Unique primed start of phage phi X174 DNA replication and mobility of the primosome in a direction opposite chain synthesis.

A specific fragment of the phi X174 viral circle sustains the primed start of complementary DNA strand synthesis in vitro, even though the intact circle permits primed starts at many sites. The 300-nucleotide fragment from restriction nuclease digestion contains the recognition site for protein n', a DNA-dependent ATPase essential for priming phi X174 DNA replication. This n' recognition site contains within it a 44-nucleotide sequence with a potential hairpin structure and may be regarded as the starting signal for replication [Shlomai, J. & Kornberg, A. (1980) Proc. Natl. Acad. Sci. USA 77, 799-803]. After initiation on the 3' side of this sequence, the priming system (primosome) repeatedly generates primers by moving processively on the DNA template in a direction opposite to chain elongation. This primosome mobility is an attractive model for the discontinuous phase of Escherichia coli chromosome replication, in which processive primosome movement with the replicating fork is proposed for repeated initiations of nascent replication fragments.     

Movement and site selection for priming by the primosome in phage phi X174 DNA replication.

The Escherichia coli priming system used for initiation of DNA chains on phage phi X174 single-stranded DNA is a multiprotein unit called the primosome [Arai, K. & Kornberg. A. (1981) Proc. Natl. Acad. Sci. USA 78, 69-73]. Assembled with participation of seven prepriming proteins and primase at a unique place on the phi X174 DNA template, the primosome is bound tightly to the DNA, yet moves rapidly and unidirectionally opposite to primer and DNA chain synthesis. Contributions of protein n' and dnaB protein, two components of the primosome, to movement and site selection for priming are considered in this report. Figuratively, the primosome can be likened to a locomotive that depends on protein n' as its engine and dnaB protein as the engineer. Protein n', a DNA-dependent ATPase (dATPase) appears to use the energy of hydrolysis of the nucleoside triphosphate for processive translocation of the primosome. dnaB protein, A DNA-dependent ribonucleosidetriphosphatase, depends on allosteric effects of a nucleoside triphosphate to induce changes in the structure of the single-stranded DNA at preferred sequences that enable primase to synthesize a short primer for initiation of DNA synthesis (unpublished data). These primosome properties have important implications for the progress of the replication fork of the E. coli chromosome.     

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.     

Heteroduplex formation by recA protein: polarity of strand exchanges.

Purified recA protein promotes strand exchanges between linear duplex DNA and homologous circular single-stranded phage phi X174 DNA that carries a short hybridized fragment [West, S. C., Cassuto, E. & Howard-Flanders, P. (1981) Proc. Natl. Acad. Sci. USA 78, 2100-2104]. In this paper we investigate the mechanism of this strand exchange reaction. We show that recA protein initiates strand exchanges by pairing the free end of the duplex fragment with the single-stranded DNA. In addition, we find that strand exchanges are polar, stable heteroduplex molecules being formed by the directional transfer transfer of the (-) strands starting at 3' termini.     

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.     

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.     

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.     

Functional division and reconstruction of a plasmid replication origin: molecular dissection of the oriV of the broad-host-range plasmid RSF1010.

Two single-stranded DNA initiation signals (designated ssi) present in the origin of vegetative DNA replication (oriV) of the broad-host-range plasmid RSF1010 are essential for the priming of replication of each complementary DNA strand of this plasmid in Escherichia coli. Each of the RSF1010 ssi signals, ssiA and ssiB, could be replaced by a primosome assembly site from plasmid pACY184 or from bacteriophage phi X174. In these chimeric origins, replication of the strand complementary to that containing the primosome assembly site was no longer dependent on the RSF1010 primase, protein RepB', but required the E. coli primase, DnaG. If both ssiA and ssiB sites of RSF1010 were replaced by primosome assembly sites, protein RepB' was no longer essential for the replication at this origin, whereas proteins RepA and RepC of RSF1010 were still required. These results strongly suggest that the two ssi sites and the RepB' protein actually direct the priming of DNA synthesis in the replication of RSF1010, and the proteins RepA and RepC are involved in the prepriming events--i.e., the opening of the DNA duplex at oriV. It is evident that the origin of RSF1010 can be separated into three functional domains and reconstructed by replacing the ssi sites with heterologous elements.     

An enzyme system for replication of duplex circular DNA: the replicative form of phage phi X174.

Viral single strands (SS) are converted to the duplex from (RF) by a soluble enzyme fraction uninfected Escherichia coli [Schekman et al. (1975) J. Biol. Chem. 250, 5859-5865]. When reactions were supplemented with a soluble enzyme fraction from phi X174-infected cells, replication of phi X174 superhelical RF I DNA was observed. The activity supplied by infected cells was absent in cells treated with chloramphenicol or in cells infected with a phi X174 phage mutant in cistron A (cis A). A host function coded by the rep gene, essential in vivo for RF replication (but not for SS leads to RF), was supplied by enzyme fractions from either infected or uninfected cells. Based on complementation assays, the cisA-dependent and the rep-dependent proteins have each been purified about 1000-fold. The synthetic products of the enzymatic reaction were identified as RF I and RF II in which viral (+) and complementary (-) strands were newly synthesized.     

Polar branch migration promoted by recA protein: effect of mismatched base pairs.

Escherichia coli recA protein makes joint molecules from single-stranded circular phage DNA (viral or plus strand) and homologous linear duplex DNA by a polar reaction that displaces the 5' end of the plus strand from the duplex molecule [Kahn, R., Cunningham, R. P., DasGupta, C. & Radding, C. M. (1981) Proc. Natl. Acad. Sci. USA 78, 4786-4790]. Growth of the heteroduplex joint, which results from strand exchange or branch migration, stopped at the borders of regions of nonhomologous DNA that were variously located 145, 630, or 1202 nucleotides from the end. Accumulation of migrating branches at heterologous borders demonstrates that their migration is not the result of random diffusion but is actively driven by recA protein. Growth of the heteroduplex joint was blocked even when a heterologous insertion was located in the single-stranded DNA, a case in which the flexible single-stranded region might conceivably fold out of the way under some condition. The recA protein did not make joint molecules from phage phi X174 and G4DNAs, which are 70% homologous, but did join phage fd and M13DNAs, which are 97% homologous. In the latter case, heteroduplex joints extended through regions containing isolated mismatched base pairs but stopped in a region where the fd and M13 sequences differ by an average of 1 base pair in 10. These results suggest that in genetic recombination the discrimination of perfect or near-perfect homology from a high degree of relatedness may be attributable in part to the mechanism by which recA protein promotes strand transfer.     

Initiation of DNA replication on single-stranded DNA templates catalyzed by purified replication proteins of bacteriophage lambda and Escherichia coli.

Initiation of bacteriophage lambda DNA replication at the chromosomal origin depends on the lambda O and P replication proteins. These two viral initiators, together with an Escherichia coli protein fraction, promote the replication in vitro of single-stranded circular DNA chromosomes such as that of bacteriophage M13. This nonspecific strand initiation reaction, which we have termed the "lambda single-strand replication reaction," has now been established with eight purified proteins, each of which is also required for replication of the phage lambda chromosome in vivo. An early rate-limiting step in the overall reaction is the ATP-dependent assembly of an activated nucleoprotein prepriming complex. In this step the lambda O and P initiators cooperate with the E. coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto M13 DNA that is coated with the single-stranded DNA-binding protein. Multiple RNA primers are synthesized on each DNA circle when isolated prepriming complex is incubated with primase and rNTPs. In the complete system, DNA polymerase III holoenzyme extends the first primer synthesized into full-length complementary strands. Because the properties of this system are closely analogous to those found for the replication of phi X174 viral DNA by E. coli proteins, we infer that a mobile prepriming or priming complex (primosome) operates in the lambda single-strand replication reaction.     

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.     

Identification of a primosome assembly site in the region of the ori 2 replication origin of the Escherichia coli mini-F plasmid.

A primosome assembly site for F plasmid DNA replication has been identified. This site, which we term rriA (F), is localized to one strand of a 385-base-pair Sau3A restriction fragment very close to ori 2 and within the 2.25-kilobase DNA sequence required for replication and incompatibility of the entire F plasmid. rriA (F) was isolated by cloning into the deletion phage vector M13 delta Elac. This phage forms very faint plaques due to a deletion of the M13 complementary strand origin but forms large wild-type plaques when DNA single-strand initiation determinants are inserted. The single-stranded viral DNA of the Sau3A F-M13 delta Elac recombinant provides an effector site of dATP hydrolysis by the primosomal protein n'. It also provides an assembly site for the Escherichia coli primosome protein complex that directs the in vitro conversion of the single-stranded DNA to a double-stranded form by the same mechanism as that used by phi X174. Homologies of the nucleotide sequence between this F DNA sequence and the previously identified primosome assembly sites in phi X174 phage DNA and in ColE1 plasmid DNA (rriA and rriB) have been found. The sequences 5' G-T-G-A-G-C-G 3' and 5' G-N-G-G-A-A-G-C 3' or variations of these sequences occur from two to five times within each assembly locus. In addition, two distinct 15-base-pair sequences in rriA (F) are perfectly homologous to corresponding sequences in rriA (ColE1).     

An Escherichia coli replication protein that recognizes a unique sequence within a hairpin region in phi X174 DNA.

Protein n', a prepriming DNA replication enzyme of Escherichia coli, is a phi X174 DNA-dependent ATPase. Restriction of phi X174 DNA have led to the identification of a 55-nucleotide fragment that carries the protein n' recognition sequence. Molecular hybridization and sequence analysis have located this sequence within the untranslated region between genes F and G, a map location analogous to that of the unique complementary strand origin of phage G4 DNA. Within the 55-nucleotide fragment is a sequence of 44 nucleotides that forms a stable hairpin structure. This duplex may be the signal for protein n' to initiate the prepriming events that led to the start of phi X174 complementary DNA strand replication.     

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.     

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.     

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.     

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.     

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.