ mutant. Highly purified enzyme preparations consist mostly of two major polypeptides, Mr 140,000 and 180,000, when analyzed by sodium dodecyl sulfate gel electrophoresis. These polypeptides cosediment with the enzyme activity through a glycerol gradient with a sedimentation coefficient of 3.6 S. DNA priming activity in extracts of E. coli strains harboring the mutant plasmids R64drd11 or ColIdrd1, which are derepressed in functions of conjugational DNA transfer, severalfold higher than the activity from strains carrying the corresponding wild-type plasmid. This correlation suggests that the enzyme may play a role in conjugational DNA synthesis.     

Analysis of the phiX DNA replication cycle by electron microscopy.

We have monitored the development of intracellular phiX DNA forms during the course of a virus life cycle that duplicates as closely as possible the normal infection of individual cells by single virions. The viral DNA was isolated in a one-step purification procedure, and quantitative electron microscopy was performed on the samples, resulting in the following conclusions: (i) Early in the life cycle, when the cells accumulate duplex rings, two types of DNA replication intermediates are observed: a rolling circle with a single-stranded tail; and a novel form, a single-stranded circle that is partially duplex. Thus, duplex ring synthesis appears to occur in two asymmetric steps, with positive strand DNA first being processed from the tail of the rolling circle and circularized, before it acts as a template for negative strand synthesis. (ii) Late in the life cycle, as single-stranded circles are synthesized and virus particles are assembled, only one replicating intermediate is observed--the rolling circle with a single-stranded tail. At this stage, the number of rolling circles reaches a level of about 35 per cell. (iii) The net rate of polymerization in the rolling circle intermediates is about 200 nucleotides per sec.     

Isolation and Function of the Gene A Initiator of Bacteriophage ?X 174, A Highly Specific DNA Endonuclease

The gene A product of Escherichia coli bacteriophage ϕX 174, necessary for initiation of ϕX DNA synthesis, has been identified and purified to near homogeneity. The gene A protein is shown to be a highly specific DNA endonuclease which breaks one phosphodiester bond in the viral strand of either double-stranded, circular ϕX replicative form I (RFI), or in single-stranded, circular ϕX viral DNA; it is inactive against all other species of DNA tested.     

phiX174 DNA-dependent DNA synthesis in vitro: requirement for P1 ban protein in dnaB mutant extracts of Escherichia coli.

Ammonium sulfate fractionation of crude extracts of E. coli yields a soluble enzyme fraction (about 25-fold purification) that catalyzes the conversion of phiX174 single-stranded DNA to duplex DNA. The reaction is rifampicin-resistant, requires single-stranded DNA, Mg++, deoxynucleoside triphosphates, and ATP, and is stimulated by KCl. Such soluble enzyme fractions were prepared from E. coli strains carrying the prophage mutant P1bac, in which the viral dnaB analog (ban) protein is expressed constitutively, or P1bacban, in which the expression of ban protein is prevented. DNA-synthesizing activity of ban protein containing fractions from wild-type or dnaB(P1bac) lysogens was more temperature-resistant than that from E. coli containing only wild-type dnaB protein, whereas that from dnaB(P1bacban) lysogens of dnaB cells was extremely thermolabile. It is suggested that the temperature-resistant DNA synthesis with fractions from P1bac lysogens is mediated by the P1 ban protein.     

Studies on In Vitro DNA Synthesis Isolation of dna B Gene Product from Escherichia coli

Formation of duplex DNA from varphiX174 single-stranded DNA by extracts of E. coli was previously shown to require the gene product of dna B. Using as an assay the stimulation of varphiX174 DNA-dependent synthesis in inactivated extracts of dna B temperature-sensitive cells, we purified the dna B gene product from wild-type E. coli as well as from a dna B temperature-sensitive mutant. The dna B temperature-sensitive gene product is more thermolabile than its wild-type counterpart.     

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.     

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.     

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.     

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.     

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.     

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.     

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.