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DiaA Dynamics Are Coupled with Changes in Initial Origin Complexes Leading to Helicase Loading

2009; Elsevier BV; Volume: 284; Issue: 37 Linguagem: Inglês

10.1074/jbc.m109.002717

ISSN

1083-351X

Autores

Kenji Keyamura, Yoshito Abe, Masahiro Higashi, Tadashi Ueda, Tsutomu Katayama,

Tópico(s)

Genomics and Chromatin Dynamics

Resumo

Chromosomal replication initiation requires the regulated formation of dynamic higher order complexes. Escherichia coli ATP-DnaA forms a specific multimer on oriC, resulting in DNA unwinding and DnaB helicase loading. DiaA, a DnaA-binding protein, directly stimulates the formation of ATP-DnaA multimers on oriC and ensures timely replication initiation. In this study, DnaA Phe-46 was identified as the crucial DiaA-binding site required for DiaA-stimulated ATP-DnaA assembly on oriC. Moreover, we show that DiaA stimulation requires only a subgroup of DnaA molecules binding to oriC, that DnaA Phe-46 is also important in the loading of DnaB helicase onto the oriC-DnaA complexes, and that this process also requires only a subgroup of DnaA molecules. Despite the use of only a DnaA subgroup, DiaA inhibited DnaB loading on oriC-DnaA complexes, suggesting that DiaA and DnaB bind to a common DnaA subgroup. A cellular factor can relieve the DiaA inhibition, allowing DnaB loading. Consistently, DnaA F46A caused retarded initiations in vivo in a DiaA-independent manner. It is therefore likely that DiaA dynamics are crucial in the regulated sequential progress of DnaA assembly and DnaB loading. We accordingly propose a model for dynamic structural changes of initial oriC complexes loading DiaA or DnaB helicase. Chromosomal replication initiation requires the regulated formation of dynamic higher order complexes. Escherichia coli ATP-DnaA forms a specific multimer on oriC, resulting in DNA unwinding and DnaB helicase loading. DiaA, a DnaA-binding protein, directly stimulates the formation of ATP-DnaA multimers on oriC and ensures timely replication initiation. In this study, DnaA Phe-46 was identified as the crucial DiaA-binding site required for DiaA-stimulated ATP-DnaA assembly on oriC. Moreover, we show that DiaA stimulation requires only a subgroup of DnaA molecules binding to oriC, that DnaA Phe-46 is also important in the loading of DnaB helicase onto the oriC-DnaA complexes, and that this process also requires only a subgroup of DnaA molecules. Despite the use of only a DnaA subgroup, DiaA inhibited DnaB loading on oriC-DnaA complexes, suggesting that DiaA and DnaB bind to a common DnaA subgroup. A cellular factor can relieve the DiaA inhibition, allowing DnaB loading. Consistently, DnaA F46A caused retarded initiations in vivo in a DiaA-independent manner. It is therefore likely that DiaA dynamics are crucial in the regulated sequential progress of DnaA assembly and DnaB loading. We accordingly propose a model for dynamic structural changes of initial oriC complexes loading DiaA or DnaB helicase. In many cellular organisms, multiple proteins form dynamic complexes on the chromosomal origin for the initiation of DNA replication. In Escherichia coli, ATP-DnaA forms a specific multimeric complex on the origin (oriC), resulting in an initiation complex that is competent in the replicational initiation (1Messer W. FEMS Microbiol. Rev. 2002; 26: 355-374PubMed Google Scholar, 2Mott M.L. Berger J.M. Nat. Rev. Microbiol. 2007; 5: 343-354Crossref PubMed Scopus (273) Google Scholar, 3Zakrzewska-Czerwiñska J. Jakimowicz D. Zawilak-Pawlik A. Messer W. FEMS Microbiol. Rev. 2007; 31: 378-387Crossref PubMed Scopus (87) Google Scholar). ATP-DnaA complexes, but not ADP-DnaA complexes, unwind the DNA duplex within the oriC DNA unwinding element (DUE) 2The abbreviations used are:DUEDNA unwinding elementAAAATPases associated with a variety of cellular activitiessssingle-strandedADLASATP-DnaA-preferential low affinity sitesHSQCheteronuclear single quantum correlationRIDAregulatory inactivation of DnaASSBsingle-strand binding protein. with the aid of superhelicity of oriC DNA and heat energy, resulting in the formation of open complexes (4Sekimizu K. Bramhill D. Kornberg A. Cell. 1987; 50: 259-265Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 5Bramhill D. Kornberg A. Cell. 1988; 52: 743-755Abstract Full Text PDF PubMed Scopus (515) Google Scholar). At the unwound region, the loading of a DnaB replicative helicase is mediated by a DnaC helicase loader, resulting in the formation of the prepriming complex (6Marszalek J. Kaguni J.M. J. Biol. Chem. 1994; 269: 4883-4890Abstract Full Text PDF PubMed Google Scholar, 7Fang L. Davey M.J. O'Donnell M. Mol. Cell. 1999; 4: 541-553Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). DnaG primase then complexes with DnaB loaded on the single-stranded (ss) region, which leads to primer synthesis and the loading of DNA polymerase III holoenzyme (8O'Donnell M. J. Biol. Chem. 2006; 281: 10653-10656Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The cellular ATP-DnaA level fluctuates during the replication cycle with a peak around the time of initiation (9Kurokawa K. Nishida S. Emoto A. Sekimizu K. Katayama T. EMBO J. 1999; 18: 6642-6652Crossref PubMed Scopus (191) Google Scholar). At the post-initiation stage, DnaA-ATP is hydrolyzed in a manner depending on ADP-Hda protein and the DNA-loaded form of the β-clamp subunit of the polymerase III holoenzyme, yielding inactive ADP-DnaA (10Katayama T. Kubota T. Kurokawa K. Crooke E. Sekimizu K. Cell. 1998; 94: 61-71Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 11Kato J. Katayama T. EMBO J. 2001; 20: 4253-4262Crossref PubMed Scopus (217) Google Scholar, 12Su'etsugu M. Shimuta T.R. Ishida T. Kawakami H. Katayama T. J. Biol. Chem. 2005; 280: 6528-6536Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 13Su'etsugu M. Nakamura K. Keyamura K. Kudo Y. Katayama T. J. Biol. Chem. 2008; 283: 36118-36131Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). This DnaA inactivation system is called RIDA (regulatory inactivation of DnaA). Hda consists of a short N-terminal region bearing a clamp-binding motif and a C-terminal AAA+ domain. This protein is activated by ADP binding, which allows interaction with ATP-DnaA in a DNA-loaded β-clamp-dependent manner. RIDA decreases the level of cellular ATP-DnaA in a replication-coordinated manner and represses extra initiation events (9Kurokawa K. Nishida S. Emoto A. Sekimizu K. Katayama T. EMBO J. 1999; 18: 6642-6652Crossref PubMed Scopus (191) Google Scholar, 10Katayama T. Kubota T. Kurokawa K. Crooke E. Sekimizu K. Cell. 1998; 94: 61-71Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 11Kato J. Katayama T. EMBO J. 2001; 20: 4253-4262Crossref PubMed Scopus (217) Google Scholar). DNA unwinding element ATPases associated with a variety of cellular activities single-stranded ATP-DnaA-preferential low affinity sites heteronuclear single quantum correlation regulatory inactivation of DnaA single-strand binding protein. The timing of chromosomal replication initiation is strictly regulated and needs to be linked to the regulation of the dynamic conformational changes in the DnaA-oriC complexes, as well as to the cellular ATP-DnaA levels. DiaA is a DnaA-binding protein that stimulates ATP-DnaA assembly on oriC and thus the initiation of replication (14Ishida 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 (94) Google Scholar, 15Keyamura 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 (102) Google Scholar). DiaA mutants show delayed initiation and even asynchronous initiations of multiple origins when cells are rapidly growing and multiple rounds of replication are progressing simultaneously. DiaA is a homotetramer, and each protomer has a DnaA-binding site, which allows the simultaneous binding of multiple DnaA molecules to the homotetramer and the stimulation of cooperative binding of ATP-DnaA molecules on oriC. DnaA consists of four functional domains as follows: the C-terminal domain IV has a DNA-binding helix-turn-helix structure (16Fujikawa N. Kurumizaka H. Nureki O. Terada T. Shirouzu M. Katayama T. Yokoyama S. Nucleic Acids Res. 2003; 31: 2077-2086Crossref PubMed Scopus (147) Google Scholar) and domain III is an AAA+ domain that contains ATP-interacting motifs, homomultimer formation sites, and specific residues, termed B/H motifs, that can interact with ssDNA of the unwound DUE (17Neuwald A.F. Aravind L. Spouge J.L. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar, 18Iyer L.M. Leipe D.D. Koonin E.V. Aravind L. J. Struct. Biol. 2004; 146: 11-31Crossref PubMed Scopus (635) Google Scholar, 19Felczak M.M. Kaguni J.M. J. Biol. Chem. 2004; 279: 51156-51162Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 20Kawakami H. Keyamura K. Katayama T. J. Biol. Chem. 2005; 280: 27420-27430Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 21Ozaki S. Kawakami H. Nakamura K. Fujikawa N. Kagawa W. Park S.Y. Yokoyama S. Kurumizaka H. Katayama T. J. Biol. Chem. 2008; 283: 8351-8362Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Domain III forms a head-to-tail homomultimer whose overall structure is altered by ATP binding. It is possible that this multimer forms a spiral shape, in which one round of the spiral contains approximately seven protomers, and the resultant central pore carries the B/H motifs on the surface (21Ozaki S. Kawakami H. Nakamura K. Fujikawa N. Kagawa W. Park S.Y. Yokoyama S. Kurumizaka H. Katayama T. J. Biol. Chem. 2008; 283: 8351-8362Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 22Erzberger J.P. Mott M.L. Berger J.M. Nat. Struct. Mol. Biol. 2006; 13: 676-683Crossref PubMed Scopus (236) Google Scholar). Domain II is a flexible, unstructured linker (23Abe 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 (86) Google Scholar, 24Nozaki S. Ogawa T. Microbiology. 2008; 154: 3379-3384Crossref PubMed Scopus (39) Google Scholar), and domain I has a compactly folded structure, which interacts with several proteins including domain I per se, DiaA, and DnaB helicase (14Ishida 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 (94) Google Scholar, 15Keyamura 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 (102) Google Scholar, 23Abe 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 (86) Google Scholar, 25Sutton M.D. Carr K.M. Vicente M. Kaguni J.M. J. Biol. Chem. 1998; 273: 34255-34262Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 26Seitz H. Weigel C. Messer W. Mol. Microbiol. 2000; 37: 1270-1279Crossref PubMed Scopus (103) Google Scholar). Domain I most likely forms homodimers in a head-to-head manner, which would line up the DnaB-interacting sites within this domain, thereby promoting DnaB loading (23Abe 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 (86) Google Scholar). E. coli oriC carries a dozen DnaA-binding sites, including the high affinity 9-mer DnaA boxes (R1 and R4 sites) and ATP-DnaA-preferential low affinity sites (ADLAS), which include the I and τ sites (20Kawakami H. Keyamura K. Katayama T. J. Biol. Chem. 2005; 280: 27420-27430Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 27McGarry K.C. Ryan V.T. Grimwade J.E. Leonard A.C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2811-2816Crossref PubMed Scopus (128) Google Scholar). The interaction of ATP-DnaA with ADLAS is specifically important for the activation of DnaA-oriC complexes. DiaA stimulates the cooperative binding of ATP-DnaA on oriC, especially on ADLAS, resulting in the formation of open complexes (15Keyamura 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 (102) Google Scholar). DnaB helicase stably complexes with DnaC, and the resulting DnaBC complexes can interact with open complexes, loading DnaB onto ssDNA of the unwound DUE. We have previously determined the tertiary structure of the DnaA domain I and found that DnaA Glu-21, within this domain, is a DnaB interaction site, specifically required for DnaB loading onto open complexes (23Abe 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 (86) Google Scholar). The fundamental complex structure, the spatial organization of oriC-DnaA multimers complexed with DiaA, and those involved in the loading of DnaB onto oriC complexes have yet to be revealed. In this study, our first step was the determination of a crucial DiaA-binding site, Phe-46, on DnaA domain I, using NMR and mutant analyses. Next we found that this site is required for DiaA-dependent stimulation of initiation complex formation and that only a subgroup of DnaA molecules, assembled on oriC, is sufficient for DiaA stimulation. Furthermore, we revealed that DnaA Phe-46 is also important for interactions with DnaB helicase. Like the DiaA stimulation, the stimulation of DnaB loading requires only a subgroup of DnaA molecules assembled on oriC. Competition analyses suggested that DiaA and DnaB interact with a common DnaA subgroup on oriC. Only a specific DnaA subgroup in an initiation complex might expose domain I to a position available for the protein loading. Cells might contain a modulator for the inhibition of DnaB loading by DiaA. Thus we infer that DiaA can regulate the initiation of replication both positively and negatively, i.e. it promotes ATP-DnaA assembly and inhibits DnaB loading, thereby ensuring the sequential and regulated progress of initiation reactions. In addition we propose a novel model for the structure of initiation complexes that includes DiaA and suggest possible modes of interactions for DiaA and DnaB on the initial complexes. The NMR spectra were recorded at 25 °C on a Varian Unity INOVA 600 spectrometer. NMR samples of the 15N-labeled DnaA N terminus were dissolved in buffer (50 mm sodium phosphate (pH 6.5), 20 mm EDTA, 40 mm KCl, 2 mm dithiothreitol, and 10% sucrose containing 100% D2O or 90% H2O, 10% D2O). The chemical shift assignments for the DnaA N terminus in the same buffer were as described previously (28Abe Y. Watanabe N. Yoshida Y. Ebata F. Katayama T. Ueda T. Biomol. NMR Assign. 2007; 1: 57-59Crossref PubMed Scopus (3) Google Scholar). DiaA P72A was prepared by triplicate dialysis against the same buffer. We recorded the 1H-15N HSQC spectra of 0.1 mm 15N-labeled DnaA in the presence of various concentrations (0, 10, 20, and 30 μm) of dialyzed DiaA. For the identification of the binding site between DiaA and DnaA, we recorded 1H-15N HSQC for 48 h at each DiaA concentration. The respective concentrations of the proteins were corrected for dilution. The E. coli K12 derivatives KH5402-1 (wild type), KA450 [ΔoriC1071::Tn10 dnaA17 (Am) rnhA199 (Am)], TK24 [dnaA204 diaA26::Tn5], KA451 [dnaA::Tn10 rnhA::cat], and YH105 [thyA rpsL ΔdiaA::FRT kan] have been described previously (15Keyamura 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 (102) Google Scholar, 29Kawakami H. Ozaki S. Suzuki S. Nakamura K. Senriuchi T. Su'etsugu M. Fujimitsu K. Katayama T. Mol. Microbiol. 2006; 62: 1310-1324Crossref PubMed Scopus (32) Google Scholar, 30Fujimitsu K. Su'etsugu M. Yamaguchi Y. Mazda K. Fu N. Kawakami H. Katayama T. J. Bacteriol. 2008; 190: 5368-5381Crossref PubMed Scopus (50) Google Scholar). The wild-type diaA in KA451 was replaced with ΔdiaA::FRT kan by P1 transduction using YH105, resulting in KK001. LB medium contained Bacto-tryptone (1%), yeast extract (0.5%), and NaCl (1%). The plasmids pKA234, M13KEW101, and pOZ18 have also been described previously (29Kawakami H. Ozaki S. Suzuki S. Nakamura K. Senriuchi T. Su'etsugu M. Fujimitsu K. Katayama T. Mol. Microbiol. 2006; 62: 1310-1324Crossref PubMed Scopus (32) Google Scholar). For the construction of pDnaAF46A, a base substitution was introduced into the wild-type dnaA allele carried on pKA234 using a QuikChange site-directed mutagenesis kit (Stratagene). The mutagenic primers used for the construction of pDnaAF46A (bearing dnaA F46A) were 5′-GTACGCGCCAAACCGCGCGGTCCTTCGATTGGGTACG-3′, together with its complementary strand. For the construction of pRF46A, a 1.5-kb EcoRI fragment containing dnaA F46A was isolated from pDnaAF46A and inserted into the corresponding site of the low copy mini-R plasmid derivative, pOZ18. For the construction of pMZ1, a 5.7-kb BglII fragment was isolated from pOZ18, followed by self-ligation and exclusion of both dnaA and rnhA genes. For the construction of pRRNH, a 6.6-kb EcoRI fragment was isolated from pOZ18, followed by self-ligation and exclusion of the dnaA gene. Hexahistidine-fused DiaA (His-DiaA), including wild-type DiaA, DiaA P72A, and DiaA F191L, was purified as described previously (15Keyamura 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 (102) Google Scholar). Hexahistidine-fused DnaB (His-DnaB) was purified using a nickel - affinity purification system (31Chang P. Marians K.J. J. Biol. Chem. 2000; 275: 26187-26195Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Biotin-tagged DnaA (bio-DnaA) was purified as described previously (15Keyamura 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 (102) Google Scholar). This assay was performed as described previously (15Keyamura 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 (102) Google Scholar). These assays were performed as described previously, with minor modifications (15Keyamura 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 (102) Google Scholar). In an oriC pulldown assay, the indicated amounts of DnaA, His-DiaA, His-DnaB, DnaC, and biotin-tagged oriC fragment (419 bp) were incubated on ice for 15 min in buffer (10 μl) containing 50 mm HEPES-KOH (pH 7.6), 1 mm EDTA, 2 mm dithiothreitol, 20% sucrose, 100 mm KCl, 0.1 mg/ml bovine serum albumin, 1 mm ATP, 10 mm magnesium acetate, and 0.01% Triton X-100. The mixture was further incubated at 4 °C for 15 min with gentle rotation in the presence of streptavidin-coated beads (Promega) equilibrated in the same buffer (12.5 μl) as described above. The beads and bound material were collected, washed in buffer (12.5 μl) containing 50 mm HEPES-KOH (pH 7.6), 1 mm EDTA, 2 mm dithiothreitol, 20% sucrose, 50 mm KCl, 1 mm ATP, 10 mm magnesium acetate, and 0.01% Triton X-100, resuspended in 10 μl of SDS sample buffer, and analyzed by SDS-10−13% PAGE and silver staining. In a DnaA pulldown assay, the indicated amounts of DnaA, His-DiaA, an oriC fragment (419 bp), and bio-DnaA were incubated on ice for 15 min in the same buffer (10 μl) as above. bio-DnaA-bound materials were analyzed as above. The staged RIDA reconstituted system was performed essentially as described previously (13Su'etsugu M. Nakamura K. Keyamura K. Kudo Y. Katayama T. J. Biol. Chem. 2008; 283: 36118-36131Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 23Abe 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 (86) Google Scholar). Briefly, the DNA-loaded β-clamps were isolated using a gel filtration spin column as described previously (12Su'etsugu M. Shimuta T.R. Ishida T. Kawakami H. Katayama T. J. Biol. Chem. 2005; 280: 6528-6536Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 13Su'etsugu M. Nakamura K. Keyamura K. Kudo Y. Katayama T. J. Biol. Chem. 2008; 283: 36118-36131Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Next, [α-32P]ATP-DnaA (0.25 pmol) was incubated at 30 °C for 20 min in buffer (12.5 μl) containing 20 mm Tris-HCl (pH 7.5), 8 mm dithiothreitol, 8 mm magnesium acetate, 0.01% Brij-58, 10% glycerol, 0.1 mg/ml bovine serum albumin, 120 mm potassium glutamate, 30 μm ADP, the indicated amounts of the C-terminally hexahistidine-fused Hda (Hda-cHis), and the DNA-loaded β-clamps. Nucleotides bound to DnaA were recovered on a nitrocellulose filter, separated by thin layer chromatography, and quantified by a BAS2500 bioimaging analyzer (Fuji Film). This analysis was performed essentially as described previously (15Keyamura 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 (102) Google Scholar, 20Kawakami H. Keyamura K. Katayama T. J. Biol. Chem. 2005; 280: 27420-27430Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 21Ozaki S. Kawakami H. Nakamura K. Fujikawa N. Kagawa W. Park S.Y. Yokoyama S. Kurumizaka H. Katayama T. J. Biol. Chem. 2008; 283: 8351-8362Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Briefly, the indicated amounts of DnaA and His-DiaA were 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, 35 mm ammonium sulfate, 23 mm potassium acetate, 4 mm dithiothreitol, 10% 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 or ADP, and 5.5 ng of 32P-end-labeled oriC fragment (419 bp), which was amplified by PCR using a M13KEW101 oriC plasmid, followed by incubation at 30 °C for 4 min in the presence of DNase I (1.3 milliunits; Invitrogen). The resultant DNA samples were purified, separated by 5% sequence gel electrophoresis, and analyzed using a BAS2500 bioimaging analyzer (Fuji Film). This assay was performed as described previously (15Keyamura 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 (102) Google Scholar). The indicated amounts of DnaA were incubated at 38 °C for 3 min in buffer (50 μl) containing 60 mm HEPES-KOH (pH 7.6), 130 mm KCl, 0.1 mm zinc acetate, 8 mm magnesium acetate, 30% glycerol, 0.32 mg/ml bovine serum albumin, 5 mm ATP, 16 ng of HU protein, 320 ng of M13KEW101 RF I (61 fmol), and the indicated amounts of His-DiaA, followed by incubation at the same temperature for 200 s in the presence of P1 nuclease (10 units; Yamasa Co.). The resultant DNA samples were purified and digested with AlwNI, following by 1% agarose gel electrophoresis and ethidium bromide staining. The DNA fragments were quantified by densitometric scanning. This assay was performed as described previously (23Abe 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 (86) Google Scholar, 29Kawakami H. Ozaki S. Suzuki S. Nakamura K. Senriuchi T. Su'etsugu M. Fujimitsu K. Katayama T. Mol. Microbiol. 2006; 62: 1310-1324Crossref PubMed Scopus (32) Google Scholar). Briefly, the indicated amounts of DnaA were incubated at 30 °C for 10 min in buffer (25 μl) containing M13-A site single-stranded DNA (220 pmol as nucleotide), 20 mm Tris-HCl (pH 7.5), 0.1 mg/ml bovine serum albumin, 8 mm dithiothreitol, 8 mm magnesium acetate, 0.01% Brij-58, 125 mm potassium glutamate, 0.5 μg of SSB, 117 ng of DnaB, 260 ng of DnaC, 72 ng of DnaG, 76 ng of DNA polymerase III*, 26 ng of β-clamp subunit, 1 mm ATP, 0.25 mm each of GTP, CTP, and UTP, 0.1 mm each of dNTP, and [α-32P]dTTP (50–100 cpm/pmol). DNA polymerase III* consists of the clamp loader complex and the DNA polymerase III core complex, which contains the catalytic center of the polymerase. [α-32P]dTTP was included to enable the subsequent measurement of DNA synthesis by liquid scintillation counting of acid-insoluble materials. This assay was performed essentially as described previously (23Abe 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 (86) Google Scholar). Briefly, the indicated amounts of ATP-DnaA were incubated at 30 °C for 30 min in buffer (12.5 μl) containing 20 mm Tris-HCl (pH 7.5), 0.1 mg/ml bovine serum albumin, 8 mm dithiothreitol, 10 mm magnesium acetate, 125 mm potassium glutamate, 2 mm ATP, 1.2 μg of SSB, 1.3 ng of HU protein, 68 ng of DnaB, 46 ng of DnaC, 90 ng of DNA gyrase A subunit, 113 ng of DNA gyrase B subunit, and 100 ng of M13KEW101 RF I (19 fmol). The reaction was stopped in the presence of SDS and EDTA, followed by 0.65% agarose gel electrophoresis and ethidium bromide staining. The produced form I* DNA was quantified by densitometry. This system was performed essentially as described previously (14Ishida 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 (94) Google Scholar, 15Keyamura 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 (102) Google Scholar). Briefly, the indicated amounts of ATP-DnaA and His-DiaA were incubated at 30 °C for 20 min in buffer (25 μl) containing 40 mm HEPES-KOH (pH 7.6), 40 mm phosphocreatine, 2 mm ATP, 0.5 mm each of GTP, CTP, and UTP, 10 mm magnesium acetate, 0.1 mg/ml creatine kinase, and 7% polyvinyl alcohol, 240 μg of crude protein extract (TK24 fraction II), 200 ng of M13KEW101 RF I (600 pmol as nucleotide), 0.1 mm each of dNTP, and [α-32P]dTTP (50–100 cpm/pmol). This analysis was performed essentially as described previously (15Keyamura 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 (102) Google Scholar, 29Kawakami H. Ozaki S. Suzuki S. Nakamura K. Senriuchi T. Su'etsugu M. Fujimitsu K. Katayama T. Mol. Microbiol. 2006; 62: 1310-1324Crossref PubMed Scopus (32) Google Scholar). Briefly, cells were grown in LB medium containing thymine (50 μg/ml) and ampicillin (50 μg/ml) exponentially for 10 generations at 30 °C until the absorbance (A660) reached 0.2. Incubation was further continued at the same temperature for 4 h in the presence of rifampicin (300 μg/ml) and cephalexin (10 μg/ml). Cells were fixed in cold 70% ethanol, washed, and stained with 2 μm SYTOX green (Invitrogen), followed by analysis using a FACSCalibur flow cytometry (BD Biosciences). This analysis was performed as described previously (15Keyamura 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 (102) Google Scholar, 29Kawakami H. Ozaki S. Suzuki S. Nakamura K. Senriuchi T. Su'etsugu M. Fujimitsu K. Katayama T. Mol. Microbiol. 2006; 62: 1310-1324Crossref PubMed Scopus (32) Google Scholar). Previously we determined the structures of the DiaA homotetramer, using x-ray analysis, and the DnaA N-terminal domains I and II (residues 1–108), using NMR analysis (15Keyamura 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 (102) Google Scholar, 23Abe 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 (86) Google Scholar). In addition, we showed that DiaA binds directly to DnaA domain I (residues 1–86) (14Ishida 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 (94) Google Scholar, 15Keyamura 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 (102) Google Scholar). Based on these results, we performed NMR analysis using DiaA and 15N-labeled DnaA domains I and II, to determine the mode of the DiaA-DnaA domain I interaction. When we used wild-type DiaA, severe signal broadening occurred, and site-specific chemical shifts were not detected, probably because of the increased molecular size because of tight binding between the two proteins (data not shown). When we used DiaA P72A, which has a considerably reduced specific affinity for DnaA (15Keyamura 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 (102) Google Scholar), specific chemical shifts were observed. As the concentration of DiaA P72A increased, signal broadening occurred in the 1H-15N HSQC spectra of the N terminus of the DnaA protein, especially in domain I (Fig. 1, A and B), consistent with an interaction between DiaA and DnaA domain I. When the molar ratio of DnaA:DiaA was 1:0.2, we found slight, but significant, chemical shift changes for several specific residues, including, in particular, Glu-21, Trp-25, and Phe-46 (Fig. 1, C and D). Based on the results above, we constructed a structural model of the DiaA-DnaA domain I complex (Fig. 1E). DnaA Glu-21 and Phe-46 are exposed on the protein surface where they form a small patch together with Trp-25 and Trp-50 (23Abe Y. Jo T. Mat

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