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DNA at preferred sequences that enable primase to synthesize a short primer for initiation of DNA synthesis (unpublished data).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.     

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.     

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.     

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.     

The priA gene encoding the primosomal replicative n'' protein of Escherichia coli.

The Escherichia coli gene encoding protein n' has been isolated and named priA for primosomal protein A. Protein n' is absolutely required for the conversion of single-stranded phi X174 DNA to the duplex replicative form in an in vitro-reconstituted system. The gene maps to 88.7 minutes on the chromosome adjacent to the cytR locus. Soluble protein extracts from cells harboring the priA gene on a multicopy plasmid contained 45-fold more n' replication activity than wild-type extracts. Enhanced overproduction of greater than 1000-fold was achieved by replacing the natural Shine-Dalgarno sequence with that of the phage T7 phi 10 gene and placing this priA under the control of the T7 phage promoter and RNA polymerase. The priA sequence reveals a 732-amino acid open reading frame and a nucleotide-binding consensus site consistent with the size and ATPase activity of the purified protein. The gene for protein n has been named priB and the putative gene for protein n", priC.     

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.     

Morphogenesis of phi X174: in vitro synthesis of infectious phage from purified viral components.

An in vitro system that synthesizes infectious phage phi X174 was developed. The synthesis depended on phi X174 supercoiled replicative form DNA, purified phi X174 gene A protein, gene C protein, gene J protein, prohead (phage head precursor composed of gene F, G, H, B, and D proteins), and uninfected host crude extract. The infectious phage synthesis was coupled with DNA synthesis. De novo initiation, elongation, and termination of phi X174 single-stranded DNA was observed. The phage synthesized in vitro cosedimented with in vivo phage in sucrose gradients and had the same buoyant density as in vivo phage in a CsCl gradient. Our results indicate that the in vitro system mimics the in vivo phi X174 assembly process.     

Escherichia coli factor Y sites of plasmid pBR322 can function as origins of DNA replication.

The Escherichia coli replication factor Y, in conjunction with other genetically undefined E. coli replication factors and the gene products of the E. coli dnaB, dnaC, and dnaG loci, is involved in de novo primer formation on the phi X174 (+) single-strand circular DNA template [(+) ss(c)DNA]. The participation of factor Y in this series of reactions is correlated with its phi X174 (+) ss(c)-specific DNA-dependent ATPase activity. Recently two factor Y effector DNA segments of the plasmid pBR322 have been identified in close proximity to the plasmid origin of DNA replication. We report here that insertion of these factor Y sites into the filamentous phage f1R229 (+) ss(c)DNA confers upon it the ability to be converted to RF DNA in vitro through a rifampicin-resistant dnaB, dnaC, and dnaG gene product-dependent pathway. Our data suggest that factor Y effector sites can function as origins of DNA replication.     

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).     

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.     

Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: evidence for bypass of pyrimidine photodimers.

Replication of UV-irradiated circular single-stranded phage M13 DNA by Escherichia coli RNA polymerase (EC 2.7.7.6) and DNA polymerase III holoenzyme (EC 2.7.7.7) in the presence of single-stranded DNA binding protein yielded full-length as well as partially replicated products. A similar result was obtained with phage G4 DNA primed with E. coli DNA primase, and phage phi X174 DNA primed with a synthetic oligonucleotide. The fraction of full-length DNA was several orders of magnitude higher than predicted if pyrimidine photodimers were to constitute absolute blocks to DNA replication. Recent models have suggested that pyrimidine photodimers are absolute blocks to DNA replication and that SOS-induced proteins are required to allow their bypass. Our results demonstrate that, under in vitro replication conditions, E. coli DNA polymerase III holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins.     

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.     

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.     

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.     

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.     

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.     

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.     

Molecular cloning and DNA sequence analysis of Escherichia coli priA, the gene encoding the primosomal protein replication factor Y.

Escherichia coli replication factor Y (protein n') functions in the assembly of a mobile multiprotein replication-priming complex called the primosome. Although the role of factor Y in primosome assembly during replication in vitro of bacteriophage phi X174 and plasmid pBR322 DNA is clear, its role in E. coli chromosomal replication is not. To address this issue, the gene for factor Y has been cloned molecularly and its DNA sequence has been determined. The cloned fragment of DNA contained an open reading frame capable of encoding a polypeptide of 81.7 kDa. This open reading frame contains amino acid sequences identical to 13 N-terminal amino acids of purified factor Y, as well as to a 10-amino acid internal sequence (from a cyanogen bromide fragment) as determined by gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame using a bacteriophage T7 transient expression system resulted in the accumulation of a polypeptide with an apparent molecular mass of 78 kDa that comigrated with bona fide factor Y during SDS/polyacrylamide gel electrophoresis. Soluble extracts made from cells overexpressing the product of the putative factor Y open reading frame showed a 2000-fold increase in factor Y activity during bacteriophage phi X174 complementary-strand DNA synthesis in vitro when compared to control extracts. The gene encoding factor Y, which maps to 88.5 min on the E. coli chromosome, has been designated primosome A (priA).     

Replication deficiencies in priA mutants of Escherichia coli lacking the primosomal replication n'' protein.

The priA gene of Escherichia coli encodes the protein that initiates assembly of the promosome, the entity essential for the replication of phage phi X174 and ColE1-like plasmids in vitro. We have prepared a null priA mutant to assess its role in vivo in replication of phages, plasmids, and the host chromosome. Extracts of this mutant are inert in the initial conversion of the phi X174 viral strand to the duplex form, confirming the absence of the PriA activity. In vivo, the priA mutant fails to produce phi X174 phage and, remarkably, is unable to maintain plasmids that depend on the E. coli chromosome origin as well as those of ColE1. Deficiencies in cell growth and cell division are also manifest.