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• W2040269512 abstract "We have expressed and purified 13 proteins predicted to be required for B. subtilis DNA replication. When combined with a circular DNA template with a 5′ unpaired flap, these proteins reconstitute replication of both the leading and lagging strands at the physiological rate. Consistent with the in vivo requirement for two DNA polymerase III replicases for B. subtilis chromosomal replication, both PolC and DnaE are required for reconstitution of the replication fork in vitro. Leading strand synthesis requires PolC plus ten proteins; lagging strand synthesis additionally requires primase and DnaE. DnaE does not serve as the lagging strand replicase, like DNA polymerase δ in eukaryotes, but instead functions like eukaryotic DNA polymerase α, adding a stretch of deoxynucleotides to the RNA primer before handoff to PolC. Primase equilibrates with the fork prior to synthesis of each Okazaki fragment, and its concentration controls the frequency of initiation and Okazaki fragment size. We have expressed and purified 13 proteins predicted to be required for B. subtilis DNA replication. When combined with a circular DNA template with a 5′ unpaired flap, these proteins reconstitute replication of both the leading and lagging strands at the physiological rate. Consistent with the in vivo requirement for two DNA polymerase III replicases for B. subtilis chromosomal replication, both PolC and DnaE are required for reconstitution of the replication fork in vitro. Leading strand synthesis requires PolC plus ten proteins; lagging strand synthesis additionally requires primase and DnaE. DnaE does not serve as the lagging strand replicase, like DNA polymerase δ in eukaryotes, but instead functions like eukaryotic DNA polymerase α, adding a stretch of deoxynucleotides to the RNA primer before handoff to PolC. Primase equilibrates with the fork prior to synthesis of each Okazaki fragment, and its concentration controls the frequency of initiation and Okazaki fragment size. The B. subtilis replisome was reconstituted with 13 purified proteins Two DNA polymerase IIIs are required: DnaE and PolC DnaE extends RNA primers by a few nucleotides before handoff to PolC PolC is responsible for the majority of DNA synthesis For several decades, E. coli has provided the prototype for biochemical understanding of the replication of a cellular chromosome (Baker and Kornberg, 1991Baker T.A. Kornberg A. Initiation of chromosomal replication.in: Eckstein F. Lilley D.M.J. Nucleic Acids and Molecular Biology. Springer-Verlag, New York1991: 84Crossref Google Scholar, Marians, 2000Marians K.J. PriA-directed replication fork restart in Escherichia coli.Trends Biochem. Sci. 2000; 25: 185-189Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Our mechanistic knowledge has been facilitated, and often led, by biochemical studies in complete systems encoded by bacteriophages λ, T4, T7, Φ29, and SV40 (Alberts, 1987Alberts B.M. Prokaryotic DNA replication mechanisms.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1987; 317: 395-420Crossref PubMed Scopus (67) Google Scholar, Benkovic et al., 2001Benkovic S.J. Valentine A.M. Salinas F. Replisome-mediated DNA replication.Annu. Rev. Biochem. 2001; 70: 181-208Crossref PubMed Scopus (265) Google Scholar, Collins et al., 1993Collins K.L. Russo A.A. Tseng B. Kelly T.J. The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication.EMBO J. 1993; 12: 4555-4566Crossref PubMed Scopus (94) Google Scholar, Nossal et al., 2007Nossal N.G. Makhov A.M. Chastain P.D. Jones C.E. Griffith J.D. Architecture of the bacteriophage T4 replication complex revealed with nanoscale biopointers.J. Biol. Chem. 2007; 282: 1098-1108Crossref PubMed Scopus (46) Google Scholar, Richardson, 1983Richardson C.C. Bacterio phage T7 minimal requirements for the replication of a duplex DNA molecule.Cell. 1983; 33: 315-317Abstract Full Text PDF PubMed Scopus (97) Google Scholar, Salas et al., 1995Salas M. Freire R. Soengas M.S. Esteban J.A. Mendez J. Bravo A. Serrano M. Blasco M.A. Lazaro J.M. Blanco L. et al.Protein-nucleic acid interactions in bacteriophage phi 29 DNA replication.FEMS Microbiol. Rev. 1995; 17: 73-82PubMed Google Scholar, Stephens and McMacken, 1997Stephens K.M. McMacken R. Functional properties of replication fork assemblies established by the bacteriophage λ O and P replication proteins.J. Biol. Chem. 1997; 272: 28800-28813Crossref PubMed Scopus (14) Google Scholar, Waga and Stillman, 1994Waga S. Stillman B. Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro.Nature. 1994; 369: 207-212Crossref PubMed Scopus (489) Google Scholar, Wang et al., 2000Wang M. Park J.S. Ishiai M. Hurwitz J. Lee S.H. Species specificity of human RPA in simian virus 40 DNA replication lies in T-antigen-dependent RNA primer synthesis.Nucleic Acids Res. 2000; 28: 4742-4749Crossref PubMed Scopus (16) Google Scholar). The viral SV40-encoded origin recognition, helicase loader, and helicase activities of the multifunctional T-antigen have provided significant knowledge related to eukaryotic replication, yet no system is available for the reconstitution of cellular eukaryotic replication forks with purified proteins. B. subtilis has provided a useful model system for understanding unique aspects of low-GC Gram-positive DNA replication, from both a genetic and biochemical perspective. Some features of E. coli replication are conserved: a PriA-mediated restart of stalled replication forks (Polard et al., 2002Polard P. Marsin S. McGovern S. Velten M. Wigley D.B. Ehrlich S.D. Bruand C. Restart of DNA replication in Gram-positive bacteria: functional characterisation of the Bacillus subtilis PriA initiator.Nucleic Acids Res. 2002; 30: 1593-1605Crossref PubMed Scopus (42) Google Scholar), a replicase comprising Pol III, β2 and a DnaX complex (Bruck and O'Donnell, 2000Bruck I. O'Donnell M.E. The DNA replication machine of a Gram-positive organism.J. Biol. Chem. 2000; 275: 28971-28983Crossref PubMed Scopus (73) Google Scholar), and a hexameric replicative helicase (Bruand et al., 1995Bruand C. Ehrlich S.D. Janniere L. Primosome assembly site in Bacillus subtilis.EMBO J. 1995; 14: 2642-2650Crossref PubMed Scopus (78) Google Scholar). Important distinctions are also apparent that suggest that E. coli uses mechanisms that are not universally conserved, even among bacteria. B. subtilis appears to use two helicase loaders, like eukaryotic cells (Velten et al., 2003Velten M. McGovern S. Marsin S. Ehrlich S.D. Noirot P. Polard P. A two-protein strategy for the functional loading of a cellular replicative DNA helicase.Mol. Cell. 2003; 11: 1009-1020Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). B. subtilis also employs a different set of proteins that intervene between the PriA initiation protein and the helicase loader (DnaD in B. subtilis versus the unrelated PriB, PriC, and DnaT in E. coli [Bruand et al., 2001Bruand C. Farache M. McGovern S. Ehrlich S.D. Polard P. DnaB, DnaD and DnaI proteins are components of the Bacillus subtilis replication restart primosome.Mol. Microbiol. 2001; 42: 245-256Crossref PubMed Scopus (77) Google Scholar]). Particularly noteworthy is the requirement for two replicases, distinct homologs of DNA polymerase III, called PolC and DnaE (Dervyn et al., 2001Dervyn E. Suski C. Daniel R. Bruand C. Chapuis J. Errington J. Janniere L. Ehrlich S.D. Two essential DNA polymerases at the bacterial replication fork.Science. 2001; 294: 1716-1719Crossref PubMed Scopus (115) Google Scholar). PolC differs from the E. coli-like DnaE in that it contains a different arrangement of conserved modules and contains the proofreading exonuclease as part of the same polypeptide chain (Hammond et al., 1991Hammond R.A. Barnes M.H. Mack S.L. Mitchener J.A. Brown N.C. Bacillus subtilis DNA polymerase III: complete sequence, overexpression, and chacterization of the polC gene.Gene. 1991; 98: 29-36Crossref PubMed Scopus (32) Google Scholar, Low et al., 1976Low R.L. Rashbaum S.A. Cozzarelli N.R. Purification and characterization of DNA polymerase III from Bacillus subtilis.J. Biol. Chem. 1976; 251: 1311-1325Abstract Full Text PDF PubMed Google Scholar). Genetic studies have demonstrated that both PolC and DnaE are required for B. subtilis replication. Under conditions of DnaE deprivation, a small amount of leading strand DNA synthesis remained while lagging strand synthesis ceased. Based on this observation, it has been proposed that PolC might be the leading strand replicase and DnaE the lagging strand polymerase (Dervyn et al., 2001Dervyn E. Suski C. Daniel R. Bruand C. Chapuis J. Errington J. Janniere L. Ehrlich S.D. Two essential DNA polymerases at the bacterial replication fork.Science. 2001; 294: 1716-1719Crossref PubMed Scopus (115) Google Scholar). Eukaryotes also use two polymerases, Pol ɛ and Pol δ, which constitute the core of the leading and lagging strand replicases, respectively (Kunkel and Burgers, 2008Kunkel T.A. Burgers P.M. Dividing the workload at a eukaryotic replication fork.Trends Cell Biol. 2008; 18: 521-527Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). A third eukaryotic polymerase, Pol α, is complexed with primase and functions to process the nascent RNA primer, adding a small number of deoxynucleotides before handing it off to the Pol δ replicase (Nethanel and Kaufmann, 1990Nethanel T. Kaufmann G. Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40.J. Virol. 1990; 64: 5912-5918Crossref PubMed Google Scholar). Estimates have been made that B. subtilis and E. coli diverged over one billion years ago, even before plants and animals (Condon, 2003Condon C. RNA processing and degradation in Bacillus subtilis.Microbiol. Mol. Biol. Rev. 2003; 67: 157-174Crossref PubMed Scopus (126) Google Scholar, and references therein). Thus, B. subtilis provides an opportunity to examine the extent to which E. coli serves as a uniform prototype for cellular replication. To enable this examination, we expressed all proteins predicted to be required for B. subtilis replication restart and used them to reconstitute a complete cellular replication fork system that will permit mechanistic-based probing of important differences, including the contribution of a second Pol III replicase. The collective work of laboratories that use genetic approaches suggested the requirement for 13 B. subtilis proteins to reconstitute a DNA replication fork. We expressed all 13 proteins, recombinantly, in E. coli. Proteins were expressed without appended tags and with the native sequence. This was done to avoid any functional perturbations that might result from unnatural sequences interfering with protein-protein interactions or other functions. All 13 proteins were purified, initially using SDS-PAGE to guide the purifications, by standard chromatographic methods (Figure 1A). We created a minicircular template, similar to those employed in other replication systems (Lee et al., 1998Lee J. Chastain P.D. Kusakabe T. Griffith J.D. Richardson C.C. Coordinated leading and lagging strand DNA synthesis on a minicircular template.Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, Nossal et al., 2007Nossal N.G. Makhov A.M. Chastain P.D. Jones C.E. Griffith J.D. Architecture of the bacteriophage T4 replication complex revealed with nanoscale biopointers.J. Biol. Chem. 2007; 282: 1098-1108Crossref PubMed Scopus (46) Google Scholar, Yang et al., 2003Yang J. Trakselis M.A. Roccasecca R.M. Benkovic S.J. The application of a minicircle substrate in the study of the coordinated T4 DNA replication.J. Biol. Chem. 2003; 278: 49828-49838Crossref PubMed Scopus (26) Google Scholar) that had a strong (50:1) GC strand bias (Figure 1B). The template was designed so that labeled dGTP was incorporated, nearly exclusively, into the lagging strand product and labeled dCTP into the lagging strand product. Using methods similar to those developed by N. Nossal (Nossal et al., 2007Nossal N.G. Makhov A.M. Chastain P.D. Jones C.E. Griffith J.D. Architecture of the bacteriophage T4 replication complex revealed with nanoscale biopointers.J. Biol. Chem. 2007; 282: 1098-1108Crossref PubMed Scopus (46) Google Scholar), we made our minicircles larger than normally employed, to minimize steric issues. Upon addition of our 13 purified B. subtilis DNA replication proteins, robust leading and lagging strand synthesis were observed (Figure 1C). The lagging strand product was shorter (∼1 Kb), corresponding to the standard size of Okazaki fragments, as observed in other systems (Chastain et al., 2000Chastain P.D. Makhov A.M. Nossal N.G. Griffith J.D. Analysis of the Okazaki fragment distributions along single long DNAs replicated by the bacteriophage T4 proteins.Mol. Cell. 2000; 6: 803-814Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, Lee et al., 2002Lee J. Chastain P.D. Griffith J.D. Richardson C.C. Lagging strand synthesis in coordinated DNA synthesis by bacteriophage T7 replication proteins.J. Mol. Biol. 2002; 316: 19-34Crossref PubMed Scopus (53) Google Scholar, Wu et al., 1992Wu C.A. Zechner E.L. Marians K.J. Coordinated leading and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size.J. Biol. Chem. 1992; 267: 4030-4044Abstract Full Text PDF PubMed Google Scholar). In assays where single proteins were omitted from an optimized replication fork assay, we observed that all 13 proteins are required for significant levels of lagging strand synthesis (Figure 2A). Primase and DnaE could be omitted from the assay with maintenance of leading strand synthesis. This observation is consistent with the role of primase in generating primers for Okazaki fragment synthesis and the proposed role of DnaE in serving as the lagging strand replicase (Dervyn et al., 2001Dervyn E. Suski C. Daniel R. Bruand C. Chapuis J. Errington J. Janniere L. Ehrlich S.D. Two essential DNA polymerases at the bacterial replication fork.Science. 2001; 294: 1716-1719Crossref PubMed Scopus (115) Google Scholar). Since DnaE could be omitted and leading strand synthesis maintained, we re-examined protein dependencies in the presence of PolC as the only DNA polymerase. Again, we observed dependence on all proteins except primase (and DnaE) for leading strand synthesis (Figure 2B). The low levels of lagging strand synthesis observed remained dependent on all proteins. In the absence of PolC, the DnaE-supported reaction lost specificity for proteins known to be required for replication in vivo. For example, we observed no leading strand requirement for the DnaC helicase, and the reaction is stimulated by omission of PriA (Figure 2C). Thus, the low level of replication observed in the absence of PolC is inauthentic, in violation of known genetic requirements. To determine whether the requirement for one DNA polymerase could be overcome by increasing concentrations of the other, we varied polymerase concentration and quantified leading and lagging strand synthesis independently. Regardless of the PolC concentration, leading strand synthesis was reduced approximately 2-fold in the absence of DnaE (Figure 3A). In the presence of the full complement of proteins, leading strand synthesis could not be established in the absence of PolC, even at elevated DnaE concentration. Lagging strand synthesis was completely dependent upon both PolC and DnaE, regardless of the polymerase concentration used (Figure 3B). DNA replication forks progress at ∼500 bp/s in B. subtilis at 30°C (Wang et al., 2007Wang J.D. Sanders G.M. Grossman A.D. Nutritional control of elongation of DNA replication by (p)ppGpp.Cell. 2007; 128: 865-875Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). We examined the elongation rate of reconstituted replication forks in the presence of PolC and/or DnaE (Figure 4). In the fully reconstituted system, forks progressed at 560 nt/s, consistent with the in vivo velocity. PolC alone supported a reaction at a nearly equivalent rate. However, the DnaE-supported reaction was very slow (25 nt/s). This latter observation is consistent with DnaE not functioning to elongate the leading strand at the replication fork, a requirement for generating the lagging strand template. We also measured the rate of elongation by DnaE on long single-stranded templates that would more closely mimic the template for lagging strand synthesis. DnaE was very slow in the absence (13 nt/s) or presence (75 nt/s) of the τ complex and β2 (Figure S2), consistent with previous observations (Bruck and O'Donnell, 2000Bruck I. O'Donnell M.E. The DNA replication machine of a Gram-positive organism.J. Biol. Chem. 2000; 275: 28971-28983Crossref PubMed Scopus (73) Google Scholar). In the only other cellular system reconstituted at the replication fork level, E. coli, it has been found that DnaG primase cycles on and off the replication fork, through association with DnaB helicase, with the synthesis of each RNA primer for Okazaki fragment synthesis (Tougu and Marians, 1996Tougu K. Marians K.J. The interaction between helicase and primase sets the replication fork clock.J. Biol. Chem. 1996; 271: 21398-21405Crossref PubMed Scopus (115) Google Scholar, Wu et al., 1992Wu C.A. Zechner E.L. Marians K.J. Coordinated leading and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size.J. Biol. Chem. 1992; 267: 4030-4044Abstract Full Text PDF PubMed Google Scholar). For this reason, the length of Okazaki fragments is inversely proportional to the DnaG primase concentration. Higher concentrations of primase lead to more frequent associations with helicase, leading to more frequent priming and Okazaki fragment formation. We also observe a decreasing Okazaki fragment size with increasing DnaG primase concentration in our B. subtilis system. Okazaki fragment length varies from ∼1.6 kb at 0.4 nM primase down to 0.5 kb at 200 nM primase (Figure 5A). PolC does not work efficiently as the sole DNA polymerase. We specifically looked at the ability of PolC and DnaE to elongate primers during Okazaki fragment synthesis by monitoring [32P]-dGTP incorporation (Figure 5B). Only DnaE could efficiently elongate RNA primers generated at the replication fork. We next examined the relative abilities of PolC and DnaE to use RNA oligonucleotides for DNA synthesis on single-stranded model templates for Okazaki fragment initiation. We observed that both PolC and DnaE could efficiently elongate a DNA primer, but under the conditions used, only DnaE could elongate an RNA primer (Figure 6A). To eliminate the potential for an artifact created by an RNA degradative contaminant in our PolC preparations, we performed two controls. In the first, we showed that mixing PolC with DnaE did not destroy the ability of DnaE to use an RNA primer (Figure 6A). In the second, we demonstrated that primer degradation did not occur during any of the PolC and/ or DnaE-containing reactions (Figure 6B). In separate experiments, we used elevated levels of polymerase and dNTPs to see if we could force PolC to use an RNA primer (see Figure S3 available online). Both PolC and DnaE used DNA-primed templates with nearly equivalent efficiency (Km for dNTPs of 16 and 18 μM, respectively). RNA primers were also elongated by DnaE efficiently (KmdNTP = 13 μM). PolC, even at 2-fold higher concentration, was inert on an RNA primer until dNTP concentrations were elevated (>25 μM; Figure S3), supporting the notion that PolC does not efficiently elongate an RNA primer. The preceding experiments suggest that DnaE extends RNA primers initially and then hands them off to PolC for more extensive, rapid elongation. To test this, we used two assays, both RNA primed to force DnaE elongation prior to any potential action by PolC. For the first assay, to closely mimic the situation on the lagging strand of the replication fork, we set up a general priming system analogous to the one first developed with E. coli proteins (Arai and Kornberg, 1979Arai K. Kornberg A. A general priming system employing only dnaB protein and primase for DNA replication.Proc. Natl. Acad. Sci. USA. 1979; 76: 4308-4312Crossref PubMed Scopus (84) Google Scholar). Like in the E. coli system, we used an SSB-coated long single-stranded template, loaded helicase using the requisite accessory proteins and generated primers by the reversible association of primase with helicase (Figures 7A and 7B). In this system, PolC and DnaE exhibited higher levels of synthesis than either polymerase alone or the total of their activities, indicating a synergistic effect, consistent with PolC rapidly elongating primers after an initial slow processing by DnaE (Figure 7A). PolC only showed significant stimulation after a kinetic lag (1.5 min under the conditions used) consistent with a limiting step preceding its action. Additional support for a handoff between DnaE and PolC was gained by exploiting HBEMAU, a PolC-specific inhibitor developed by Brown, Wright, and colleagues that binds protein and primed DNA forming a dead-end ternary complex (Low et al., 1974Low R.L. Rashbaum S.A. Cozzarelli N.R. Mechanism of inhibition of Bacillus subtilis DNA polymerase III by the arylhydrazinopyrimidine antimicrobial agents.Proc. Natl. Acad. Sci. USA. 1974; 71: 2973-2977Crossref PubMed Scopus (20) Google Scholar, Tarantino et al., 1999Tarantino Jr., P.M. Zhi C. Gambino J.J. Wright G.E. Brown N.C. 6-Anilinouracil-based inhibitors of Bacillus subtilis DNA polymerase III: antipolymerase and antimicrobial structure-activity relationships based on substitution at uracil N3.J. Med. Chem. 1999; 42: 2035-2040Crossref PubMed Scopus (40) Google Scholar). PolC alone cannot efficiently use RNA primers, but if it normally gains access to primers by a handoff mechanism after minimal extension by DnaE, addition of HBEMAU would be expected to inhibit the reaction markedly. Indeed, we observe such inhibition (Figure 7B). HBEMAU does not affect the reaction with DnaE by itself but nearly completely inhibits reactions that contain PolC, indicating PolC obtains access to primer termini from DnaE. Control experiments show the same effect is observed, even when DnaE and accessory proteins are incubated with RNA-primed DNA before the addition of PolC (Figure S4). In a second assay, we annealed synthetic RNA primers, again to mimic the initiation reaction on the lagging strand. We performed these assays at a constant level of PolC (if present) and varied DnaE for a set time. In spite of PolC exhibiting little activity on RNA primers by itself, it afforded significant stimulation in the presence of DnaE, especially at low DnaE levels (Figure 7C). PolC also stimulated reactions containing saturating levels of DnaE, strongly supporting a cooperative reaction where a handoff occurs between polymerases. As with the general priming assays, inclusion of the PolC-specific inhibitor HBEMAU resulted in nearly complete inhibition of the reaction, indicating PolC normally gains access to primer termini early in the Okazaki fragment reaction cycle after limited synthesis by DnaE and a handoff to PolC (Figure 7D). Guided largely by genetics-based predictions, we have expressed 13 B. subtilis proteins in E. coli and found that, when added together on a synthetic rolling circle template, they reconstitute a functional DNA replication fork that moves at the same rate observed in vivo. We observe synthesis of a long leading strand and discontinuous synthesis of Okazaki fragments of the predicted size on the lagging strand. In sharp contrast to the model derived from E. coli, synthesis is dependent upon two distinct DNA polymerase IIIs, PolC and DnaE. Lagging strand synthesis is dependent on the presence of primase and, as would be predicted, leading strand synthesis is not. We expect, in future studies guided by proteomic and functional biochemical approaches, that additional auxiliary proteins will be discovered. But for now, it appears that the 13 proteins already identified enable all of the major features required for rapid, processive replication fork progression. This B. subtilis rolling circle replication fork system represents the second cellular system reconstituted from purified proteins to date. Although advanced knowledge is available in archaeal and eukaryotic systems, this feat has not been accomplished yet, perhaps due to missing components or complications imposed by complex regulation. The availability of this second cellular replication system from a divergent organism provides an opportunity to explore which features of the E. coli-based model are conserved and what variations can occur. As described in more detail in the introduction, many of the basic features of low-GC Gram-positive DNA replication are conserved, but important distinctions indicate that the applicability of the E. coli model is not universal. Our studies establish that proteins predicted by partial DNA replication reactions and from genetic studies (see the Introduction) are sufficient to reconstitute a full replication fork reaction, initiated on a flapped rolling circle template. Our work demonstrates, as expected from the E. coli paradigm (Marians, 2000Marians K.J. PriA-directed replication fork restart in Escherichia coli.Trends Biochem. Sci. 2000; 25: 185-189Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), that primase is required exclusively for lagging strand synthesis and that it equilibrates with the replication fork between synthesis of successive Okazaki fragments. Decreasing primase concentration leads to less-frequent associations and longer Okazaki fragments. This study supports the notion that PolC serves as the leading strand replicase. Rapid, efficient leading strand synthesis continues in the absence of DnaE. Consistent with previous predictions (Dervyn et al., 2001Dervyn E. Suski C. Daniel R. Bruand C. Chapuis J. Errington J. Janniere L. Ehrlich S.D. Two essential DNA polymerases at the bacterial replication fork.Science. 2001; 294: 1716-1719Crossref PubMed Scopus (115) Google Scholar), lagging strand synthesis is dependent upon DnaE. Lagging strand synthesis is also dependent upon the presence of PolC, but that could be due to an indirect role: PolC is required to generate the template for lagging strand synthesis. However, certain characteristics of DnaE suggest it is not the sole lagging strand replicase and that its role more closely mimics that of Pol α in eukaryotes (Stillman, 2008Stillman B. DNA polymerases at the replication fork in eukaryotes.Mol. Cell. 2008; 30: 259-260Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). First, DnaE is a very slow polymerase, elongating at a maximal rate of 75 nt/s under the experimental conditions used for reconstitution of replication forks. This is significantly slower than the in vivo rate of fork progression (∼500 nt/s; Wang et al., 2007Wang J.D. Sanders G.M. Grossman A.D. Nutritional control of elongation of DNA replication by (p)ppGpp.Cell. 2007; 128: 865-875Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) and could not support lagging strand synthesis, at least in a coupled system. Second, DnaE, like Pol α, lacks an intrinsic proofreading nuclease and has been demonstrated to be error prone in vitro (Bruck et al., 2003Bruck I. Goodman M.F. O'Donnell M.E. The essential C family DnaE polymerase is error-prone and efficient at lesion bypass.J. Biol. Chem. 2003; 278: 44361-44368Crossref PubMed Scopus (40) Google Scholar, Le Chatelier et al., 2004Le Chatelier E. Becherel O.J. D'Alencon E. Canceill D. Ehrlich S.D. Fuchs R.P. Janniere L. Involvement of DnaE, the second replicative DNA polymerase from Bacillus subtilis, in DNA mutagenesis.J. Biol. Chem. 2004; 279: 1757-1767Crossref PubMed Scopus (46) Google Scholar), which is inconsistent with an extensive role in replication that would lead to unacceptable mutation rates on the lagging strand template. DnaE has the ability to preferentially use RNA primers under the experimental conditions employed in our reconstituted fork assay. At low concentrations of polymerase and dNTPs that may mimic concentrations available to replication forks in vivo, we observe almost exclusive use of RNA primers by DnaE. In control experiments, the efficiency of priming by DNA primers with a sequence equivalent to the RNA primers is comparable between the two polymerases. A similar situation has recently been observed in a two-polymerase herpes virus replication reaction, where it was proposed that Pol α serves to process RNA primers, adding deoxynucleotides before handing off to a herpes-encoded DNA polymerase that cannot use RNA primers at physiological dNTP concentration (Cavanaugh and Kuchta, 2009Cavanaugh N.A. Kuchta R.D. Initiation of new DNA strands by the herpes simplex virus-1 primase-helicase complex and either herpes DNA polymerase or human DNA polymerase α.J. Biol. Chem. 2009; 284: 1523-1532Crossref PubMed Scopus (25) Google Scholar). Consistent with a handoff mechanism whereby DnaE initially extends an RNA primer followed by more extensive rapid elongation by PolC, we observe a synergistic effect if both polymerases are present simultaneously in reactions in which RNA primers are used. Blockage of DNA replication by the PolC-specific inhibitor HBEMAU provides additional evidence for a handoff mechanism whereby PolC gains access to 3′ termini of elongating strands early in the normal reaction. HBEMAU forms a ternary complex with primed DNA and PolC, arresting synthesis and denying access to other polymerases (Low et al., 1974Low R.L. Rashbaum S.A. Cozzarelli N.R. Mechanism of inhibition of Bacillus subtilis DNA polymerase III by the arylhydrazinopyri" @default.
• W2040269512 created "2016-06-24" @default.
• W2040269512 creator A5005151768 @default.
• W2040269512 creator A5047173466 @default.
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• W2040269512 date "2010-01-01" @default.
• W2040269512 modified "2023-10-12" @default.
• W2040269512 title "Reconstitution of the B. subtilis Replisome with 13 Proteins Including Two Distinct Replicases" @default.
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