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• W2000412385 abstract "Specific binding of the plasmid-encoded protein, TrfA, and the Escherichia coli DnaA protein to the origin region (oriV) is required for the initiation of replication of the broad host range plasmid RK2. It has been shown that the DnaA protein which binds to DnaA boxes upstream of the TrfA-binding sites (iterons) cannot by itself form an open complex, but it enhances the formation of the open complex by TrfA (Konieczny, I., Doran, K. S., Helinski, D. R., Blasina, A. (1997) J. Biol. Chem. 272, 20173). In this study an in vitroreplication system is reconstituted from purified TrfA protein and E. coli proteins. With this system, a specific interaction between the DnaA and DnaB proteins is required for delivery of the helicase to the RK2 origin region. Although the DnaA protein directs the DnaB-DnaC complex to the plasmid replication origin, it cannot by itself activate the helicase. Both DnaA and TrfA proteins are required for DnaB-induced template unwinding. We propose that specific changes in the nucleoprotein structure mediated by TrfA result in a repositioning of the DnaB helicase within the open origin region and an activation of the DnaB protein for template unwinding. Specific binding of the plasmid-encoded protein, TrfA, and the Escherichia coli DnaA protein to the origin region (oriV) is required for the initiation of replication of the broad host range plasmid RK2. It has been shown that the DnaA protein which binds to DnaA boxes upstream of the TrfA-binding sites (iterons) cannot by itself form an open complex, but it enhances the formation of the open complex by TrfA (Konieczny, I., Doran, K. S., Helinski, D. R., Blasina, A. (1997) J. Biol. Chem. 272, 20173). In this study an in vitroreplication system is reconstituted from purified TrfA protein and E. coli proteins. With this system, a specific interaction between the DnaA and DnaB proteins is required for delivery of the helicase to the RK2 origin region. Although the DnaA protein directs the DnaB-DnaC complex to the plasmid replication origin, it cannot by itself activate the helicase. Both DnaA and TrfA proteins are required for DnaB-induced template unwinding. We propose that specific changes in the nucleoprotein structure mediated by TrfA result in a repositioning of the DnaB helicase within the open origin region and an activation of the DnaB protein for template unwinding. The initiation of replication of a plasmid that is able to be maintained in a diverse group of bacteria (broad host range) is considerably less well understood than the initiation of replication of other prokaryotic replicons. It is of particular interest to determine the nature of the interactions between a broad host plasmid origin of replication, the plasmid-encoded replication initiation protein and the host specific replication proteins that are responsible for initiating replication. The broad host range plasmid RK2 requires for its replication in Escherichia coli an origin of replication (oriV) (Fig. 1) and a plasmid encoded initiation protein (TrfA) (1Figurski D.H. Helinski D.R. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1648-1652Crossref PubMed Scopus (2442) Google Scholar, 2Thomas C.M. Meyer R. Helinski D.R. J. Bacteriol. 1980; 141: 213-222Crossref PubMed Google Scholar, 3Thomas C.M. Stalker D.M. Helinski D.R. Mol. Gen. Genet. 1981; 181: 1-7Crossref PubMed Scopus (53) Google Scholar) which binds as a monomer to 17-bp 1The abbreviations used are: bp, base pair(s); SSB, single-stranded binding protein; ssDNA, single-stranded DNA. 1The abbreviations used are: bp, base pair(s); SSB, single-stranded binding protein; ssDNA, single-stranded DNA. direct repeats (iterons) at this origin (4Perri S. Helinski D.R. J. Biol. Chem. 1993; 268: 3662-3669Abstract Full Text PDF PubMed Google Scholar, 5Toukdarian A.E. Helinski D.R. Perri S. J. Biol. Chem. 1996; 271: 7072-7078Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In addition, RK2 replication in E. coli requires the host specified proteins DnaA, DnaB, DnaC, DNA gyrase, DnaG primase, DNA polymerase III holoenzyme, and SSB (6Pinkney M. Diaz R. Lanka E. Thomas C.M. J. Mol. Biol. 1988; 203: 927-938Crossref PubMed Scopus (44) Google Scholar, 7Kittell B.L. Helinski D.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1389-1393Crossref PubMed Scopus (60) Google Scholar). It has been shown recently that the E. coli DnaA protein binds to four DnaA consensus sequences that potentially can form a cruciform structure within the RK2 origin (8Konieczny I. Doran K.S. Helinski D.R. Blasina A. J. Biol. Chem. 1997; 272: 20173-20178Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In this same study it was found that the TrfA initiation protein in the presence of HU produces an opening of a set of 13-mers located in the A + T-rich region of the RK2 oriV. The DnaA protein enhances and/or stabilizes this open complex formation, but cannot on its own form an open complex. Initiation of replication at the E. coli chromosome origin (oriC) has been studied extensively and the formation of several distinct nucleoprotein complexes has been described (9Messer W. Weigel C. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. II. ASM Press, Washington, D.C.1996: 1579-1601Google Scholar). The initial step involves binding of the DnaA protein to DnaA boxes localized within oriC (10Fuller R.S. Funnell B.E. Kornberg A. Cell. 1984; 38: 889-900Abstract Full Text PDF PubMed Scopus (460) Google Scholar, 11Matsui M. Oka A. Takanami M. Yasuda S. Hirota Y. J. Mol. Biol. 1985; 184: 529-533Crossref PubMed Scopus (97) Google Scholar). This binding results in destabilization of the duplex DNA at the A + T-rich region and open complex formation (12Bramhill D. Kornberg A. Cell. 1988; 52: 743-755Abstract Full Text PDF PubMed Scopus (510) Google Scholar, 13Gille H. Messer W. EMBO J. 1991; 10: 1579-1584Crossref PubMed Scopus (108) Google Scholar, 14Woelker B. Messer W. Nucleic Acids Res. 1993; 21: 5025-5033Crossref PubMed Scopus (46) Google Scholar). The DnaB helicase (15LeBowitz J.H. McMacken R. J. Biol. Chem. 1986; 261: 4738-4748Abstract Full Text PDF PubMed Google Scholar) in the form of a DnaB-DnaC complex is specifically loaded at the open region of the origin (16Kobori J.A. Kornberg A. J. Biol. Chem. 1982; 257: 13770-13775Abstract Full Text PDF PubMed Google Scholar, 17Baker T.A. Funnell B.E. Kornberg A. J. Biol. Chem. 1987; 262: 6877-6885Abstract Full Text PDF PubMed Google Scholar, 18Baker T.A. Sekimizu K. Funnell B.E. Kornberg A. Cell. 1986; 45: 53-64Abstract Full Text PDF PubMed Scopus (134) Google Scholar). Cross-linking, enzyme-linked immunosorbent assay, and monoclonal antibody interference studies have shown that a physical interaction between the DnaA protein and the helicase is required for loading (19Marszalek J. Kaguni J.M. J. Biol. Chem. 1994; 269: 4883-4890Abstract Full Text PDF PubMed Google Scholar). An E. coli prepriming complex consisting of oriC DNA and the DnaA, DnaB, DnaC, and HU proteins can be isolated in vitro (19Marszalek J. Kaguni J.M. J. Biol. Chem. 1994; 269: 4883-4890Abstract Full Text PDF PubMed Google Scholar, 20Funnell B.E. Baker T.A. Kornberg A. J. Biol. Chem. 1987; 262: 10327-10334Abstract Full Text PDF PubMed Google Scholar). Two stages of prepriming complex formation at oriC have been described (9Messer W. Weigel C. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. II. ASM Press, Washington, D.C.1996: 1579-1601Google Scholar). During the first stage (prepriming complex I) helicase is loaded but is not active. It has been proposed that the activation of prepriming complex I is the result of the repositioning of the helicase leading to the formation of prepriming complex II. At this stage helicase can unwind template DNA thus allowing the priming reaction to occur. This study utilizes an in vitro replication system reconstituted from purified components to address the mechanism of helicase loading during the initiation of plasmid RK2 replication in E. coli and the role of the DnaA and TrfA proteins in this process. We show that not unlike that observed with oriC(19Marszalek J. Kaguni J.M. J. Biol. Chem. 1994; 269: 4883-4890Abstract Full Text PDF PubMed Google Scholar), a specific interaction between DnaA and DnaB is required for helicase delivery to the initiation complex. The TrfA protein activates the DnaB helicase for template unwinding presumably by forming a specific nucleoprotein structure and an open complex at the A + T-rich region of the origin. These results are discussed in the context of the broad host range replication properties of the RK2 plasmid. Purified proteins were used for the various assays. DnaB (21Arai K. Yasuda S. Kornberg A. J. Biol. Chem. 1981; 256: 5247-5252Abstract Full Text PDF PubMed Google Scholar), DnaC (22Kobori J.A. Kornberg A. J. Biol. Chem. 1982; 257: 13763-13769Abstract Full Text PDF PubMed Google Scholar), and the copy-up mutant TrfA-33 254D/267L (23Blasina A. Kittell B.L. Toukdarian A.E. Helinski D.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3559-3564Crossref PubMed Scopus (56) Google Scholar) proteins were purified as described previously. Preparations of DnaA protein and histidine tagged version of the copy-up mutant TrfA protein designated His6-TrfA 254D/267L were provided by Dr. Aresa Toukdarian (University California, San Diego) and Dr. Alessandra Blasina (Scripps Research Institute). DNA polymerase III holoenzyme was kindly provided by Dr. Michael O'Donnell (Cornell University Medical College). The anti-DnaA monoclonal antibody M7, the anti-DnaC antibody and pBSoriC plasmid containing oriC DNA fragment (24Baker T.A. Kornberg A. Cell. 1988; 55: 113-123Abstract Full Text PDF PubMed Scopus (170) Google Scholar) were kindly provided by Dr. Jon Kaguni (Michigan State University). Polyclonal anti-DnaA antibody was provided by Dr. Walter Messer (Max Plank Institute, Berlin). Anti-DnaB antibody was provided by Dr. Jaroslaw Marszalek (University of Gdansk). pTJS42 is a mini-replicon of plasmid RK2 and contains the five iteron minimal oriV (25Schmidhauser T.J. Filutowicz M. Helinski D.R. Plasmid. 1983; 9: 325-330Crossref PubMed Scopus (52) Google Scholar). Commercially available proteins and chemicals used in this study were: DNA gyrase, DnaG primase, HU, and SSB from Enzyco, Inc.; bovine serum albumin (fraction V), creatine phosphate, creatine kinase, and rNTPs from Sigma; dNTPs and Sepharose CL-4B from Pharmacia; [methyl-3H]dTTP from ICN Radiochemicals and goat anti-rabbit IgG from Bio-Rad. The RK2 oriV DNA replication reaction was established with purified components similar to those required for in vitro replication of oriC (26Kaguni J.M. Kornberg A. Cell. 1984; 38: 138-190Abstract Full Text PDF Scopus (134) Google Scholar,27Marszalek J. Kaguni J.M. J. Biol. Chem. 1992; 267: 19334-19340Abstract Full Text PDF PubMed Google Scholar). The in vitro replication mixture (25 μl) contained: 40 mm Hepes/KOH pH 8.0; 25 mm Tris/HCl, pH 7.4; 80 μg/ml bovine serum albumin; 4% sucrose; 4 mmdithiothreitol; 11 mm magnesium acetate; 2 mmATP; 50 μm of each dNTP; [methyl-3H]TTP (150 cpm/pmol); 500 μm (each) CTP, GTP, and UTP; 8 mm creatine phosphate; 20 μg/ml creatine kinase; 230 ng of SSB; 120 ng of DNA gyrase; 1600 ng of DnaB; 100 ng of DnaG primase; 55 ng of DNA polymerase III core subunit; 55 ng of τ subunit; 15 ng of β subunit; 10 ng of ψ complex; 600 ng of DnaA; 800 ng of DnaC; 300 ng of RK2 oriV (pTJS42); and 400 ng of His6-TrfA 254D/267L. Reactions were assembled on ice and then incubated at 32 °C for 30 min. Reactions were stopped by placing on ice and adding 1 ml of 0.1m sodium pyrophosphate in 10% trichloroacetic acid. Total nucleotide incorporation (picomoles) was measured by liquid scintillation counting after filtration onto Whatman GF/C glass fiber filters. The reaction mixture for oriC plasmid replication was assembled as described for RK2 oriV DNA replication except that the TrfA protein and RK2 oriV plasmid DNA were omitted and the reaction was supplemented with 200 ng of pBSoriC DNA. Reaction mixtures were incubated for 30 min at 32 °C. Column gel filtration was used to isolate RK2 prepriming complexes. The reaction mixture (total volume 100 μl: 40 mm Hepes/KOH pH 8.0, 40 mm potassium glutamate, 10 mm magnesium acetate, 50 μg/ml bovine serum albumin, 4% sucrose, 4 mm dithiothreitol, and 2 mm ATP) contained the amount of proteins equivalent to four standard in vitro replication reactions except that SSB, DNA gyrase, primase, and DNA polymerase III holoenzyme, CTP, GTP, UTP, dNTP's, and the ATP regeneration system were omitted. The TrfA-33 254D/267L protein was used instead of His6-TrfA 254D/267L and HU protein (100 ng) was added. The reaction mixtures were incubated for 20 min at 32 °C. After incubation the reactions ware run through a Sepharose CL-4B column (0.5 × 12 cm), equilibrated at room temperature with the incubation buffer and 0.01% Brij 58. Fractions (80 μl) were collected and a portion of each (40 μl) was analyzed by SDS-polyacrylamide gel electrophoresis, followed by a semi-dry protein transfer and immunoblot with rabbit antisera specific against DnaA, TrfA, DnaB, and DnaC proteins. Bound rabbit antibody was detected by a colorimetric reaction with an alkaline phosphatase conjugate goat anti-rabbit IgG. The replication reaction assay was similar to the RK2 in vitro replication reaction reconstituted with purified components described above. A 20-μl portion of each fraction collected from the Sepharose CL-4B column was supplemented by the addition of a mixture (10 μl) containing all other replication components in a standard replication buffer. After 30 min incubation at 32 °C, reactions were stopped by placing on ice followed by the addition of 0.1 m sodium pyrophosphate and 10% trichloroacetic acid. The total nucleotide incorporation (picomoles) was measured as described above. The reaction mixture is identical to the RK2 in vitro replication system reconstituted with purified components except that the DnaG primase and DNA Pol III holoenzyme components were omitted. The reaction mixtures were incubated for 30 min at 32 °C and the reactions were stopped by the addition of EDTA and SDS at a final concentration of 10 mm and 2%, respectively, followed by 2 min incubation at 65 °C. Sucrose and bromphenol blue were then added to the reaction mixture to a final concentration of 10 and 0.05%, respectively. The mixture was then analyzed on a 1% agarose gel in TBE buffer (0.09m Tris borate, 0.002 m EDTA). The samples were electrophoresed at 25 V for 20 h and the gel was stained with ethidium bromide solution. The RK2 in vitro replication system described in this study was established with highly purified proteins. As for the RK2 in vitro replication system using E. coli crude extract (6Pinkney M. Diaz R. Lanka E. Thomas C.M. J. Mol. Biol. 1988; 203: 927-938Crossref PubMed Scopus (44) Google Scholar, 7Kittell B.L. Helinski D.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1389-1393Crossref PubMed Scopus (60) Google Scholar), the replication mixture contained an ATP regeneration system, rNTPs, dNTPs, and MgOAC. The RK2oriV replication template was the supercoiled DNA form of plasmid pTJS42 which contains a 393-bp RK2 minimal origin (Fig.1). The replication reaction demonstrated a stringent dependence on oriV containing DNA and the copy-up mutant His6-TrfA 254D/267L protein (TableI). The largely dimeric His6-TrfA protein was unable to support replication in the purified system (data not shown). The monomer form of TrFA is the active form for binding to the iterons at the RK2 origin (5Toukdarian A.E. Helinski D.R. Perri S. J. Biol. Chem. 1996; 271: 7072-7078Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and since the His6-TrfA 254D/267L protein is largely in the form of a monomer, 2A. Toukdarian and D. R. Helinski, manuscript in preparation. this protein was used for these and also previous studies (8Konieczny I. Doran K.S. Helinski D.R. Blasina A. J. Biol. Chem. 1997; 272: 20173-20178Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar).Table IProtein requirements for RK2 oriV replication in the reconstituted systemComponent omittedDNA synthesispmol%None295100oriV DNA2<1TrfA52DnaA2<1DnaB31DnaC62DnaG primase176DNA gyrase4013DNA pol III41SSB83 Open table in a new tab While there is a strong dependence on DnaB, DnaC, DNA gyrase, DnaG primase, SSB, and polymerase III holoenzyme in the reconstituted system (Table I), deleting the τ subunit of polymerase III holoenzyme only slightly lowered in vitro replication activity (data not shown). The HU protein was found to be dispensable for RK2 replication in the purified system although a previous study showed it was essential for open complex formation in the absence of the DnaA protein (8Konieczny I. Doran K.S. Helinski D.R. Blasina A. J. Biol. Chem. 1997; 272: 20173-20178Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Replication of intact RK2 plasmid or RK2 mini-replicons in E. coli is DnaA dependent (6Pinkney M. Diaz R. Lanka E. Thomas C.M. J. Mol. Biol. 1988; 203: 927-938Crossref PubMed Scopus (44) Google Scholar, 7Kittell B.L. Helinski D.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1389-1393Crossref PubMed Scopus (60) Google Scholar, 28Gaylo P. Turjman N. Bastia D. J. Bacteriol. 1987; 169: 4703-4709Crossref PubMed Google Scholar). In the reconstituted RK2 replication system there is a stringent requirement for the E. coli DnaA protein (Table I). It is of interest that the amount of DnaA protein required for E. coli oriC and RK2oriV replication in vitro differs (Fig.2). In comparison to oriC, RK2oriV replication in vitro requires at least 5-fold less DnaA protein for maximum DNA synthesis (with molar ratios approximately 1:75 for oriC:DnaA and 1:15 for oriV:DnaA). This may indicate differences in affinity for the DnaA boxes in the two origins, or differences in the nucleoprotein structures of oriV-DnaA and oriC-DnaA. Unlike with oriC (29Hwang D.S. Kaguni J.M. J. Biol. Chem. 1988; 263: 10625-10632Abstract Full Text PDF PubMed Google Scholar, 30Yung B.Y.M. Crooke E. Kornberg A. J. Biol. Chem. 1990; 265: 1282-1285Abstract Full Text PDF PubMed Google Scholar) an excess of DnaA protein at the highest concentrations used did not result in an inhibition of oriVreplication (Fig. 2). The kinetics of the replication reactions with supercoiled pTJS42 or pBSoriC DNA as templates were not found to be significantly different (data not shown). The initiation of replication of E. coli oriC and bacteriophage λ DNA requires that the helicase be delivered to and loaded on ssDNA to convert the open complex into an activated prepriming complex (18Baker T.A. Sekimizu K. Funnell B.E. Kornberg A. Cell. 1986; 45: 53-64Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 31Mallory J.B. Alfano C. McMacken R. J. Biol. Chem. 1990; 265: 13297-13307Abstract Full Text PDF PubMed Google Scholar). During the initiation of replication of oriC DNA in vitro, DnaA, DnaB, and DnaC proteins interact and associate with oriC to form a prepriming complex (32Ogawa T. Baker T.A. van der Ende A. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3562-3566Crossref PubMed Scopus (57) Google Scholar, 33van der Ende A. Baker T.A. Ogawa T. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3954-3958Crossref PubMed Scopus (91) Google Scholar). It has been shown that a specific interaction between DnaB and DnaA proteins is critical for the formation of this complex and helicase delivery at oriC (19Marszalek J. Kaguni J.M. J. Biol. Chem. 1994; 269: 4883-4890Abstract Full Text PDF PubMed Google Scholar). To investigate the mechanism of helicase delivery to oriV during the initiation of RK2 replication, prepriming complexes were formed and then isolated by gel filtration using a Sepharose CL-4B column. When the DnaB helicase was incubated with supercoiled oriV plasmid DNA (pTJS42) and the DnaC, DnaA, and TrfA proteins, all four proteins were detected along with pTJS42 DNA in the column void volume (Fig.3 A). When oriVtemplate DNA was incubated only with DnaA, DnaB, and DnaC proteins, and not with the TrfA protein, again DnaA, DnaB, and DnaC were found in the void volume with pTJS42 DNA. Although under the conditions of this experiment these three proteins together do not open pTJS42 at the origin in the absence of the TrfA protein (8Konieczny I. Doran K.S. Helinski D.R. Blasina A. J. Biol. Chem. 1997; 272: 20173-20178Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), it is clear that DnaB and DnaC form a prepriming complex with the DnaA protein and oriV DNA (Fig. 3 B). When both the SSB and HU proteins were added to the preincubation mixture containing oriV and the DnaA, DnaB, and DnaC proteins, the DnaB helicase and DnaC proteins were once again observed in the void volume along with the DnaA protein (data not shown). In contrast, when the experiment was carried out in the absence of the DnaA protein but in the presence of the TrfA protein and under conditions that promote origin opening by the TrfA protein (i.e. the presence of HU), we did not observe DnaB helicase and DnaC in the void volume, indicating that the TrfA protein by itself cannot deliver the helicase to the RK2 origin (Fig. 3 C). Finally, incubation of only DnaB and DnaC with the supercoiled pTJS42 DNA did not result in the presence of DnaB or DnaC in the void volume (Fig. 3 D). We also tested the Sepharose CL-4B void fractions for replication activity. The complex of oriV, TrfA, DnaA, DnaB, and DnaC found in the void volume (Fig. 3 A) was active under complete in vitro replication assay conditions, i.e. when gyrase, DnaG primase, SSB, Pol III holoenzyme, rNTPs, and dNTPs were added (Fig. 4). The recovery of replication activity was estimated to be about 80% of the activity of an equivalent amount of incubation mixture that was not subjected to Sepharose CL-4B chromatography. The in vitro replication activity found for the prepriming complex formed from oriVand the DnaA, DnaB, and DnaC proteins without the TrfA protein (Fig. 4) was approximately at a background level, i.e. similar to that obtained when both the TrfA and DnaA proteins were not present during the formation of the complex. 3I. Konieczny and D. R. Helinski, unpublished observation. The relatively high background level observed for the complex formed in the absence of the TrfA protein was probably due to degradation of a portion of the template during the course of the experiment and, consequently, nonspecific nucleotide incorporation. The subsequent addition of TrfA to the complex formed in the absence of TrfA restored replication activity to a level approaching that seen with the complex formed from all four proteins (Fig. 4). These results indicate that the TrfA protein is still required for replication even after the helicase is delivered to the RK2 origin and that the formation of a prepriming complex that includes oriV, DnaA, DnaB, and DnaC can precede the formation of a TrfA-dependent open complex. We determined the kinetics of the oriV in vitro replication reaction using different preincubation conditions. Preincubation of TrfA, DnaA, DnaB, DnaC, and gyrase proteins with pTJS42 in the presence of HU and SSB resulted in an approximately 2-min advance in the time for significant nucleotide incorporation (Fig. 5). Presumably under these conditions, a prepriming complex consisting of oriV, TrfA, DnaA, DnaB, and DnaC is formed. By comparison, a 1-min time difference was observed when either the TrfA or the DnaA protein was omitted during the preincubation period (Fig. 5). These results may indicate that the DnaA and TrfA proteins act independently in the formation of a prepriming complex during the preincubation period. Several template unwinding assays have been developed for measuring helicase unwinding activity (17Baker T.A. Funnell B.E. Kornberg A. J. Biol. Chem. 1987; 262: 6877-6885Abstract Full Text PDF PubMed Google Scholar, 18Baker T.A. Sekimizu K. Funnell B.E. Kornberg A. Cell. 1986; 45: 53-64Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 34Zylicz M. Ang D. Liberek K. Georgopoulos C. EMBO J. 1989; 8: 1601-1608Crossref PubMed Scopus (200) Google Scholar, 35Marszalek J. Zhang W. Hupp T.R. Margulies C. Carr K.M. Cherry S. Kaguni J.M. J. Biol. Chem. 1996; 271: 18535-18542Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In this study the agarose TBE-electrophoresis method was used to determine unwinding of the supercoiled pTJS42 DNA template (35Marszalek J. Zhang W. Hupp T.R. Margulies C. Carr K.M. Cherry S. Kaguni J.M. J. Biol. Chem. 1996; 271: 18535-18542Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The formation of an electrophoretically distinct form of DNA, designated FI*, has been shown to be produced as a result of the unwinding activity of a helicase in the presence of gyrase and SSB proteins (18Baker T.A. Sekimizu K. Funnell B.E. Kornberg A. Cell. 1986; 45: 53-64Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 34Zylicz M. Ang D. Liberek K. Georgopoulos C. EMBO J. 1989; 8: 1601-1608Crossref PubMed Scopus (200) Google Scholar). This covalently closed circular DNA exhibits more rapid electrophoretic mobility and, as shown by electron microscopy (18Baker T.A. Sekimizu K. Funnell B.E. Kornberg A. Cell. 1986; 45: 53-64Abstract Full Text PDF PubMed Scopus (134) Google Scholar), is extensively single-stranded. Using RK2 in vitroreplication conditions including ATP, TrfA, DnaA, DnaB, DnaC, gyrase, HU, and SSB proteins, we observed unwinding of a substantial fraction of the oriV supercoiled DNA template molecules as determined by the appearance of the FI* form of pTJS42 DNA (Fig.6). Not unlike that found for the initiation of oriC and λ DNA replication, the formation of extensively unwound DNA is gyrase dependent (18Baker T.A. Sekimizu K. Funnell B.E. Kornberg A. Cell. 1986; 45: 53-64Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 34Zylicz M. Ang D. Liberek K. Georgopoulos C. EMBO J. 1989; 8: 1601-1608Crossref PubMed Scopus (200) Google Scholar). In comparison to unreacted supercoiled pTJS42 DNA, and not unexpectedly, we observed that this extensively single-stranded reaction product was very sensitive to nuclease P1 digestion (data not shown). Omission of TrfA or DnaA protein resulted in a failure to produce the fast migrating FI* form (Fig. 6). Thus, DnaB helicase activity on the oriV template requires both the DnaA and TrfA proteins. These results suggest that helicase remains inactive in the prepriming complex consisting of oriV, DnaA, DnaC, and DnaB and that activation requires the TrfA protein. A specific interaction between the DnaA and DnaB proteins has been shown to be required for helicase loading at oriC (19Marszalek J. Kaguni J.M. J. Biol. Chem. 1994; 269: 4883-4890Abstract Full Text PDF PubMed Google Scholar). Interference of this protein-protein interaction using monoclonal antibody M7 results in an inhibition of in vitro oriC replication (19Marszalek J. Kaguni J.M. J. Biol. Chem. 1994; 269: 4883-4890Abstract Full Text PDF PubMed Google Scholar). We examined the effect of the M7 antibody on RK2 replication in vitro. OriVreplication was inhibited by approximately 50% with 50 ng and 90% with 200 ng of M7 antibody (Fig. 7). These results indicate a similar requirement for a specific DnaA-DnaB interaction for the formation of an RK2 prepriming complex as was found for the E. coli replication origin. Altering the order of addition of the M7 monoclonal antibody and the DnaA, DnaB, DnaC, and TrfA proteins during the assay for helicase activity allowed us to obtain information as to what stage the antibody was inhibitory. When M7 antibody was added to the incubation mixture with DnaA but before DnaB, DnaC, and TrfA were added, we observed an approximately 75% inhibition of the formation of the FI*form (Fig. 8). This is consistent with the M7 antibody blocking the DnaA-DnaB interaction and, therefore, delivery of the helicase to the template. In contrast, the addition of M7 after incubation of DnaA, DnaB, DnaC, and TrfA had little or no inhibitory effect on helicase activity (Fig. 8). Surprisingly, when the order of addition was changed and the TrfA protein was added at the last step together with M7 antibody, we also observed reduced helicase activity (Fig. 8). This result may indicate instability of the prepriming complex involving the DnaA, DnaB, and DnaC proteins in which case the M7 monoclonal antibody can continue to compete with the DnaB protein for binding to DnaA even if added after the addition of both the DnaA and DnaB proteins to the supercoiled plasmid DNA template. If this is the case, then when the TrfA protein is present along with the DNA, DnaB, and DnaC proteins, the DnaA-DnaB interaction is stabilized or the DnaB protein is repositioned on the template so that it no longer is associate with the DnaA protein. We have demonstrated that plasmid RK2 can be replicated in vitro using purified proteins if the plasmid-specific initiation protein, TrfA, is added. This reconstituted replication system, similar to the system described previously for oriC(26Kaguni J.M. Kornberg A. Cell. 1984; 38: 138-190Abstract Full Text PDF Scopus (134) Google Scholar, 27Marszalek J. Kaguni J.M. J. Biol. Chem. 1992; 267: 19334-19340Abstract Full Text PDF PubMed Google Scholar), thus, can support the replication of supercoiled RK2oriV and E. coli oriC DNA. In the case of RK2oriV replication, we found that a much lower concentration of the DnaA protein is required for maximum replication of oriV when compared with oriC (Fig. 2). We also found that the excess of DnaA protein at the highest concentrations used in our experiments inhibited oriC replication" @default.
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• W2000412385 title "Helicase Delivery and Activation by DnaA and TrfA Proteins during the Initiation of Replication of the Broad Host Range Plasmid RK2" @default.
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• W2000412385 doi "https://doi.org/10.1074/jbc.272.52.33312" @default.
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