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• W2014966950 abstract "Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex. Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex. Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. In Escherichia coli, DnaA forms a stable complex with ATP or ADP and binds to 9-mer sequences called DnaA boxes within the replication origin oriC, resulting in the formation of homomultimeric complexes (1Kaguni J.M. Annu. Rev. Microbiol. 2006; 60: 351-375Crossref PubMed Scopus (163) Google Scholar, 2Messer W. FEMS Microbiol. Rev. 2002; 26: 355-374PubMed Google Scholar, 3Mott M.L. Berger J.M. Nat. Rev. Microbiol. 2007; 5: 343-354Crossref PubMed Scopus (263) Google Scholar, 4Zakrzewska-Czerwińska J. Jakimowicz D. Zawilak-Pawlik A. Messer W. FEMS Microbiol. Rev. 2007; 31: 378-387Crossref PubMed Scopus (87) Google Scholar). A DnaA-binding protein, DiaA, directly stimulates formation of ATP-DnaA multimers on oriC (5Ishida T. Akimitsu N. Kashioka T. Hatano M. Kubota T. Ogata Y. Sekimizu K. Katayama T. J. Biol. Chem. 2004; 279: 45546-45555Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 6Keyamura K. Fujikawa N. Ishida T. Ozaki S. Su'etsugu M. Fujimitsu K. Kagawa W. Yokoyama S. Kurumizaka H. Katayama T. Genes Dev. 2007; 21: 2083-2099Crossref PubMed Scopus (98) Google Scholar). ATP-DnaA multimers, but not ADP-DnaA multimers, promote specific inter-DnaA interactions on oriC, resulting in the adoption of an activated conformation as the initiation complexes, which interact with ATP-DnaA-specific low affinity sites within oriC (7Kawakami H. Keyamura K. Katayama T. J. Biol. Chem. 2005; 280: 27420-27430Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 8McGarry K.C. Ryan V.T. Grimwade J.E. Leonard A.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2811-2816Crossref PubMed Scopus (127) Google Scholar, 9Speck C. Messer W. EMBO J. 2001; 20: 1469-1476Crossref PubMed Scopus (164) Google Scholar). This conformational change triggers duplex unwinding of the AT-rich 13-mer repeats (DNA unwinding element (DUE) 4The abbreviations used are:DUEDNA-unwinding elementAAAATPases associated with a variety of cellular activitiessssingle-strandedEMSAelectrophoretic mobility shift assayRIDAregulatory inactivation of DnaA. 4The abbreviations used are:DUEDNA-unwinding elementAAAATPases associated with a variety of cellular activitiessssingle-strandedEMSAelectrophoretic mobility shift assayRIDAregulatory inactivation of DnaA.) within oriC with the aid of the superhelicity of DNA and heat energy, creating open complexes (10Sekimizu K. Bramhill D. Kornberg A. Cell. 1987; 50: 259-265Abstract Full Text PDF PubMed Scopus (345) Google Scholar, 11Bramhill D. Kornberg A. Cell. 1988; 52: 743-755Abstract Full Text PDF PubMed Scopus (510) Google Scholar). The mechanisms and functional structures within DnaA directly responsible for the ATP-DnaA-specific duplex unwinding remain unexplored. DNA-unwinding element ATPases associated with a variety of cellular activities single-stranded electrophoretic mobility shift assay regulatory inactivation of DnaA. DNA-unwinding element ATPases associated with a variety of cellular activities single-stranded electrophoretic mobility shift assay regulatory inactivation of DnaA. Open complex formation is a critical regulatory point for determining whether replicational initiation will occur during the cell cycle (1Kaguni J.M. Annu. Rev. Microbiol. 2006; 60: 351-375Crossref PubMed Scopus (163) Google Scholar, 2Messer W. FEMS Microbiol. Rev. 2002; 26: 355-374PubMed Google Scholar). DnaB helicase is loaded onto the single-stranded (ss) region in open complexes in a manner depending on a DnaA-DnaB interaction and the DnaC helicase loader. The loaded helicase expands the ssDNA region, which leads to the assembly of replication machineries, including DnaG primase and DNA polymerase III holoenzyme, thereby initiating DNA synthesis (12O'Donnell M. J. Biol. Chem. 2006; 281: 10653-10656Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). After the initiation of replication, ATP-DnaA is converted to ADP-DnaA by the promotion of DnaA-ATP hydrolysis in a manner depending on Hda protein and the DNA-loaded form of the clamp subunit of DNA polymerase III holoenzyme (13Camara J.E. Breier A.M. Brendler T. Austin S. Cozzarelli N.R. Crooke E. EMBO Rep. 2005; 6: 736-741Crossref PubMed Scopus (69) Google Scholar, 14Katayama T. Kubota T. Kurokawa K. Crooke E. Sekimizu K. Cell. 1998; 94: 61-71Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 15Kato J. Katayama T. EMBO J. 2001; 20: 4253-4262Crossref PubMed Scopus (212) Google Scholar, 16Su'etsugu M. Shimuta T. Ishida T. Kawakami H. Katayama T. J. Biol. Chem. 2005; 280: 6528-6536Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). This DnaA-inactivating system, termed RIDA (regulatory inactivation of DnaA) is required for repressing extra initiation events. The DnaA protein consists of four functional domains (1Kaguni J.M. Annu. Rev. Microbiol. 2006; 60: 351-375Crossref PubMed Scopus (163) Google Scholar, 2Messer W. FEMS Microbiol. Rev. 2002; 26: 355-374PubMed Google Scholar). Domain I is required for DnaA self-oligomerization and functional interactions with other proteins such as DnaB helicase and DiaA (5Ishida T. Akimitsu N. Kashioka T. Hatano M. Kubota T. Ogata Y. Sekimizu K. Katayama T. J. Biol. Chem. 2004; 279: 45546-45555Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 17Abe Y. Jo T. Matsuda Y. Matsunaga C. Katayama T. Ueda T. J. Biol. Chem. 2007; 282: 17816-17827Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 18Felczak M.M. Simmons L.A. Kaguni J.M. J. Biol. Chem. 2005; 280: 24627-24633Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 19Weigel C. Schmidt A. Seitz H. Tüngler D. Welzeck M. Messer W. Mol. Microbiol. 1999; 34: 53-66Crossref PubMed Scopus (89) Google Scholar). Domain II is a flexible linker (17Abe Y. Jo T. Matsuda Y. Matsunaga C. Katayama T. Ueda T. J. Biol. Chem. 2007; 282: 17816-17827Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Domain III has specific ATP recognition motifs that are characteristic of the AAA+ superfamily, of which DnaA is a member (20Iyer L.M. Leipe D.D. Koonin E.V. Aravind L. J. Struct. Biol. 2004; 146: 11-31Crossref PubMed Scopus (615) Google Scholar, 21Neuwald A.F. Aravind L. Spouge J.L. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar). Domain IV is a DNA-binding region that contains a helix-turn-helix motif for specific recognition of the DnaA box (22Erzberger J.P. Pirruccello M.M. Berger J.M. EMBO J. 2002; 21: 4763-4773Crossref PubMed Scopus (188) Google Scholar, 23Fujikawa N. Kurumizaka H. Nureki O. Terada T. Shirouzu M. Katayama T. Yokoyama S. Nucleic Acids Res. 2003; 31: 2077-2086Crossref PubMed Scopus (142) Google Scholar). The AAA+ superfamily includes various proteins that can induce ATP binding/hydrolysis-dependent conformational changes (21Neuwald A.F. Aravind L. Spouge J.L. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar, 24Hanson P.I. Whiteheart S.W. Nat. Rev. Mol. Cell. Biol. 2005; 6: 519-529Crossref PubMed Scopus (871) Google Scholar, 25Ogura T. Wilkinson A.J. Genes Cells. 2001; 6: 575-597Crossref PubMed Scopus (820) Google Scholar). Of the AAA+-specific ATP-interacting motifs, the DnaA sensor 1 Asp-269 residue supports exceptionally tight affinity for ATP/ADP (26Kawakami H. Ozaki S. Suzuki S. Nakamura K. Senriuchi T. Su'etsugu M. Fujimitsu K. Katayama T. Mol. Microbiol. 2006; 62: 1310-1324Crossref PubMed Scopus (31) Google Scholar). The DnaA sensor 2 Arg-334 is specifically required for RIDA-dependent ATP hydrolysis (27Nishida S. Fujimitsu K. Sekimizu K. Ohmura T. Ueda T. Katayama T. J. Biol. Chem. 2002; 277: 14986-14995Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 28Su'etsugu M. Kawakami H. Kurokawa K. Kubota T. Takata M. Katayama T. Mol. Microbiol. 2001; 40: 376-386Crossref PubMed Scopus (31) Google Scholar). The DnaA Box VII arginines Arg-281 and Arg-285 most likely interact in different manners with an adjacent DnaA protomer in DnaA multimers assembled on oriC. Arg-281 stabilizes the DnaA multimers (29Felczak M.M. Kaguni J.M. J. Biol. Chem. 2004; 279: 51156-51162Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Notably Arg-285 plays a crucial role in the ATP-specific conformational change of the initiation complex, which is required for open complex formation (7Kawakami H. Keyamura K. Katayama T. J. Biol. Chem. 2005; 280: 27420-27430Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). This residue is specifically required for binding of ATP-DnaA protomers to the specific low affinity sites within oriC. On the basis of common features of AAA+ proteins, it has been suggested that Arg-285 interacts with ATP bound to an adjacent DnaA protomer in the complex, which leads to conformational activation of the complex. In well characterized AAA+ proteins, a typical functional conformation is ring- or spiral-shaped oligomers (consisting of 5–7 protomers) with a central cavity or pore (24Hanson P.I. Whiteheart S.W. Nat. Rev. Mol. Cell. Biol. 2005; 6: 519-529Crossref PubMed Scopus (871) Google Scholar). Certain residues within the pore are crucial for specific functions of proteases, chaperones, SV40 T-Ag helicase, and DNA polymerase III clamp-loader subassembly (30Enemark E.J. Joshua-Tor L. Nature. 2006; 442: 270-275Crossref PubMed Scopus (410) Google Scholar, 31Kumar A. Meinke G. Reese D.K. Moine S. Phelan P.J. Fradet-Turcotte A. Archambault J. Bohm A. Bullock P.A. J. Virol. 2007; 81: 4808-4818Crossref PubMed Scopus (29) Google Scholar, 32Goedken E.R. Kazmirski S.L. Bowman G.D. O'Donnell M. Kuriyan J. Nat. Struct. Mol. Biol. 2005; 12: 183-190Crossref PubMed Scopus (35) Google Scholar). By analogy to these structures, we hypothesized that DnaA molecules oligomerize to form a central pore on oriC and that specific residues on the pore surface play a crucial role in duplex unwinding. However, DnaA does not carry residues in its primary sequence that directly correspond to the important residues within the pores of the AAA+ proteins previously characterized. DnaA is structurally classified into a subgroup different from the proteases, chaperones, helicases, and clamp-loaders in the AAA+ superfamily (20Iyer L.M. Leipe D.D. Koonin E.V. Aravind L. J. Struct. Biol. 2004; 146: 11-31Crossref PubMed Scopus (615) Google Scholar). Furthermore, unlike the well characterized AAA+ proteins, DNA-free DnaA molecules are monomers. In this study, we first constructed homology models of the DnaA oligomer that forms a pore using a crystal structure of the AAA+ domain of the hyperthermophilic eubacterium Thermotoga maritima DnaA ortholog (tmaDnaA). Next, we used this model to select candidates for the crucial residues on the pore surface of DnaA and analyzed the corresponding mutant proteins of tmaDnaA and E. coli DnaA. The candidate residues were highly conserved in DnaA orthologs. The results of our in vitro analyses are in good agreement with this structure. We found that E. coli DnaA V211A and R245A proteins are inactive in in vivo initiation. Moreover, we revealed using in vitro reconstituted systems that these mutant proteins are specifically inactive in origin unwinding, whereas these retain activities for binding to ATP and DnaA boxes and for the formation of the ATP-DnaA-specific multimer conformation on oriC. Furthermore, using a newly constructed system of an electrophoretic mobility shift assay (EMSA), we revealed that the ATP form of these mutant DnaA multimers on oriC, unlike that of wild-type DnaA, are inactive in direct binding to ssDUE. Thus, we have revealed specific roles for the previously unexpected residues in open complex formation. These residues reside on the putative pore of the DnaA multimer on oriC and play crucial and specific roles in oriC unwinding. Thus we propose a novel mechanism for ATP-dependent regulation of open complex formation. Strains, Plasmids, and Oligonucleotides—E. coli strains KH5402-1 (wild type), KA450 (ΔoriC1071::Tn10 rnhA199(Am) dnaA17(Am)), KA451 (dnaA::Tn10 rnhA::cat), and KA413 (dnaA46) were previously described (26Kawakami H. Ozaki S. Suzuki S. Nakamura K. Senriuchi T. Su'etsugu M. Fujimitsu K. Katayama T. Mol. Microbiol. 2006; 62: 1310-1324Crossref PubMed Scopus (31) Google Scholar, 33Shimuta T. Nakano K. Yamaguchi Y. Ozaki S. Fujimitsu K. Matsunaga C. Noguchi K. Emoto A. Katayama T. Genes Cells. 2004; 9: 1151-1166Crossref PubMed Scopus (16) Google Scholar). pKA234, M13KEW101, pTHMA-1, pOZ14, and pOZ18 were also previously described (26Kawakami H. Ozaki S. Suzuki S. Nakamura K. Senriuchi T. Su'etsugu M. Fujimitsu K. Katayama T. Mol. Microbiol. 2006; 62: 1310-1324Crossref PubMed Scopus (31) Google Scholar, 34Ozaki S. Fujimitsu K. Kurumizaka H. Katayama T. Genes Cells. 2006; 11: 425-438Crossref PubMed Scopus (28) Google Scholar). For construction of the derivatives of pKA234 and pTHMA-1, mutations were introduced in the dnaA region using a QuikChange site-directed mutagenesis kit (Stratagene) and mutagenic primers. For the sequences of these primers, see supplemental Table S1. The derivatives of pOZ18 were constructed as we previously described (26Kawakami H. Ozaki S. Suzuki S. Nakamura K. Senriuchi T. Su'etsugu M. Fujimitsu K. Katayama T. Mol. Microbiol. 2006; 62: 1310-1324Crossref PubMed Scopus (31) Google Scholar). Assays for DnaA Activities—Binding activities of DnaA for ATP and DnaA boxes were assessed by a filter-retention assay, surface plasmon resonance analysis, or EMSA as we described previously (7Kawakami H. Keyamura K. Katayama T. J. Biol. Chem. 2005; 280: 27420-27430Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 26Kawakami H. Ozaki S. Suzuki S. Nakamura K. Senriuchi T. Su'etsugu M. Fujimitsu K. Katayama T. Mol. Microbiol. 2006; 62: 1310-1324Crossref PubMed Scopus (31) Google Scholar, 34Ozaki S. Fujimitsu K. Kurumizaka H. Katayama T. Genes Cells. 2006; 11: 425-438Crossref PubMed Scopus (28) Google Scholar). The P1 nuclease assay using E. coli DnaA was performed essentially as previously described (6Keyamura K. Fujikawa N. Ishida T. Ozaki S. Su'etsugu M. Fujimitsu K. Kagawa W. Yokoyama S. Kurumizaka H. Katayama T. Genes Dev. 2007; 21: 2083-2099Crossref PubMed Scopus (98) Google Scholar, 11Bramhill D. Kornberg A. Cell. 1988; 52: 743-755Abstract Full Text PDF PubMed Scopus (510) Google Scholar, 17Abe Y. Jo T. Matsuda Y. Matsunaga C. Katayama T. Ueda T. J. Biol. Chem. 2007; 282: 17816-17827Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 26Kawakami H. Ozaki S. Suzuki S. Nakamura K. Senriuchi T. Su'etsugu M. Fujimitsu K. Katayama T. Mol. Microbiol. 2006; 62: 1310-1324Crossref PubMed Scopus (31) Google Scholar). The resultant DNA samples were analyzed by 1% agarose gel electrophoresis and ethidium bromide staining. The intensities of DNA bands were quantified by densitometric scanning. When tmaDnaA was used, unwinding reactions were incubated at 48 °C in the presence of pOZ14 (400 ng) instead of M13KEW101 (34Ozaki S. Fujimitsu K. Kurumizaka H. Katayama T. Genes Cells. 2006; 11: 425-438Crossref PubMed Scopus (28) Google Scholar). The minichromosome replication and ABC primosome systems were reconstituted in vitro with purified proteins as previously described (17Abe Y. Jo T. Matsuda Y. Matsunaga C. Katayama T. Ueda T. J. Biol. Chem. 2007; 282: 17816-17827Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 26Kawakami H. Ozaki S. Suzuki S. Nakamura K. Senriuchi T. Su'etsugu M. Fujimitsu K. Katayama T. Mol. Microbiol. 2006; 62: 1310-1324Crossref PubMed Scopus (31) Google Scholar, 35Masai H. Arai K. Eur. J. Biochem. 1995; 230: 384-395Crossref PubMed Scopus (15) Google Scholar, 36Masai H. Nomura N. Arai K. J. Biol. Chem. 1990; 265: 15134-15144Abstract Full Text PDF PubMed Google Scholar). DNase I Footprint Analysis—This analysis was essentially performed as described previously (6Keyamura K. Fujikawa N. Ishida T. Ozaki S. Su'etsugu M. Fujimitsu K. Kagawa W. Yokoyama S. Kurumizaka H. Katayama T. Genes Dev. 2007; 21: 2083-2099Crossref PubMed Scopus (98) Google Scholar, 7Kawakami H. Keyamura K. Katayama T. J. Biol. Chem. 2005; 280: 27420-27430Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). DnaA was incubated at 30 °C for 10 min in buffer (10 μl) containing 25 mm Hepes-KOH (pH 7.6), 5 mm calcium acetate, 2.8 mm magnesium acetate, 45 mm ammonium sulfate, 4 mm dithiothreitol, 10% (v/v) glycerol, 0.2% Triton X-100, 0.5 mg/ml bovine serum albumin, 14 μg/ml poly(dA-dT)-(dA-dT), 14 μg/ml poly(dI-dC)-(dI-dC), 3 mm ATP/ADP, and a 419-bp 32P-end-labeled oriC fragment (5.5 ng), followed by incubation for 4 min at the same temperature in the presence of DNase I (0.63 milliunit). After the reaction was stopped by addition of 0.5% SDS, DNA was extracted with phenol/chloroform, precipitated with ethanol, and analyzed by 5% sequencing gel electrophoresis. ssDUE Binding Analysis by EMSA—DnaA was incubated at 0 °C for 15 min in the presence of 3 mm ATP or ADP as we previously described (26Kawakami H. Ozaki S. Suzuki S. Nakamura K. Senriuchi T. Su'etsugu M. Fujimitsu K. Katayama T. Mol. Microbiol. 2006; 62: 1310-1324Crossref PubMed Scopus (31) Google Scholar). ATP-DnaA and ADP-DnaA were incubated at 30 °C for 10 min in 10 μl of buffer G containing 13 nm (130 fmol) oriCΔLMR DNA and 2 nm (20 fmol) 32P-end-labeled M28 ssDNA (5′-GATCTGTTCTATTGTGATCTCTTATTAG), its complementary M28-rev ssDNA, or its derivatives. Buffer G contains 20 mm Hepes-KOH (pH 7.6), 1 mm EDTA (pH 8.0), 4 mm dithiothreitol, 5 mm magnesium acetate, 10% (v/v) glycerol, 0.1 mg/ml bovine serum albumin, and 1 mm ATP or ADP. The mixtures were then separated by 4% polyacrylamide gel electrophoresis at room temperature. DNA was visualized using GelStar (Cambrex) staining, and ssDNA was detected using a BAS-2500 Bio-imaging analyzer. The oriCΔLMR fragment was prepared by PCR using M13KEW101 and primers ori-1 (5′-ATCGCACTGCCCTGTGG) and ori-2 (5′-CAAATAAGTATACAGATCGTGCG). ssDUE Binding Analysis Using Pull-down System—DnaA and 32P-end-labeled M28 ssDNA were incubated under the same conditions described for EMSA except that 5′-biotinylated oriCΔLMR (bio-oriCΔLMR) was used instead of unmodified oriCΔLMR. The reaction was further incubated for 10 min at room temperature with gentle rotation in the presence of streptavidin-beads (Promega) equilibrated in the buffer G (10 μl). The beads and bound materials were collected and washed in the same buffer (20 μl). M28 ssDNA complexed with bio-oriCΔLMR and DnaA was eluted in buffer (10 μl) containing 10 mm Tris-HCl (pH 7.5 at 1 m), 1 mm EDTA and 0.1% SDS, spotted on the 3MM paper (Whatman) and analyzed using a BAS-2500 Bio-imaging analyzer. The bio-oriCΔLMR fragment was prepared by PCR using primers ori-1 and 5′-biotinylated ori-2. Structural Models of the Initiation Complex—By analogy to some AAA+ protein multimers, we hypothesized that DnaA multimers on oriC have a central pore that plays a crucial role in DUE unwinding. To predict which residues comprise the putative pore, we first identified the crystal structure of tmaDnaA AAA+ domain (Fig. 1A, Table 1, and supplemental “Experimental Procedures”) and then constructed homology models of the multimeric structure using this crystal structure (Fig. 1, C and D). The crystal structure of tmaDnaA AAA+ domain bound to ADP was solved at 3.05 Å and was basically identical to that of Aquifex aeolicus DnaA AAA+ domain (Fig. 1B) (22Erzberger J.P. Pirruccello M.M. Berger J.M. EMBO J. 2002; 21: 4763-4773Crossref PubMed Scopus (188) Google Scholar).TABLE 1Data collection and refinement statistics of tmaDnaA crystalsSpace groupR32C2Unit cella = b = 221.82 Å, c = 55.60 Å, α, β = 90°, γ = 120°a = 133.34 Å, b = 221.79 Å, c = 55.60 Å, β = 106.13°Resolution (Å)50.0-3.0050.0-3.05Unique reflections10,29027,730Redundancy3.72.3Completeness (%) (last shell)96.3 (74.6)94.1 (68.3)I/σ(I) (last shell)13.4 (2.46)13.7 (2.31)Rsym (%) (last shell)aRsym = Σ|Iavg — Ii|/ΣIi.7.8 (35.1)7.0 (29.7)Rwork (%)23.423.2Rfree (%)29.126.4a Rsym = Σ|Iavg — Ii|/ΣIi. Open table in a new tab Using the model of a homohexamer ring (Fig. 1C), we searched for the residues that are exposed on the pore surface. We particularly focused on residues bearing hydrophobic or basic side chains that would interact favorably with DNA. tmaDnaA Val-176, Met-179, Lys-180, and Lys-209 residues were found to be strong candidates (Fig. 1E). Identical or chemically similar residues corresponding to these are highly conserved among eubacterial DnaA homologs (Fig. 1F). During the course of this study, a multimeric crystal structure was reported for the A. aeolicus DnaA AAA+ domain (37Erzberger J.P. Mott M.L. Berger J.M. Nat. Struct. Mol. Biol. 2006; 13: 676-683Crossref PubMed Scopus (228) Google Scholar). Although this complex does not include the cognate oriC DNA, the multimers form a spiral helix in crystals. We therefore used this structure as a second model (Fig. 1D). The residues indicated above are also exposed on the pore of this spiral model (Fig. 1E). Analyses of tmaDnaA Mutant Proteins—Using tmaDnaA and the predicted oriC (tma-oriC) of T. maritima, we previously determined the minimal oriC region and ATP-tmaDnaA-specific DUE in vitro (34Ozaki S. Fujimitsu K. Kurumizaka H. Katayama T. Genes Cells. 2006; 11: 425-438Crossref PubMed Scopus (28) Google Scholar). ATP-tmaDnaA-dependent unwinding within tma-oriC DUE can be assessed by P1 nuclease. To elucidate the functions of the selected residues, we analyzed a set of tmaDnaA mutant proteins with single-alanine substitutions at Val-176, Met-179, Lys-180, or Lys-209. All of the mutant tmaDnaA proteins were purified by the method reported previously for wild-type tmaDnaA. The ATP binding activities of the mutant proteins were similar to that of wild-type tmaDnaA (Table 2). In an EMSA, the tmaDnaA box binding activity of each mutant protein was similar to that of wild-type tmaDnaA (data not shown). In contrast to these activities, tma-oriC unwinding was severely inhibited in each tmaDnaA mutant protein (Fig. 1G).TABLE 2ATP binding activity of the mutant DnaAPositiontmaDnaAE. coli DnaAProteinKDStoichiometryProteinKDStoichiometrynmnmWT240.22WT490.45iV176A240.20V211A210.45iiM179A230.24iiiK180A220.23ivK223A220.30vK209A320.31K243A720.37viR245A410.27 Open table in a new tab E. coli DnaA Mutants Inactive in in Vivo Initiation—To assess the in vivo significance of these residues, we constructed plasmids encoding E. coli DnaA mutant proteins that have Ala substitutions at the positions corresponding to the tmaDnaA residues indicated above (Fig. 1, E and F). The E. coli DnaA residues Val-211 to Ile-219 reside in a region corresponding to that of tmaDnaA Val-176 to Lys-180 or in its flanking region (positions i–iii in Fig. 1F). E. coli DnaA Lys-243 and Arg-245 residues reside in a region corresponding to tmaDnaA Lys-209 and Gly-211 (positions v and vi, respectively, in Fig. 1F). tma-DnaA Gly-211 is exposed on the pore surface (Fig. 1E). Some DnaA orthologs conserve the basic moiety corresponding to E. coli DnaA Arg-245. In addition, we analyzed E. coli DnaA Lys-223 and Arg-224 (Lys-223 corresponds to position iv in Fig. 1F). These residues have basic side chains that are conserved in this region of DnaA orthologs. To determine the importance of these residues in vivo, we performed plasmid complementation tests (Table 3). First, we used as a host a dnaA46 mutant strain, KA413, that is defective in colony formation at 42 °C. The initiation activity of the DnaA46 protein is temperature-sensitive, and the protein is labile at 42 °C, with a shortened half-life in vivo (33Shimuta T. Nakano K. Yamaguchi Y. Ozaki S. Fujimitsu K. Matsunaga C. Noguchi K. Emoto A. Katayama T. Genes Cells. 2004; 9: 1151-1166Crossref PubMed Scopus (16) Google Scholar, 38Hwang D.S. Kaguni J.M. J. Biol. Chem. 1988; 263: 10625-10632Abstract Full Text PDF PubMed Google Scholar). The first series of DnaA-producing plasmids bearing wild-type or mutant dnaA alleles was introduced into KA413 cells, and the transformant cells were incubated overnight at 30 °C or 42 °C (Table 3, experiment A). At 30 °C, colonies formed with a similar efficiency among the plasmids, whereas the plasmids bearing the dnaA V211A allele (pKW44-1) and dnaA K223A allele (pKW47-1) did not support colony formation at 42 °C. In these experiments, the inducer arabinose was not included in the medium, thereby allowing only leaky expression. Immunoblot analysis indicated that DnaA proteins were expressed from the plasmids at a similar level even at 42 °C (supplemental Fig. S1).TABLE 3Plasmid complementation testsExperimentHostPlasmidAlleleTransformation efficiencyaCFU, colony-forming units; WT, wild-type.30 °C42 °C42 °C/30 °CCFU/μg DNAAbFor experiment A, KA413 (dnaA46) cells were transformed with plasmid bearing the indicated dnaA allele and incubated on LB agar plates containing thymine (50 μg/ml) and ampicillin (50 μg/ml) at 30 °C for 21 h or at 42 °C for 12 h. Transformation efficiencies and the ratio are shown.KA413pKA234WT1.3 × 1061.5 × 1061.2pKW44-1V211A9.6 × 105<1.0 × 103<0.001pKW46-1K212A5.2 × 1055.1 × 1050.98pL214RL214R2.4 × 1062.6 × 1061.1pQ215AQ215A4.2 × 1065.6 × 1061.3pN216AN216A1.6 × 1062.1 × 1061.3pN217AN217A2.1 × 1062.2 × 1061.0pI219AI219A2.4 × 1062.4 × 1061.0pKW47-1K223A7.7 × 1052.0 × 1030.003pKW48-1R224A1.0 × 1068.5 × 1050.85pING1 (vector)None1.1 × 106<1.0 × 103<0.001BcFor experiment B, KH5402-1 (wild-type dnaA) cells and KA451 (dnaA::Tn10 rnhA::cat) cells were transformed with mini-R derivative plasmid bearing wild-type rnhA and the indicated dnaA allele, and incubated as above at 30 °C for 20 h.KH5402-1pOZ18WT3.4 × 105pMNR-N216AN216A1.1 × 105pMNR-N217AN217A1.3 × 105pMNR-I219AI219A1.6 × 105pOZ20K243A5.6 × 105pOZ21R245A2.6 × 105KA451pOZ18WT3.4 × 105pMNR-N216AN216A<10pMNR-N217AN217A5.9 × 104pMNR-I219AI219A7.5 × 104pOZ20K243A<25pOZ21R245A<25a CFU, colony-forming units; WT, wild-type.b For experiment A, KA413 (dnaA46) cells were transformed with plasmid bearing the indicated dnaA allele and incubated on LB agar plates containing thymine (50 μg/ml) and ampicillin (50 μg/ml) at 30 °C for 21 h or at 42 °C for 12 h. Transformation efficiencies and the ratio are shown.c For experiment B, KH5402-1 (wild-type dnaA) cells and KA451 (dnaA::Tn10 rnhA::cat) cells were transformed with mini-R derivative plasmid bearing wild-type rnhA and the indicated dnaA allele, and incubated as above at 30 °C for 20 h. Open table in a new tab Next, we used as a host a dnaA-disru" @default.
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• W2014966950 title "A Common Mechanism for the ATP-DnaA-dependent Formation of Open Complexes at the Replication Origin" @default.
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