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).     

Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair

Studies of recombination-dependent replication (RDR) in the T4 system have revealed the critical roles played by mediator proteins in the timely and productive loading of specific enzymes onto single-stranded DNA (ssDNA) during phage RDR processes. The T4 recombination mediator protein, uvsY, is necessary for the proper assembly of the T4 presynaptic filament (uvsX recombinase cooperatively bound to ssDNA), leading to the recombination-primed initiation of leading strand DNA synthesis. In the lagging strand synthesis component of RDR, replication mediator protein gp59 is required for the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA. Together, uvsY and gp59 mediate the productive coupling of homologous recombination events to the initiation of T4 RDR. UvsY promotes presynaptic filament formation on 3' ssDNA-tailed chromosomes, the physiological primers for T4 RDR, and recent results suggest that uvsY also may serve as a coupling factor between presynapsis and the nucleolytic resection of double-stranded DNA ends. Other results indicate that uvsY stabilizes uvsX bound to the invading strand, effectively preventing primosome assembly there. Instead, gp59 directs primosome assembly to the displaced strand of the D loop/replication fork. This partitioning mechanism enforced by the T4 recombination/replication mediator proteins guards against antirecombination activity of the helicase component and ensures that recombination intermediates formed by uvsX/uvsY will efficiently be converted into semiconservative DNA replication forks. Although the major mode of T4 RDR is semiconservative, we present biochemical evidence that a conservative "bubble migration" mode of RDR could play a role in lesion bypass by the T4 replication machinery.     

Assembly and subunit stoichiometry of the functional helicase-primase (primosome) complex of bacteriophage T4

Physical biochemical techniques are used to establish the structure, subunit stoichiometry, and assembly pathway of the primosome complex of the bacteriophage T4 DNA replication system. Analytical ultracentrifugation and fluorescence anisotropy methods show that the functional T4 primosome consists of six gp41 helicase subunits that assemble into a hexagon, driven by the binding of six NTPs (or six nonhydrolyzable GTPγS analogues) that are located at and stabilize the intersubunit interfaces, together with a single tightly bound gp61 primase subunit. Assembling the components of the primosome onto a model DNA replication fork is a multistep process, but equilibrium cannot be reached along all mixing pathways. Producing a functional complex requires that the helicase hexamer be assembled in the presence of the DNA replication fork construct prior to the addition of the primase to avoid the formation of metastable DNA-protein aggregates. The gp41 helicase hexamer binds weakly to fork DNA in the absence of primase, but forms a much more stable primosome complex that expresses full and functional helicase (and primase) activities when bound to a gp61 primase subunit at a helicase:primase subunit ratio of 61. The presence of additional primase subunits does not change the molecular mass or helicase activity of the primosome, but significantly inhibits its primase activity. We develop both an assembly pathway and a minimal mechanistic model for the structure and function of the T4 primosome that are likely to be relevant to the assembly and function of the replication primosome subassemblies of higher organisms as well.     

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.     

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.     

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.     

Conversion of ϕX174 Viral DNA to Double-Stranded Form by Purified Escherichia coli Proteins

The E. coli proteins that catalyze the conversion of varphiX174 single-stranded DNA to duplex DNA have now been purified extensively. The reaction depends on dnaB, dnaC(D), dnaE, and dnaG gene products, DNA elongation factors I and II, E. coli DNA binding protein, and two additional E. coli proteins, replication factors X and Y. DNA synthesis by these proteins requires varphiX174 viral DNA, dNTPs, Mg(+2), and ATP. The product synthesized is full-length linear varphiX174 DNA. The reaction has been resolved into two steps. The first step involves the interaction of ATP and varphiX174 DNA with dnaB and dnaC(D) gene products, E. coli DNA binding protein, and replication factors X and Y in the absence of dNTPs. Subsequent dNMP incorporation requires the addition of DNA polymerase III, DNA elongation factors I and II, dnaG gene product, and dNTPs.     

Bovine papilloma virus (BPV)-encoded E1 protein contains multiple activities required for BPV DNA replication.

Replication of bovine papilloma virus (BPV) DNA requires two virus-encoded proteins, E1 and E2, while all other proteins are supplied by the host cell. Here, we describe the isolation of the E1 protein and show that it is a multifunctional protein. Purified E1 protein was required for the in vitro replication of BPV origin-containing DNA by extracts of mouse cells, as reported [Yang, L., Li, R., Mohr, I. J., Clark, R. & Botchan, M. R. (1991) Nature (London) 353, 628-632]. In addition, the E1 protein cosedimented with a number of other activities including (i) DNA helicase activity, (ii) BPV origin-containing DNA-specific binding activity, (iii) DNA-dependent ATPase activity, and (iv) BPV origin-specific unwinding of superhelical DNA. The E1 protein, acting as a helicase, moved in the 3'-->5' direction, like simian virus 40 (SV40) large tumor antigen, which plays a pivotal role in SV40 DNA replication. However, unlike the SV40 large tumor antigen, the helicase activity of E1 was stimulated 5-fold by the presence of a fork structure at the junction between single-stranded and double-stranded DNA and was supported efficiently by all eight nucleoside triphosphates. The E1-catalyzed ATPase activity required the presence of single-stranded or double-stranded DNAs.     

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).     

RAD3 protein of Saccharomyces cerevisiae is a DNA helicase.

The Saccharomyces cerevisiae RAD3 gene, which is required for cell viability and excision repair of damaged DNA, encodes an 89-kDa protein that has a single-stranded DNA-dependent ATPase activity. We now show that the RAD3 protein also possesses a helicase activity that unwinds duplex regions in DNA substrates constructed by annealing DNA fragments of 71-851 nucleotides to circular, single-stranded M13 DNA. The DNA helicase activity is dependent on the hydrolysis of ATP, has a pH optimum of approximately 5.6, and is inhibited by antibodies raised against a truncated RAD3 protein produced in Escherichia coli. The RAD3 helicase translocates along single-stranded DNA in the 5'----3' direction. The direction of RAD3 helicase movement is consistent with the possibility that it unwinds DNA duplexes in advance of the replication fork during DNA replication.     

Deletion mutants defining the Escherichia coli replication factor Y effector site sequences in pBR322 DNA.

The Escherichia coli DNA replication factor Y, along with other genetically undefined replication proteins, is involved in a dnaB-, dnaC-, and dnaG-dependent pathway of primer formation on phi X174 single-stranded circular DNA. In addition, replication factor Y has a site-specific, single-stranded DNA-dependent ATPase activity. We have previously demonstrated the presence of two factor Y effector sites on pBR322 DNA. When inserted into the filamentous phage f1R229, these sites can function as rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent origins of DNA replication. We report here the construction of deletion mutants of the two pBR322 factor Y effector sites. These deleted sites no longer function as effectors for factor Y ATPase activity nor as templates for rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent DNA synthesis. We conclude that the DNA sequences required for factor Y ATPase activity and origin function are likely to be identical.     

The 2B domain of the Escherichia coli Rep protein is not required for DNA helicase activity

The Escherichia coli Rep protein is a 3' to 5' SF1 DNA helicase required for replication of bacteriophage phiX174 in E. coli, and is structurally homologous to the E. coli UvrD helicase and the Bacillus stearothermophilus PcrA helicase. Previous crystallographic studies of Rep protein bound to single-stranded DNA revealed that it can undergo a large conformational change consisting of an approximately 130 degrees rotation of its 2B subdomain about a hinge region connected to the 2A subdomain. Based on crystallographic studies of PcrA, its 2B subdomain has been proposed to form part of its duplex DNA binding site and to play a role in duplex destabilization. To test the role of the 2B subdomain in Rep-catalyzed duplex DNA unwinding, we have deleted its 2B subdomain, replacing it with three glycines, to form the RepDelta2B protein. This RepDelta2B protein can support phiX174 replication in a rep(-) E. coli strain, although the growth rate of E. coli containing the repDelta2B gene is approximately 1.5-fold slower than with the wild-type rep gene. Pre-steady-state, single-turnover DNA unwinding kinetics experiments show that purified RepDelta2B protein has DNA helicase activity in vitro and unwinds an 18-bp DNA duplex with rates at least as fast as wild-type Rep, and with higher extents of unwinding and higher affinity for the DNA substrate. These studies show that the 2B domain of Rep is not required for DNA helicase activity in vivo or in vitro, and that it does not facilitate DNA unwinding in vitro.     

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.     

Model system for DNA replication of a plasmid DNA containing the autonomously replicating sequence from Saccharomyces cerevisiae.

A negatively supercoiled plasmid DNA containing autonomously replicating sequence (ARS) 1 from Saccharomyces cerevisiae was replicated with the proteins required for simian virus 40 DNA replication. The proteins included simian virus 40 large tumor antigen as a DNA helicase, DNA polymerase alpha.primase, and the multisubunit human single-stranded DNA-binding protein from HeLa cells; DNA gyrase from Escherichia coli, which relaxes positive but not negative supercoils, was included as a "swivelase." DNA replication started from the ARS region, proceeded bidirectionally with the synthesis of leading and lagging strands, and resulted in the synthesis of up to 10% of the input DNA in 1 h. The addition of HeLa DNA topoisomerase I, which relaxes both positive and negative supercoils, to this system inhibited DNA replication, suggesting that negative supercoiling of the template DNA is required for initiation. These results suggest that DNA replication starts from the ARS region where the DNA duplex is unwound by torsional stress; this unwound region can be recognized by a DNA helicase with the assistance of the multisubunit human single-stranded DNA-binding protein.     

Identification of two Escherichia coli factor Y effector sites near the origins of replication of the plasmids (ColE1 and pBR322.

The Escherichia coli replication factor Y has been characterized as a phi X174 (+) strand specific DNA-dependent phosphohydrolase. In conjunction with other E. coli replication proteins, factor Y is involved in the formation of heterogeneous primers that are elongated by the E. coli DNA polymerase III elongation machinery. We report here that the heat-denatured DNAs of plasmids pBR322 and ColE1 serve as effectors for the hydrolysis of ATP by factor Y. The DNA sequences of pBR322 responsible for factor Y effector activity have been localized. Two separate regions of the pBR322 chromosome support Y ATPase activity. These sequences are near the replication origin and are located on opposite DNA strands.     

Autoinhibition of Escherichia coli Rep monomer helicase activity by its 2B subdomain

DNA helicases catalyze separation of double-helical DNA into its complementary single strands, a process essential for DNA replication, recombination, and repair. The Escherichia coli Rep protein, a superfamily 1 DNA helicase, functions in DNA replication restart and is required for replication of several bacteriophages. Monomers of Rep do not display helicase activity in vitro; in fact, DNA unwinding requires Rep dimerization. Here we show that removal of the 2B subdomain of Rep to form RepDelta2B activates monomer helicase activity, albeit with limited processivity. Although both full length Rep and RepDelta2B monomers can translocate with 3' to 5' directionality along single-stranded DNA, the 2B subdomain inhibits the helicase activity of full length Rep. This suggests an autoregulatory mechanism for Rep helicase, which may apply to other nonhexameric helicases, whereby helicase activity is regulated by the rotational conformational state of the 2B subdomain; formation of a Rep dimer may relieve autoinhibition by altering the 2B subdomain orientation.     

Helicase properties of the Escherichia coli UvrAB protein complex.

The Escherichia coli UvrA protein has an associated ATPase activity with a turnover number affected by the presence of UvrB protein as well as by DNA. Specifically, the structure of DNA significantly influences the turnover rate of the UvrAB ATPase activity. Double-stranded DNA maximally activates the turnover rate 10-fold whereas single-stranded DNA maximally activates the turnover rate 20-fold, suggesting that the mode of interaction of UvrAB protein with different DNAs is distinctive. We have previously shown that the UvrAB protein complex, driven by the binding energy of ATP, can locally unwind supercoiled DNA. The nature of the DNA unwinding activity and single-stranded DNA activation of ATPase activity suggests potential helicase activity. In the presence of a number of helicase substrates, the UvrAB complex, indeed, manifests a strand-displacement activity--unwinding short duplexes and D-loop DNA, thereby generating component DNA structures. The energy for the activity is derived from ATP or dATP hydrolysis. Unlike the E. coli DnaB, the UvrAB helicase is sensitive to UV-induced photoproducts.     

Processive DNA helicase activity of the minichromosome maintenance proteins 4, 6, and 7 complex requires forked DNA structures

The minichromosome maintenance (Mcm) proteins 2-7 are required for both the initiation and elongation steps of chromosomal DNA replication. Previous studies have shown that the Mcm complex consisting of the Mcm 4, 6, and 7 proteins contains 3' to 5' DNA helicase activity with limited processivity (displacing duplex DNA regions up to 30 nt). In this report, we show that the presence of both 5' and 3' single-stranded tails in DNA helicase substrates is essential for the processive helicase activity of the Mcm complex. The presence of both 5' and 3' tails facilitated the formation of double heterohexameric complexes of Mcm4/6/7 on substrate DNA, which appeared to be essential for the processive helicase activity. The double heterohexameric complex of Mcm4/6/7, in the presence of a single-strand DNA binding protein, is capable of unwinding duplex DNA region of about 600 bp in length. These results support the hypothesis that the Mcm4/6/7 complex can function as a replication helicase.