Early Steps of Bacillus subtilis Primosome Assembly
2001; Elsevier BV; Volume: 276; Issue: 49 Linguagem: Inglês
10.1074/jbc.m101996200
ISSN1083-351X
AutoresStéphanie Marsin, S. Mcgovern, S. Dusko Ehrlich, Claude Bruand, Patrice Polard,
Tópico(s)Bacteriophages and microbial interactions
ResumoPrimosomes are nucleoprotein assemblies designed for the activation of DNA replication forks. Their primary role is to recruit the replicative helicase onto single-stranded DNA. The "replication restart" primosome, defined in Escherichia coli, is involved in the reactivation of arrested replication forks. Binding of the PriA protein to forked DNA triggers its assembly. PriA is conserved in bacteria, but its primosomal partners are not. InBacillus subtilis, genetic analysis has revealed three primosomal proteins, DnaB, DnaD, and DnaI, that have no obvious homologues in E. coli. Interestingly, they are involved in primosome function both at arrested replication forks and at the chromosomal origin. Our biochemical analysis of the DnaB and DnaD proteins unravels their role in primosome assembly. They are both multimeric and bind individually to DNA. Furthermore, DnaD stimulates DnaB binding activities. DnaD alone and the DnaD/DnaB pair interact specifically with PriA of B. subtilis on several DNA substrates. This suggests that the nucleoprotein assembly is sequential in the PriA, DnaD, DnaB order. The preferred DNA substrate mimics an arrested DNA replication fork with unreplicated lagging strand, structurally identical to a product of recombinational repair of a stalled replication fork. Primosomes are nucleoprotein assemblies designed for the activation of DNA replication forks. Their primary role is to recruit the replicative helicase onto single-stranded DNA. The "replication restart" primosome, defined in Escherichia coli, is involved in the reactivation of arrested replication forks. Binding of the PriA protein to forked DNA triggers its assembly. PriA is conserved in bacteria, but its primosomal partners are not. InBacillus subtilis, genetic analysis has revealed three primosomal proteins, DnaB, DnaD, and DnaI, that have no obvious homologues in E. coli. Interestingly, they are involved in primosome function both at arrested replication forks and at the chromosomal origin. Our biochemical analysis of the DnaB and DnaD proteins unravels their role in primosome assembly. They are both multimeric and bind individually to DNA. Furthermore, DnaD stimulates DnaB binding activities. DnaD alone and the DnaD/DnaB pair interact specifically with PriA of B. subtilis on several DNA substrates. This suggests that the nucleoprotein assembly is sequential in the PriA, DnaD, DnaB order. The preferred DNA substrate mimics an arrested DNA replication fork with unreplicated lagging strand, structurally identical to a product of recombinational repair of a stalled replication fork. single-stranded DNA polymerase chain reaction polyacrylamide gel electrophoresis double-stranded DNA Chromosomal replication depends on the initial assembly of replication forks at defined origins and on the re-assembly of the ongoing replication forks in case of their arrest. Extensive genetic and biochemical studies in the Gram-negative bacteriumEscherichia coli have unraveled two mechanisms for activating and reactivating DNA replication. The first occurs at the unique origin (oriC) of the circular chromosome and ensures the accurate timing of replication within the cell cycle (for reviews see Refs. 1Marians K.J. Neidhardt F.C. Escherichia coli and Salmonella. American Society of Microbiology Press, Washington, D. C.1996: 749-763Google Scholar and 2Messer W. Weigel C. Neidhardt F.C. Escherichia coli and Salmonella. American Society for Microbiology Press, Washington, D. C.1996: 1579-1601Google Scholar). The triggering factor of this highly regulated initiation process is the DnaA protein, which specifically recognizesoriC. The second reactivation process has been described more recently (3Kogoma T. Microbiol. Mol. Biol. Rev. 1997; 61: 212-238Crossref PubMed Scopus (432) Google Scholar, 4Cox M.M. Prog. Nucleic Acids Res. Mol. Biol. 1999; 63: 311-366Crossref PubMed Google Scholar, 5Cox M.M. Goodman M.F. Kreuzer K.N. Sherratt D.J. Sandler S.J. Marians K.J. Nature. 2000; 404: 37-41Crossref PubMed Scopus (856) Google Scholar, 6Marians K.J. Trends Biochem. Sci. 2000; 25: 185-189Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 7Sandler S.J. Marians K.J. J. Bacteriol. 2000; 182: 9-13Crossref PubMed Scopus (175) Google Scholar). Its initiator is the PriA protein, which promotes replication restart by binding to particular DNA structures (8Marians K.J. Prog. Nucleic Acids Res. Mol. Biol. 1999; 63: 39-67Crossref PubMed Google Scholar). The replication routes opened by DnaA and PriA in E. colihave been reproduced in vitro with model DNA substrates and purified protein components (for review see Ref. 9Masai H. Arai K.I. Front. Biosci. 1996; 1: 48-58Crossref Scopus (22) Google Scholar). In both cases, these specialized proteins promote the recruitment of the replicative DnaB helicase on ssDNA.1 This enzyme can be viewed as the keystone of the replication machinery, because it melts the DNA double helix, is tightly associated with the τ subunit of the DNA polymerase III holoenzyme, and interacts distributively with the DnaG primase that primes DNA synthesis (1Marians K.J. Neidhardt F.C. Escherichia coli and Salmonella. American Society of Microbiology Press, Washington, D. C.1996: 749-763Google Scholar). The proteins required for the recruitment of the DnaB-DnaG pair are known as primosomal proteins, and the nucleoprotein complex resulting from their assembly is designated the primosome. The DnaA-dependent primosome forms at oriC and includes DnaA, DnaB, and DnaG. DnaB loading onto ssDNA is assisted by the DnaC primosomal protein (10Wahle E. Lasken R.S. Kornberg A. J. Biol. Chem. 1989; 264: 2469-2475Abstract Full Text PDF PubMed Google Scholar). The PriA-dependent primosome assembles on two distinct DNA substrates recognized by PriA. The first is a sequence designated pas (forprimosome assembly site), discovered in the genome of the bacteriophage φX174 and required for the conversion of circular ssDNA to dsDNA (8Marians K.J. Prog. Nucleic Acids Res. Mol. Biol. 1999; 63: 39-67Crossref PubMed Google Scholar). The second is a "D-loop" structure, which mimics the proposed product of recombinational repair of a stalled replication fork (4Cox M.M. Prog. Nucleic Acids Res. Mol. Biol. 1999; 63: 311-366Crossref PubMed Google Scholar, 5Cox M.M. Goodman M.F. Kreuzer K.N. Sherratt D.J. Sandler S.J. Marians K.J. Nature. 2000; 404: 37-41Crossref PubMed Scopus (856) Google Scholar, 11Jones J.M. Nakai H. EMBO J. 1997; 16: 6886-6895Crossref PubMed Google Scholar, 12McGlynn P. Al-Deib A.A. Liu J. Marians K.J. Lloyd R.G. J. Mol. Biol. 1997; 270: 212-221Crossref PubMed Scopus (164) Google Scholar, 13Liu J. Marians K.J. J. Biol. Chem. 1999; 274: 25033-25041Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The PriA-dependent primosome, also designated the "replication restart primosome" (7Sandler S.J. Marians K.J. J. Bacteriol. 2000; 182: 9-13Crossref PubMed Scopus (175) Google Scholar), contains the proteins PriA, PriB, PriC, and DnaT, which assemble sequentially in the order given to recruit the helicase DnaB, also with the help of DnaC, and the primase DnaG. PriA is a helicase moving on ssDNA with the 3′ → 5′ direction (8Marians K.J. Prog. Nucleic Acids Res. Mol. Biol. 1999; 63: 39-67Crossref PubMed Google Scholar). Its DNA melting activity is dispensable for in vitroprimosome assembly on the pas sequence and on an artificial D-loop structure (13Liu J. Marians K.J. J. Biol. Chem. 1999; 274: 25033-25041Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 14Zavitz K.H. Marians K.J. J. Biol. Chem. 1993; 268: 4337-4346Abstract Full Text PDF PubMed Google Scholar) but is essential on a nearly fully double-stranded forked DNA (15Jones J.M. Nakai H. J. Mol. Biol. 1999; 289: 503-516Crossref PubMed Scopus (86) Google Scholar). In this particular case, it has been proposed that the PriA helicase activity generates the ssDNA substrate required for the installation of the DnaB helicase. Nevertheless, the helicase activity of PriA appears dispensable for its pivotal role in the "coordinated processing of damaged replication forks" (5Cox M.M. Goodman M.F. Kreuzer K.N. Sherratt D.J. Sandler S.J. Marians K.J. Nature. 2000; 404: 37-41Crossref PubMed Scopus (856) Google Scholar, 14Zavitz K.H. Marians K.J. J. Biol. Chem. 1993; 268: 4337-4346Abstract Full Text PDF PubMed Google Scholar,16Sandler S.J. Genetics. 2000; 155: 487-497PubMed Google Scholar). DnaA, PriA, the replicative helicase, and the primase are conserved in bacteria, arguing for generalization of the E. coli DNA replication initiation schemes to these microorganisms. Genetic and biochemical analyses conducted on DnaA and PriA of the Gram-positive bacterium Bacillus subtilis have confirmed their primosomal function (17Moriya S. Imai Y. Hassan A.K. Ogasawara N. Plasmid. 1999; 41: 17-29Crossref PubMed Scopus (60) Google Scholar). 2P. Polard, S. Marsin, S. McGovern, M. Velten, D. Wigley, S. D. Ehrlich, and C. Bruand, submitted for publication.2P. Polard, S. Marsin, S. McGovern, M. Velten, D. Wigley, S. D. Ehrlich, and C. Bruand, submitted for publication. However, there is ample evidence for a striking difference in the composition of B. subtilis primosomes relative to their E. colicounterparts. First, no obvious homologues of PriB, PriC, and DnaT are encoded by the B. subtilis genome (19Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessieres P. Bolotin A. Borchert S. Borriss R. Boursier L. Brans A. Braun M. Brignell S.C. Bron S. Brouillet S. Bruschi C.V. Caldwell B. Capuano V. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3088) Google Scholar). Second, threeB. subtilis essential genes,dnaB, 3Note that the B. subtilis DnaB protein is distinct from the E. coli DnaB helicase. TheB. subtilis helicase is named DnaC.3Note that the B. subtilis DnaB protein is distinct from the E. coli DnaB helicase. TheB. subtilis helicase is named DnaC. dnaD, and dnaI, are required for the PriA-dependent primosome activity (20Bruand C. Ehrlich S.D. Jannière L. EMBO J. 1995; 14: 2642-2650Crossref PubMed Scopus (78) Google Scholar). Two of these,dnaB and dnaD, encode proteins that have no homologues in E. coli. The product of the third, DnaI, has a marginal sequence similarity with the E. coli DnaC (21Koonin E.V. Nucleic Acids Res. 1992; 20: 1997Crossref PubMed Scopus (45) Google Scholar, 22Bruand C. Ehrlich S.D. Microbiology. 1995; 141: 1199-1200Crossref PubMed Scopus (24) Google Scholar), interacts with the B. subtilis DnaC3 helicase and could be the counterpart of the E. coli helicase loader (23Imai Y. Ogasawara N. Ishigo-oka D. Kadoya R. Daito T. Moriya S. Mol. Microbiol. 2000; 36: 1037-1048Crossref PubMed Scopus (57) Google Scholar). However, suppressors of PriA deficiency in E. coli map in the C-terminal moiety of DnaC (24Sandler S.J. Samra H.S. Clark A.J. Genetics. 1996; 143: 5-13Crossref PubMed Google Scholar), whereas in B. subtilis they map in the C-terminal part of DnaB and not in DnaI (25Bruand C. Farache M. McGovern S. Ehrlich S.D. Polard P. Mol. Microbiol. 2001; 42: 245-255Crossref PubMed Scopus (78) Google Scholar). These observations point to the intriguing differences between the E. coli and B. subtilis systems, further underlined by the fact that none of the PriA-dependent primosome components is required for initiation of DNA replication inE. coli, whereas all of the components but PriA are essential for this process in B. subtilis(25Bruand C. Farache M. McGovern S. Ehrlich S.D. Polard P. Mol. Microbiol. 2001; 42: 245-255Crossref PubMed Scopus (78) Google Scholar).2 We report an in vitro study of the assembly of the B. subtilis replication restart primosome. We have purified the DnaB and DnaD proteins and studied their individual and concerted activities in the presence of various DNA substrates related to arrested DNA forks. We present evidence that DnaB and DnaD are multimeric and display affinity for DNA. Furthermore, DnaD stimulates the binding of DnaB to ssDNA and to DNA molecules carrying a 5′ ssDNA tail. In the presence of PriA, which is a better ssDNA and forked DNA-binding protein, DnaD alone and the DnaD/DnaB pair assemble specifically and preferentially on the fork structures with 5′ ssDNA tails. These molecules include the correct ssDNA strand for the proper loading of the replicative helicase. Therefore, we suggest that the assembly of PriA, DnaD, and DnaB promote loading of the replicative helicase DnaC, possibly assisted by DnaI, at the arrested replication forks. Finally, we speculate that DnaD and DnaB play similar roles in the DnaA-dependent primosome that promotes initiation of chromosomal replication. E. colistrain MiT898 (26Polard P. Ton-Hoang B. Haren L. Bétermier M. Walczak R. Chandler M. J. Mol. Biol. 1996; 264: 68-81Crossref PubMed Scopus (52) Google Scholar) was used for all plasmid constructions except that of the PriA Ec expressing plasmid, for which the E. coli strain JC19008 priA2::kanharboring the suppressive mutation dnaC809 (24Sandler S.J. Samra H.S. Clark A.J. Genetics. 1996; 143: 5-13Crossref PubMed Google Scholar) was used. Strains were grown in Luria broth supplemented with 25 mg·ml−1 of thymine (27Miller J.H. A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory Press, New York1992Google Scholar) and ampicillin (100 μg·ml−1). PriA BS and DnaB overexpression was carried out in MiT898, PriA Ec in JC19006 (24Sandler S.J. Samra H.S. Clark A.J. Genetics. 1996; 143: 5-13Crossref PubMed Google Scholar), and DnaD inE. coli strain B834(DE3) (28Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4772) Google Scholar). All standard DNA manipulations were carried out as described in Sambrook et al. (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Radiolabeled nucleotide [γ-32P]ATP was from ICN (specific activity of 4500 Ci mmol−1). Oligonucleotides were from Genset (France). All enzymes used for DNA cloning and modification were purchased from Roche Molecular Biochemicals or New England Biolabs and used as recommended. Chitin resin was from New England Biolabs. For constructing pSMG24,priA Ec was PCR-amplified from plasmid pAPJ422 with Osmg1-Osmg49 as primers, cleaved byNdeI, and inserted between the NdeI andSapI sites of pCYB1 (New England Biolabs). TheSapI site was filled in with Klenow polymerase prior to ligation. Most of the cloned priA Ec gene was exchanged by that of plasmid pAPJ422 byNdeI/RsrII restriction. The remaining sequence ofpriA Ec carried by pSMG24 originating from the PCR fragment has been verified as follows: Osmg1, 5′ GAGCGGATAACAATTTCACACAGG 3′; Osmg49, 5′ ACCCTCAATCGGATCAACATCCA3 ′. For constructing pSMG6, dnaB was PCR-amplified from chromosomal DNA of B. subtilis strain 168 with Osmg9-Osmg10 as primers, cleaved by the NdeI and SapI, and inserted in pCYB1 cleaved by the two enzymes. dnaB sequence cloned in pSMG6 has been verified as follows: Osmg9, 5′ GAATTCCATATGGCTGACTATTGGAAAGAT 3′; Osmg10, 5′ GAATTCGCTCTTCCGCAATAGGCAGAGTATTTTTTCAGTT 3′. Construction of pSMG22 was in two steps. First, dnaDwas PCR-amplified from chromosomal DNA of B. subtilis strain 168 with Osmg13 and Osmg14 primers, digested with NdeI andSapI, and inserted in pCYB1 cleaved by the two enzymes. This generated plasmid pSMG8. The sequence of dnaD cloned in pSMG8 has been verified. DnaD expression from this plasmid in MiT898 strain gave mainly the expected protein accompanied, however, by two proteins originating from internal initiation of translation withindnaD (not shown). To reduce synthesis of these two products, DnaD translation was placed under the control of the stronger translational signals of the φ10 gene carried by the pTYB1 vector (New England Biolabs). The exchange (usingNdeI-SapI restriction), which gave pSMG22, placed the expression of DnaD under the transcriptional control of the T7 RNA polymerase. As expected, the synthesis of the shorter DnaD derivatives was highly diminished. Osmg13, 5′ GAATTCCATATGAAAAAACAGCAATTTATTG 3′; Osmg14, 5′ GAATTCGCTCTTCCGCATTGTTCAAGCCAATTGTAAAAAG 3′. All protein manipulations were at 4 °C. The purification columns used were all Hitrap from Amersham Pharmacia Biotech, as was the desalting PD10 column. The four proteins were purified from fusion proteins with the in vitro excisable Intein-Chitin-Binding-Domain Tag (New England Biolabs). PriA Bs purification will be described elsewhere.2 The same procedure was used for PriA Ec, except that expression was in strain JC19008 carrying plasmid pSMG24. DnaD overexpression was in E. coli strain B834 (DE3), cultivated at 30 °C in 1 liter of LB untilA 600 reached ∼1, and then transferred to 24 °C for 4 h in the presence of 0.5 mmisopropyl-1-thio-β-d-galactopyranoside to induce protein expression. Cells were harvested, resuspended in 12 ml of ice-cold HEN500-T buffer (20 mm Hepes, 0.1 mm EDTA, 500 mm NaCl, 0.1% Triton X-100, pH 7.6), and broken by sonication (Vibracell 72408 sonicator from Bioblock was used). The lysate was centrifuged at 20,000 × gfor 1 h, and supernatant was loaded onto 2 ml of chitin beads and washed with HEN500; the protein was separated from intein by addition of 30 mm dithiothreitol. After overnight incubation, the eluted proteins were further purified by conventional chromatography. DnaD was diluted three times with buffer Q50 (buffer Q: 50 mm Tris, 0.1 mm EDTA, 1 mmdithiothreitol, pH 8, supplemented with 50 mm NaCl) and applied to a Q column equilibrated with the same buffer. The flow-through containing DnaD was bound to a heparin column equilibrated with buffer Q50. DnaD was eluted with a linear NaCl gradient in buffer Q and collected at 250 mm NaCl. To produce DnaB, E. coli strain MiT898 harboring pSMG6 was grown at 30 °C for 15 h without isopropyl-1-thio-β-d-galactopyranoside induction. The first steps of DnaB purification were as for DnaD, except that the sonication was carried out in L buffer (20 mm Tris, 500 mm NaCl, 0.1 mm EDTA, 0.1% Triton X-100, pH 8). DnaB was eluted from chitin beads, precipitated with 45% saturation ammonium sulfate, resuspended in buffer Q50, and desalted on PD10 column equilibrated in the same buffer. DnaB was bound to a Q column and washed with Q buffer supplemented with 120 mmNaCl, and DnaB was eluted in Q buffer with 250 mm NaCl. DnaB-containing fractions were diluted twice with Q50 and loaded on a heparin column. DnaB was eluted in Q buffer supplemented with 400 mm NaCl, desalted on PD10 in Q50, and finally bound on a SP column. DnaB was eluted with a linear NaCl gradient in Q buffer and collected at 400 mm NaCl. A polypeptide of about 110 kDa, co-purified with DnaB, represented less than 2% of the total protein preparation. This unknown protein could not be separated from DnaB either by gel filtration or by sucrose gradient centrifugation at high ionic strength (data not shown), suggesting a stable interaction of DnaB with an unknown E. coli protein. Purified PriA Bs, PriA Ec, DnaB, and DnaD proteins were stored at −20 °C in the presence of 50% glycerol. They kept their characteristic "protein-DNA" and "protein-protein" interacting activities for at least 6 months. Protein concentrations were estimated by Bradford analysis using the Bio-Rad Protein Assay. Electrophoretic analysis of protein samples was carried out by the SDS-PAGE method of Laemmli (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Coomassie staining of the gels was performed as described before (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Experiments to determine Stokes radius (Å) and the sedimentation coefficient (s) were carried out in TM buffer (20 mm Tris, 0.1 mmEDTA, 50 mm NaCl, 0.01% Triton X-100, pH 8). The Stokes radius of the different proteins was determined using a Superose 12 HR 10/30 gel filtration column (Amersham Pharmacia Biotech). The column was standardized with proteins of known Stokes radius: thyroglobulin (669 kDa, 85 Å), ferritin (444 kDa, 61 Å), catalase (232 kDa, 52 Å), bovine serum albumin (67 kDa, 35 Å), and ovalbumin (43 kDa, 30 Å). Sedimentation coefficients were estimated using a 12-ml linear 5–24% sucrose gradient centrifuged for 16 h at 38,000 rpm in a Beckman SW40 rotor. Internal standards included for calculating sedimentation coefficients were catalase (11.3 s), aldolase (7.4s), bovine serum albumin (4.2 s), and ovalbumin (3.5 s). Sedimentation coefficients, Stokes radius, and molecular weights were estimated as previously described (31Kolb S.J. Hudmon A. Ginsberg T.R. Waxham M.N. J. Biol. Chem. 1998; 273: 31555-31564Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), using the equation from Siegel and Monty (32Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1542) Google Scholar). Nine DNA substrates were used for the gel shift assays as follows: a ssDNA fragment of 90 nucleotides designated Ost4; five forked DNA molecules FI, FII, FIII, FIV, and FV; three dsDNA substrates harboring a ssDNA tail O-5′, O-3′, and O-3′40; a dsDNA of 98-base ds98. The forked and tailed molecules were prepared by annealing of the following purified oligonucleotides: Ost4, 5′ GCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTG 3′; Ost6, 5′ CAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCCGCCAGCCACAGTCGTGGCCATTGCCATATGGCCCG 3′; Ost7, 5′ GGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGC 3′; Ost9, 5′ CAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCCGCCAGCCACAGTCGTGGCCATTGCCATATGGCCCGGTCTAC 3′; Ost24, 5′ CGGGCCATATGGCAATGGCCACGACTGTGGCTGGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGC 3′; Ost25, 5′ CGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTG 3′; Ost26, 5′ CAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCCG 3′; and Oflo7, 5′ CGGGCCATATGGCAATGGCCACGACTGTGGCTGG 3′. FI results from the annealing of Ost4 and Ost6; FII from Ost4, Ost6, and Oflo7; FIII from Ost4, Ost6, and Ost7; FIV from Ost4, Ost6, Ost7, and Oflo7; and FV from Ost4, Ost6, and Ost24. O-5′ results from the annealing of Ost4 and Ost26; O-3′ from that of Ost6 and Ost25; and O-3′40 from that of Ost9 and Ost25. Prior to annealing, one oligonucleotide was labeled at the 5′ end with [γ-32P]ATP and T4 polynucleotide kinase (New England Biolabs as recommended by the supplier). Ost4 was labeled prior to construction of FI, FII, FIII, FIV, and O-5′; Ost24 prior to construction of FV; Ost6 prior to construction of O-3′; and Ost9 prior to construction of O-3′40. Unlabeled oligonucleotides were added in a 5-fold excess in the annealing buffer A (10 mm Tris, 1 mm EDTA, 100 mm NaCl, pH 8). The mixture was heated at 95 °C for 5 min and cooled slowly (∼3 h) to room temperature. The annealed products were loaded on an 8% PAGE (30:1) and electrophoresed in 1× TBE buffer. The properly assembled products were eluted in buffer E (10 mm Tris, 1 mm EDTA, 0.2% SDS, 300 mm NaCl, pH 8) overnight at 37 °C and recovered by ethanol precipitation. They were resuspended and stored in buffer A. The correct oligonucleotide assembly was ascertained by radiolabeling of the final substrates and separation on a sequencing gel in denaturing conditions. ds98 was made by PCR amplification of pUC19 plasmid with 1201 and 1211 primers (New England Biolabs) using Taq polymerase (Promega), and the obtained fragment was blunted with Klenow fragment and T4DNAPol (Promega). DNA was loaded on an 8% polyacrylamide (30:1) gel, migrated in 1× TBE buffer, and purified by passive elution (33Maxam A.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 560-564Crossref PubMed Scopus (5387) Google Scholar). ds98 was then labeled as Ost4. The concentrations of DNA substrates were estimated by monitoring the specific activity of radiolabeled Ost4 and the final ratio activity of purified substrates. Different proteins were incubated at concentration indicated in the figures with labeled DNA substrates (0.1 nm) in 20 μl of R buffer (50 mm Hepes, 1 mm dithiothreitol, 1 mm EDTA, 0.1 mg/ml bovine serum albumin, 50 mm NaCl, 12.5% glycerol, pH 7.4) at 30 °C for 10 min. At the end of incubation, 5 μl of loading buffer (50% glycerol, 0.4% cyanol, 0.1 mg/ml bovine serum albumin) were added, and the samples were loaded on a 5% polyacrylamide (30:1) gel containing 5% glycerol and 0.25× TBE and migrated in 0.25× TBE. After electrophoresis, gels were dried under vacuum, revealed with a Storm Apparatus (Molecular Dynamics), and the results were quantified with the ImageQuant Software and the apparent K D value was determined according to Riggs et al. (34Riggs A.D. Suzuki H. Bourgeoss S. J. Mol. Biol. 1970; 48: 67-83Crossref PubMed Scopus (548) Google Scholar). PriA Bs (92 kDa) has been previously overproduced in E. coli and purified.2 By using a similar procedure, DnaB (55 kDa), DnaD (28 kDa), and PriA Ec (82 kDa) were purified to a level exceeding 95% (Fig.1; see "Experimental Procedures"). The native molecular weights of PriA Ec, PriA Bs, DnaB, and DnaD have been estimated by the combination of the Stokes radius and sedimentation coefficient values, determined by gel filtration chromatography and sucrose gradient centrifugation, respectively. These experiments have shown that the four purified polypeptide preparations were composed of a single homogeneous protein form. PriA Bs and PriA Ec were monomeric, as it was already known for PriA Ec (Table I; see Ref. 35Shlomai J.M. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 799-803Crossref PubMed Scopus (89) Google Scholar). In contrast, DnaB and DnaD were multimeric, apparently self-associating as a tetramer and between a dimer and a trimer, respectively (Table I). These results are consistent with the recent report about DnaD multimerization state as a dimer (36Ishigo-oka D. Ogasawara N. Moriya S. J. Bacteriol. 2001; 183: 2148-2150Crossref PubMed Scopus (42) Google Scholar), and the multimerization of DnaB was predictable from reported double-hybrid interaction (23Imai Y. Ogasawara N. Ishigo-oka D. Kadoya R. Daito T. Moriya S. Mol. Microbiol. 2000; 36: 1037-1048Crossref PubMed Scopus (57) Google Scholar). However, it should be noted that seemingly incoherent results were obtained for DnaB, which appeared to be trimer by sedimentation coefficient and close to a heptamer by the Stokes radius. The Siegel and Monty equation (32Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1542) Google Scholar), which includes both the Stokes radius value and the sedimentation coefficient values, indicates that DnaB is a tetramer. Glutaraldehyde cross-linking of DnaB generated a homogeneous form composed of at least four monomers, which had a sedimentation coefficient identical to that of untreated DnaB (data not shown). We conclude that DnaB is a tetramer, of the shape greatly different from globular.Table IPhysical characteristics of PriA Bs, PriA Ec, DnaB, and DnaDProteinStokes radiusSedimentation coefficientEstimated native massNo. of subunitsCalculated mass of a monomerCalculated massÅS 10 −13 skDakDaPriA Bs354.671191.391.3PriA Ec325.881181.881.8DnaB658.0227454.9220DnaD394.3732–327.655.2–82.8The Stokes radius and sedimentation coefficient values of each protein were determined from gel filtration and sucrose gradient analyses. Native molecular masses were calculated from the estimated values (see "Experimental Procedures"). Open table in a new tab The Stokes radius and sedimentation coefficient values of each protein were determined from gel filtration and sucrose gradient analyses. Native molecular masses were calculated from the estimated values (see "Experimental Procedures"). To determine whether the three B. subtilis proteins interact with each other, the appropriate mixtures were made with pairs of proteins and analyzed by gel filtration, sucrose gradient sedimentation, and cross-link experiments. No protein-protein interaction was detected, even at low ionic strength (data not shown). We then tested whether the three proteins interact in the presence of different synthetic forked DNA. These substrates supposedly mimic the chromosomal sites targeted by PriA to promote DNA replication restart. Before carrying out the mixing experiments, proteins were individually assayed with various DNA substrates. The substrates used were the 90-nucleotide single-stranded oligonucleotide Ost4 and the five Y-shaped DNA structures presented in Fig. 2. FI, FII, FIII, and FIV mimic particular forks that may be encountered during chromosomal DNA replication. FI represents a half of an open duplex, which could form at oriC by the action of DnaA. FII, FIII, and FIV have a sequence identical to FI but with the leading, the lagging, or both strands replicated, respectively. FII mimics the forked structure on which PriA Ec promotes replication in vitro (13Liu J. Marians K.J. J. Biol. Chem. 1999; 274: 25033-25041Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). A double-stranded DNA of 98 base pairs (ds98) and the FV fork, which is identical to FIV but with no strand interruption, were also used as controls and known to interact poorly with PriA Ec (12McGlynn P. Al-Deib A.A. Liu J. Marians K.J. Lloyd R.G. J. Mol. Biol. 1997; 270: 212-221Crossref PubMed Scopus (164) Google Scholar). We have observed previously a stable and specific binding of PriA Bs to an artificial D-loop structure.2 Here we observed PriA Bs binding to all substrates with almost the same apparentK D, except to ds98 and to FV (TableII and Fig. 2 C). Two nucleoprotein complexes were detected, apparently form
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