Title: DiaA, a Novel DnaA-binding Protein, Ensures the Timely Initiation of Escherichia coli Chromosome Replication
Abstract: The DnaA protein is the initiator of Escherichia coli chromosomal replication. In this study, we identify a novel DnaA-associating protein, DiaA, that is required for the timely initiation of replication during the cell cycle. DiaA promotes the growth of specific temperature-sensitive dnaA mutants and ensures stable minichromosome maintenance, whereas DiaA does not decrease the cellular DnaA content. A diaA::Tn5 mutation suppresses the cold-sensitive growth of an overinitiation type dnaA mutant independently of SeqA, a negative modulator of initiation. Flow cytometry analyses revealed that the timing of replication initiation is disrupted in the diaA mutant cells as well as wild-type cells with pBR322 expressing the diaA gene. Gel filtration and chemical cross-linking experiments showed that purified DiaA forms a stable homodimer. Immunoblotting analysis indicated that a single cell contains about 280 DiaA dimers. DiaA stimulates minichromosome replication in an in vitro system especially when the level of DnaA included is limited. Moreover, specific and direct binding between DnaA and DiaA was observed, which requires a DnaA N-terminal region. DiaA binds to both ATP- and ADP-bound forms of DnaA with a similar affinity. Thus, we conclude that DiaA is a novel DnaA-associating factor that is crucial to ensure the timely initiation of chromosomal replication. The DnaA protein is the initiator of Escherichia coli chromosomal replication. In this study, we identify a novel DnaA-associating protein, DiaA, that is required for the timely initiation of replication during the cell cycle. DiaA promotes the growth of specific temperature-sensitive dnaA mutants and ensures stable minichromosome maintenance, whereas DiaA does not decrease the cellular DnaA content. A diaA::Tn5 mutation suppresses the cold-sensitive growth of an overinitiation type dnaA mutant independently of SeqA, a negative modulator of initiation. Flow cytometry analyses revealed that the timing of replication initiation is disrupted in the diaA mutant cells as well as wild-type cells with pBR322 expressing the diaA gene. Gel filtration and chemical cross-linking experiments showed that purified DiaA forms a stable homodimer. Immunoblotting analysis indicated that a single cell contains about 280 DiaA dimers. DiaA stimulates minichromosome replication in an in vitro system especially when the level of DnaA included is limited. Moreover, specific and direct binding between DnaA and DiaA was observed, which requires a DnaA N-terminal region. DiaA binds to both ATP- and ADP-bound forms of DnaA with a similar affinity. Thus, we conclude that DiaA is a novel DnaA-associating factor that is crucial to ensure the timely initiation of chromosomal replication. In Escherichia coli, the timing of initiation of chromosomal replication during the cell cycle is strictly regulated (1Helmstetter C. Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2. American Society for Microbiology, Washington, D. C.1996: 1627-1639Google Scholar). Only a few protein factors related to this regulation have been identified to date. The DnaA protein is the initiator of replication, and its nucleotide-bound forms change in coordination with the replication cycle (2Boye E. Løbner-Olesen A. Skarstad K. EMBO Rep. 2000; 1: 479-483Crossref PubMed Scopus (130) Google Scholar, 3Katayama T. Mol. Microbiol. 2001; 41: 9-17Crossref PubMed Scopus (66) Google Scholar, 4Messer W. FEMS Microbiol. Rev. 2002; 26: 355-374PubMed Google Scholar). The cellular content of ATP-bound DnaA increases temporarily around the time of replication initiation (5Kurokawa K. Nishida S. Emoto A. Sekimizu K. Katayama T. EMBO J. 1999; 18: 6642-6652Crossref PubMed Scopus (191) Google Scholar). DnaA in its active ATP-bound form unwinds duplex in the initiation complex, which includes the origin (oriC) and DnaA multimers, so that DnaB helicase, DnaG primase, and DNA polymerase III holoenzyme can assemble replisomes on single-stranded DNA (4Messer W. FEMS Microbiol. Rev. 2002; 26: 355-374PubMed Google Scholar, 6Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman and Company, NY1992Google Scholar). The level of DnaA-ATP decreases in a DNA replication-dependent manner due to hydrolysis of DnaA-bound ATP. The resultant ADP-DnaA is inactive. The DNA-loaded sliding clamp subunit of DNA polymerase III holoenzyme and Hda protein functionally interact with ATP-DnaA to induce hydrolysis (7Katayama T. Kubota T. Kurokawa K. Crooke E. Sekimizu K. Cell. 1998; 94: 61-71Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 8Kato J. Katayama T. EMBO J. 2001; 20: 4253-4262Crossref PubMed Scopus (217) Google Scholar). This system for regulatory inactivation of DnaA is designated "RIDA 1The abbreviations used are: RIDA, regulatory inactivation of DnaA; ORF, open reading frame; DAD, DnaA domain." and limits initiation to once per oriC per cell cycle. In dnaA mutant cells, the timing of initiation at oriC is disrupted during the cell cycle, even under conditions that allow the cells to grow (9Skarstad K. Boye E. Steen H.B. EMBO J. 1986; 5: 1711-1717Crossref PubMed Scopus (299) Google Scholar, 10Skarstad K. von Meyenburg K. Hansen F.G. Boye E. J. Bacteriol. 1988; 170: 852-858Crossref PubMed Google Scholar). This finding indicates that timely initiation requires strict regulation of DnaA activity In E. coli, when the growth rate of cells is above a certain level, the next round of replication is initiated while the present round is still ongoing (1Helmstetter C. Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2. American Society for Microbiology, Washington, D. C.1996: 1627-1639Google Scholar). These cells contain two or four copies, or four or eight copies of oriC, depending on the growth conditions. In wild-type cells, the initiation steps at multiple oriC sites are synchronized (9Skarstad K. Boye E. Steen H.B. EMBO J. 1986; 5: 1711-1717Crossref PubMed Scopus (299) Google Scholar). In seqA mutant cells, this synchrony is disturbed, resulting in odd numbers of oriC copies in a cell (11Lu M. Campbell J.L. Boye E. Kleckner N. Cell. 1994; 77: 413-426Abstract Full Text PDF PubMed Scopus (421) Google Scholar). The dam gene product, Dam, methylates the adenine residue in the palindromic sequence GATC that is repeated 11 times within the minimal oriC region (12Zyskind J.W. Smith D.W. Cell. 1986; 46: 489-490Abstract Full Text PDF PubMed Scopus (59) Google Scholar). SeqA preferentially binds to these hemimethylated GATC sequences, which inhibits re-initiation at oriC (11Lu M. Campbell J.L. Boye E. Kleckner N. Cell. 1994; 77: 413-426Abstract Full Text PDF PubMed Scopus (421) Google Scholar, 13Slater S. Wold S. Lu M. Boye E. Skarstad K. Kleckner N. Cell. 1995; 82: 927-936Abstract Full Text PDF PubMed Scopus (246) Google Scholar, 14Wold S. Boye E. Slater S. Kleckner N. Skarstad K. EMBO J. 1998; 17: 4158-4165Crossref PubMed Scopus (65) Google Scholar, 15Skarstad K. Lueder G. Lurz R. Speck C. Messer W. Mol. Microbiol. 2000; 36: 1319-1326Crossref PubMed Scopus (58) Google Scholar, 16Taghbalout A. Landoulsi A. Kern R. Yamazoe M. Hiraga S. Holland B. Kohiyama M. Malki A. Genes Cells. 2000; 5: 873-884Crossref PubMed Scopus (42) Google Scholar). Semi-conservative replication of fully methylated oriC DNA yields hemimethylated oriC, which is sustained for a while by SeqA until Dam re-methylates these sites. Lack of SeqA accelerates the timing of re-methylation of oriC (11Lu M. Campbell J.L. Boye E. Kleckner N. Cell. 1994; 77: 413-426Abstract Full Text PDF PubMed Scopus (421) Google Scholar). Asynchronous initiation is also observed in dam mutants (17Boye E. Løbner-Olesen A. Cell. 1990; 62: 981-989Abstract Full Text PDF PubMed Scopus (175) Google Scholar). Moreover, cells expressing mutants of dnaC and Histone-like proteins, HU, IHF, and FIS, display an asynchronous replication phenotype during exponential growth (18Boye E. Løbner-Olesen A. Skarstad K. Biochim. Biophys. Acta. 1988; 951: 359-364Crossref PubMed Scopus (46) Google Scholar, 19Boye E. Lyngstadaas A. Løbner-Olesen A. Skarstad K. Wold S. Fanning E. DNA Replication and the Cell Cycle. Springer-Verlag, Berlin, Germany1993: 15-26Crossref Google Scholar, 20Withers H. Bernander R. J. Bacteriol. 1998; 180: 1624-1631Crossref PubMed Google Scholar, 21Ryan V.T. Grimwade J.E. Nievera C.J. Leonard A.C. Mol. Microbiol. 2002; 46: 113-124Crossref PubMed Scopus (85) Google Scholar). These histone-like proteins may participate in initiation complex formation at oriC (21Ryan V.T. Grimwade J.E. Nievera C.J. Leonard A.C. Mol. Microbiol. 2002; 46: 113-124Crossref PubMed Scopus (85) Google Scholar, 22Cassler M.R. Grimwade J.E. Leonard A.C. EMBO J. 1995; 14: 5833-5841Crossref PubMed Scopus (110) Google Scholar, 23Grimwade J.E. Ryan V.T. Leonard A.C. Mol. Microbiol. 2000; 35: 825-844Crossref PubMed Scopus (65) Google Scholar). As specified above, strict regulation of oriC and DnaA functions is necessary to ensure precise initiation timing. In this study, we isolated a suppressor gene from an overinitiation-type dnaA mutant, dnaAcos (24Kellenberger-Gujer G. Podhajska A.J. Caro L. Mol. Gen. Genet. 1978; 162: 9-16Crossref PubMed Scopus (86) Google Scholar, 25Katayama T. Kornberg A. J. Biol. Chem. 1994; 269: 12698-12703Abstract Full Text PDF PubMed Google Scholar, 26Katayama T. Akimitsu T. Mizushima T. Miki T. Sekimizu K. Mol. Microbiol. 1997; 25: 661-670Crossref PubMed Scopus (19) Google Scholar, 27Su'etsugu M. Emoto A. Fujimitsu K. Keyamura K. Katayama T. Genes Cells. 2003; 8: 731-745Crossref PubMed Scopus (25) Google Scholar). Our data show that this previously uncharacterized gene is required to ensure the timely initiation of chromosomal replication. A mutant bearing an inactive form of this gene displays an asynchronous replication phenotype. Moreover, the encoded protein directly and specifically binds DnaA. We designate this novel gene diaA, representing "DnaA initiator-associating factor." Strains, Plasmids, Media, and Buffer—E. coli K-12 strains used for genetic experiments are described in Table I. The recA::Tn10 mutation was introduced via P1 transduction. Transductants of recipient strains bearing wild-type diaA and diaA::Tn5 mutations were obtained with similar efficiency. The recA mutation was confirmed by measuring its sensitivity to UV. pKP1673, a gift from Dr. Takeyoshi Miki, is a low copy mini-R vector containing genes for active partition and chloramphenicol resistance (30Miki T. Park J.A. Nagao K. Murayama N. Horiuchi T. J. Mol. Biol. 1992; 225: 39-52Crossref PubMed Scopus (114) Google Scholar). For plasmid complementation experiments, a 3.3-kb HpaI fragment bearing the diaA gene (yraO) was isolated from a Kohara λ phage #518 (31Kohara Y. Akiyama K. Isono K. Cell. 1987; 50: 495-508Abstract Full Text PDF PubMed Scopus (1110) Google Scholar) and inserted into the StuI site of pKP1673, resulting in pNA095. A 1.2-kb HpaI-BssHII fragment containing the entire diaA gene was similarly isolated, the BssHII end was filled in, and the resultant fragment was inserted to the StuI site of pKP1673, generating pNA102. The diaA gene within pNA102 was digested with MluI, filled in, and self-ligated to produce pNA121. The 1.2-kb HpaI-BssHII fragment was similarly filled in and inserted into the HincII site of pUC19. From the resultant plasmid (pNA159), a 1.2-kb EcoRI-PstI fragment containing diaA was isolated and inserted into the corresponding restriction sites of pT7-5, generating pKA251. For protein purification, BL21(DE3) cells containing pKA251 were used for the overproduction of DiaA. The filled-in 1.2-kb HpaI-BssHII fragment was additionally inserted into the EcoRV site of pBR322 and designated pNA135. pTOA5 (2.7 kb) and pTOA24 (6.1 kb) minichromosomes bearing oriC-mioC and gidA-oriC-mioC regions, respectively, in addition to the β-lactamase gene, were kindly provided by Dr. Tohru Ogawa (32Kano Y. Ogawa T. Ogura T. Hiraga S. Okazaki T. Imamoto F. Gene (Amst.). 1991; 103: 25-30Crossref PubMed Scopus (37) Google Scholar). The media used are described in a previous report (24Kellenberger-Gujer G. Podhajska A.J. Caro L. Mol. Gen. Genet. 1978; 162: 9-16Crossref PubMed Scopus (86) Google Scholar, 26Katayama T. Akimitsu T. Mizushima T. Miki T. Sekimizu K. Mol. Microbiol. 1997; 25: 661-670Crossref PubMed Scopus (19) Google Scholar, 33Katayama T. Takata M. Sekimizu K. Mol. Microbiol. 1997; 26: 687-697Crossref PubMed Scopus (35) Google Scholar). M9 medium was supplemented with 0.2% glucose, 2 μg/ml thiamine, 20 μg/ml tyrosine, and 40 μg/ml each of isoleucine, valine, threonine, methionine, and tryptophan. Unless otherwise indicated, the medium was supplemented with 50 μg/ml thymine. M13oriCE10 and pBSoriC (or pTB101) are derivatives of M13mp10 and pBluescript vectors, respectively, containing a minimal oriC region (34Nishida 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 (107) Google Scholar). Buffer C contained 40 mm Hepes-KOH (pH 7.6), 1 mm EDTA, 2 mm dithiothreitol, and 15% glycerol. Buffer C′ represents buffer C containing 10% glycerol. Buffer C″ contained 50 mm Hepes-KOH (pH 7.6), 1 mm EDTA, 2 mm dithiothreitol, and 20% sucrose.Table IStrains used in this studyStrainRelevant genotypeSource or referenceKH5402-1thyA(26Katayama T. Akimitsu T. Mizushima T. Miki T. Sekimizu K. Mol. Microbiol. 1997; 25: 661-670Crossref PubMed Scopus (19) Google Scholar)KA452KH5402-1 dnaA46 tnaA::Tn10(26Katayama T. Akimitsu T. Mizushima T. Miki T. Sekimizu K. Mol. Microbiol. 1997; 25: 661-670Crossref PubMed Scopus (19) Google Scholar)KA466KH5402-1 dnaA508 tnaA::Tn10 diaA26::Tn5This workNKN1KH5402-1 dnaA508 tnaA::Tn10(27Su'etsugu M. Emoto A. Fujimitsu K. Keyamura K. Katayama T. Genes Cells. 2003; 8: 731-745Crossref PubMed Scopus (25) Google Scholar)NKN211KH5402-1 dnaA5 tnaA::Tn10(27Su'etsugu M. Emoto A. Fujimitsu K. Keyamura K. Katayama T. Genes Cells. 2003; 8: 731-745Crossref PubMed Scopus (25) Google Scholar)NKN212KH5402-1 dnaA508 tnaA::Tn10(27Su'etsugu M. Emoto A. Fujimitsu K. Keyamura K. Katayama T. Genes Cells. 2003; 8: 731-745Crossref PubMed Scopus (25) Google Scholar)NKN241KH5402-1 dnaA601 tnaA::Tn10(27Su'etsugu M. Emoto A. Fujimitsu K. Keyamura K. Katayama T. Genes Cells. 2003; 8: 731-745Crossref PubMed Scopus (25) Google Scholar)NA001KH5402-1 dnaAcos(26Katayama T. Akimitsu T. Mizushima T. Miki T. Sekimizu K. Mol. Microbiol. 1997; 25: 661-670Crossref PubMed Scopus (19) Google Scholar)NA026KH5402-1 dnaAcos diaA26::Tn5This workNA141KH5402-1 diaA26::Tn5This workNA169KH5402-1 recA::Tn10This workNA173KH5402-1 recA::Tn10 diaA26::Tn5This workNA181KH5402-1 dnaA46 tnaA::Tn10 diaA26::Tn5This workNA183KH5402-1 dnaA508 tnaA::Tn10 diaA26::Tn5This workNA184KH5402-1 dnaA5 tnaA::Tn10 diaA26::Tn5This workNA185KH5402-1 dnaA601 tnaA::Tn10 diaA26::Tn5This workWM433dnaA204(28Fuller R.S. Kaguni J.M. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7370-7374Crossref PubMed Scopus (240) Google Scholar)TK24WM433 diaA26::Tn5This workMC1061araΔ139 Δ(ara-leu)7696(29Kubota T. Katayama T. Ito Y. Mizushima T. Sekimizu K. Biochem. Biophys. Res. Commun. 1997; 232: 130-135Crossref PubMed Scopus (38) Google Scholar) Open table in a new tab Isolation of dnaAcos Suppressors Using Transposon Mutagenesis and Determination of the Insertion Site—A previously described procedure was employed (26Katayama T. Akimitsu T. Mizushima T. Miki T. Sekimizu K. Mol. Microbiol. 1997; 25: 661-670Crossref PubMed Scopus (19) Google Scholar, 27Su'etsugu M. Emoto A. Fujimitsu K. Keyamura K. Katayama T. Genes Cells. 2003; 8: 731-745Crossref PubMed Scopus (25) Google Scholar). Briefly, defective λ phage containing Tn5 (λ467) was introduced into NA001 (dnaAcos) at 42 °C, and Tn5 was transposed onto the chromosome. After the strain was cured of λ phage, kanamycin-resistant colonies were isolated and purified at 30 °C. Tn5 in isolated suppressor mutants was transduced back into NA001 using P1 phage for confirmation of growth at 30 °C. One of the resultant transductants was designated NA026. The BamHI fragment of NA026 chromosomal DNA, which includes part of Tn5 encoding the kanamycin-resistant gene and the border region between Tn5 and the chromosome, was cloned into pUC18. The border region was sequenced, as described previously (26Katayama T. Akimitsu T. Mizushima T. Miki T. Sekimizu K. Mol. Microbiol. 1997; 25: 661-670Crossref PubMed Scopus (19) Google Scholar, 27Su'etsugu M. Emoto A. Fujimitsu K. Keyamura K. Katayama T. Genes Cells. 2003; 8: 731-745Crossref PubMed Scopus (25) Google Scholar). Immunoblotting Analysis—Immunoblot analysis for DiaA was basically similar to that for DnaA (25Katayama T. Kornberg A. J. Biol. Chem. 1994; 269: 12698-12703Abstract Full Text PDF PubMed Google Scholar), except that polyclonal rabbit anti-DiaA antiserum and NA141 (diaA::Tn5) were used. Briefly, cells were grown exponentially at 37 °C in LB medium. At an optical density (A595) of 0.8, 1-ml aliquots were withdrawn, and 5% trichloroacetic acid immediately added. Precipitates formed on ice were collected by brief centrifugation and used for SDS-polyacrylamide (12%) gel electrophoresis. Separated proteins were blotted onto Immobilon-P membrane (Millipore) and detected using polyclonal rabbit anti-DiaA antiserum and alkaline phosphate-conjugated anti-rabbit antiserum (Bio-Rad). Flow Cytometry Analysis—Flow cytometry was performed according to an earlier report (26Katayama T. Akimitsu T. Mizushima T. Miki T. Sekimizu K. Mol. Microbiol. 1997; 25: 661-670Crossref PubMed Scopus (19) Google Scholar, 27Su'etsugu M. Emoto A. Fujimitsu K. Keyamura K. Katayama T. Genes Cells. 2003; 8: 731-745Crossref PubMed Scopus (25) Google Scholar, 33Katayama T. Takata M. Sekimizu K. Mol. Microbiol. 1997; 26: 687-697Crossref PubMed Scopus (35) Google Scholar). Briefly, cells were grown exponentially for about 10 generations at 30 °C in the indicated media with serial dilutions until an optical density (A660) of 0.2. Incubation was further continued in the presence of rifampicin (150 μg/ml) and cephalexin (10 μg/ml) for 4 h at the same temperature. Cells were collected by brief centrifugation and suspended in cold 70% ethanol. After washing and suspension of cells in cold buffer containing 10 mm Tris-HCl (pH 7.5) and 20 mm magnesium sulfate, chromosomal DNA was stained with mithramycin (27 μg/ml) and ethidium bromide (5 μg/ml). Cells were analyzed with a BRYTE-HS flow cytometer (Bio-Rad). dnaA46 cells incubated at 42 °C and containing one or two chromosomes per cell were used to calibrate the chromosome number. Overproduction and Purification of DiaA—BL21(DE3) cells bearing pKA251 were grown at 37 °C in LB medium (4.8 liters) containing ampicillin (50 μg/ml). Isopropyl-1-thio-β-d-galactosidase (1 mm) was added at an optical density (A660) of 0.8, and incubation continued for 4 h. Cells were harvested by centrifugation at 4 °C, resuspended to an optical density (A595) of 200 in cold buffer containing 25 mm Hepes-KOH (pH7.6), 1 mm EDTA, and 2 mm dithiothreitol, and frozen in liquid nitrogen for storage at –80 °C. Frozen cells were thawed at 4–8 °C, incubated on ice for 30 min in the presence of 100 mm KCl and 300 μg/ml lysozyme, and frozen in liquid nitrogen. All the procedures described below were performed at 0–6 °C. Frozen cell paste was thawed, and supernatant fractions obtained by centrifugation in a Beckman type 50.2 Ti rotor at 40,000 rpm for 20 min. Proteins in the supernatant (fraction I, 21.5 ml) were precipitated with 0.22 g/ml ammonium sulfate, collected by centrifugation, and resuspended in chilled buffer C (fraction II, 1.5 ml). The solution was dialyzed against buffer C and diluted in the same buffer to 2 mg protein/ml. Supernatant (44 ml) was obtained by centrifugation, and a 40-ml aliquot was loaded onto a hydroxylapatite column (18 ml) equilibrated with buffer C at a flow rate of 18 ml/h. The column was washed with three volumes of buffer C, three volumes of buffer C containing 1 m KCl, and two volumes of buffer C containing 1 mm potassium phosphate. Proteins were eluted with a linear gradient (180 ml) from 1 to 300 mm potassium phosphate in buffer C. DiaA was detected in fractions containing 10–20 mm potassium phosphate (fraction III, 10 ml). Next, fraction III was dialyzed against buffer C containing 50 mm KCl and loaded onto a Mono Q column (bed volume of 1 ml, Amersham Biosciences) equilibrated with the same buffer at a flow rate of 0.2 ml/min. The column was washed with three volumes of buffer C containing 50 mm KCl, followed by five volumes of buffer C containing 200 mm KCl. Proteins were eluted with a linear gradient (10 ml) from 200 to 600 mm KCl in buffer C. DiaA protein was eluted at around 350 mm KCl (fraction IV, 1.6 ml). Chemical Cross-linking of DiaA—DiaA (5 μg) was incubated at room temperature for 3 h in buffer (25 μl) containing 40 mm Hepes-KOH (pH7.6), 40 mm KCl, 1 mm EDTA, 2 mm dithiothreitol, 20% sucrose, and the indicated amounts of glutaraldehyde. Protein was precipitated by incubation on ice in the presence of 10% trichloroacetic acid. Precipitates were collected, dissolved in 40 μl of standard SDS sample buffer, and incubated at 100 °C for 5 min. Aliquots (10 μl) were subjected to SDS-polyacrylamide (12%) gel electrophoresis, and proteins were stained with Coomassie Brilliant Blue. Gel Filtration of DiaA Protein—DiaA protein (200 μl of fraction IV, 3.1 mg/ml) was loaded on a Superdex 75 HR 10/30 column (Amersham Biosciences) equilibrated with buffer C containing 150 mm KCl at 4–6 °C at a flow rate of 0.3 ml/min. Using the same buffer and flow rate, DiaA was eluted. The elution profile was monitored by absorbance at 280 nm. In Vitro Minichromosome Replication and RIDA Assay—A replicative crude extract was prepared from WM433 (dnaA204) and TK24 (WM433 diaA::Tn5), using the method described previously (7Katayama T. Kubota T. Kurokawa K. Crooke E. Sekimizu K. Cell. 1998; 94: 61-71Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 25Katayama T. Kornberg A. J. Biol. Chem. 1994; 269: 12698-12703Abstract Full Text PDF PubMed Google Scholar, 28Fuller R.S. Kaguni J.M. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7370-7374Crossref PubMed Scopus (240) Google Scholar). Using the indicated amounts of extracts and DnaA protein, M13oriCE10 minichromosome RFI (200 ng; 600 pmol as nucleotide) was incubated in reaction (25 μl), as described previously (25Katayama T. Kornberg A. J. Biol. Chem. 1994; 269: 12698-12703Abstract Full Text PDF PubMed Google Scholar, 28Fuller R.S. Kaguni J.M. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7370-7374Crossref PubMed Scopus (240) Google Scholar, 35Katayama T. Crooke E. J. Biol. Chem. 1995; 270: 9265-9271Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The RIDA reaction was promoted as above, except that [α-32P]dTTP was excluded and [α-32P]ATP-DnaA was added (7Katayama T. Kubota T. Kurokawa K. Crooke E. Sekimizu K. Cell. 1998; 94: 61-71Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 8Kato J. Katayama T. EMBO J. 2001; 20: 4253-4262Crossref PubMed Scopus (217) Google Scholar, 35Katayama T. Crooke E. J. Biol. Chem. 1995; 270: 9265-9271Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Nucleotide-bound forms of DnaA were assessed by immunoprecipitation and thinlayer chromatography, in accordance with earlier reports (7Katayama T. Kubota T. Kurokawa K. Crooke E. Sekimizu K. Cell. 1998; 94: 61-71Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 8Kato J. Katayama T. EMBO J. 2001; 20: 4253-4262Crossref PubMed Scopus (217) Google Scholar). Assays for Specific Binding of DiaA to DNA-bound DnaA—Gel filtration experiments were performed as follows: DnaA protein (11.5 pmol) and pBSoriC (0.43 pmol) were incubated for 5 min at 30 °C in buffer C (25 μl) containing 50 mm KCl. Fraction II (1 μg) prepared from DiaA-overproducing cells was added. After further incubation for 5 min at 30 °C, the reaction was loaded onto a Microspin S-400HR spin column (Amersham Biosciences) equilibrated with the same buffer, and the void fraction was obtained by centrifugation (3000 rpm, 2 min) at room temperature. Control experiments in which DnaA, pBSoriC, or DiaA were excluded from the reaction, were additionally performed. Eluted proteins were analyzed by SDS-polyacrylamide (12%) gel electrophoresis and silver staining. Similar results were obtained in the presence of 150 mm KCl. A pull-down assay using a biotin-conjugated polynucleotide was performed as we described previously (36Obita T. Iwura T. Su'etsugu M. Yoshida Y. Tanaka Y. Katayama T. Ueda T. Imoto T. Biochem. Biophys. Res. Commun. 2002; 299: 42-48Crossref PubMed Scopus (17) Google Scholar), except that DiaA was included in the binding reaction. Briefly, the polynucleotide used was doublestrand DNA (29-mer) containing DnaA box R1 with a 5′-biotin-conjugated 30-mer T-stretch tag. DnaA protein (10 pmol) and biotin-tagged DnaA box DNA (5 pmol) were incubated for 5 min on ice in buffer C′ containing 100 mm KCl (50 μl). DiaA protein (2.4 pmol of dimer or the indicated amounts) was added, and incubation continued for 5 min on ice. Streptavidin-coated magnetic beads in suspension (50 μl; Promega) were washed twice in buffer C′ and resuspended in an equal volume of buffer C′ containing 100 mm KCl and bovine serum albumin (100 μg/ml), then added to the reaction mixture, and incubation continued for 1 h at 4 °C with gentle rotation. Streptavidin-coated beads and bound materials were collected using magnetic force, washed in buffer C′ containing 100 mm KCl and 100 μg/ml bovine serum albumin, and suspended in standard SDS sample buffer (20 μl). Beads were excluded as above, and proteins were analyzed using SDS-polyacrylamide gel electrophoresis. The buffer for washing included the same nucleotide at a concentration of 1 μm when nucleotide-bound DnaA was used. When a cleared soluble extract (fraction I) prepared from WM433 was used instead of DiaA, the above method was applied, except for the addition of buffer C″ containing 150 mm KCl (instead of buffer C′ containing 100 mm KCl). Briefly, DnaA protein (20 pmol) and biotin-tagged DnaA box DNA (20 pmol) were incubated for 5 min on ice in buffer C″ (200 μl) containing 150 mm KCl. DnaA bound to DNA was isolated using streptavidin beads as above, suspended in buffer C″ (160 μl) containing 400 μg of WM433 fraction I and 40 mm KCl, and incubated for 1 h at 4 °C with gentle rotation. Beads bearing bound materials were collected, washed in 160 μl of buffer C″ containing 150 mm KCl and bovine serum albumin (100 μg/ml), and suspended in SDS sample buffer (80 μl). Aliquots (20 and 50 μl) of this sample were subjected to SDS-polyacrylamide gel electrophoresis as above and immunoblotted using anti-DiaA antiserum, respectively. The WM433 fraction I was obtained with the method of Fuller et al. (28Fuller R.S. Kaguni J.M. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7370-7374Crossref PubMed Scopus (240) Google Scholar), except that buffer C″ containing 250 mm KCl, 20 mm spermidine-HCl, and 300 μg/ml lysozyme was employed for cell lysis. Construction of Overproducing Plasmids of Biotin-tagged DnaA Proteins—The full-length DnaA-coding region (1.4 kb) was amplified by PCR from pKA234 (29Kubota T. Katayama T. Ito Y. Mizushima T. Sekimizu K. Biochem. Biophys. Res. Commun. 1997; 232: 130-135Crossref PubMed Scopus (38) Google Scholar) using primers, 5′-GCGGATCCGTGTCACTTTCGCTTTG (TAKU5) and 5′-GCGGTACCCTTACGATGACAATGTTCTG (TAKU6). The resultant fragment was ligated to pAN6 vector (Avidity) using the BamHI and KpnI sites, resulting in pTKM12. A 1.5-kb region was amplified by PCR from pTKM12 using primers, 5′-CTAGTCTAGATTTAAGAAGGAGATATACATATGTCCGGCCTGAACGAC (KWdN5XSA) and 5′-CTTCTCTCATCCGCCAAAACAG (sim16). The resultant fragment was ligated to pBAD18 using the XbaI and HindIII sites, resulting in pST11-2, which was an overproducer of the DnaA conjugated with the biotin ligase recognition peptide (bioDnaA). A 0.45-kb region was amplified by PCR from pST11-2 using primers, KWdN5XSA, and 5′-CGGAATTCTTAACGATAGGTCGGTTCTGCC (TAKU12). The resultant fragment was digested with XbaI and ligated to pBAD18 using the NheI and SmaI sites, resulting in pTKM13, which was an overproducer of the DnaA domains I–II conjugated with the biotin ligase recognition peptide (bioDAD I–II). A 5.6-kb region was amplified by PCR from pST11-2 using primers, 5′-GGATCCTCGAGCTCCCGGCG (TAKU68) and 5′-TCTAACGTAAACGTCAAACACACG (TAKU69). The resultant fragment was self-ligated, resulting in pTKM14, which was an overproducer of the DnaA domains III–IV conjugated with the biotin ligase recognition peptide (bioDAD III-IV). All constructions were confirmed by nucleotide sequencing. Overproduction and Purification of Biotin-tagged DnaA Proteins— MC1061 cells bearing pBirA (Avidity) and pST11-2 were grown at 37 °C in 2.4 liter of LB medium containing ampicillin (100 μg/ml) and chloramphenicol (20 μg/ml). When an optical density (A660) of the culture reached 0.4–0.6, 1% l(+)-arabinose, 1 mm isopropyl-1-thio-β-d-galactosidase, and 50 μm (+)-biotin were included and incubation was further continued for 3 h. Cells were harvested by centrifugation and resuspended in buffer C″ as described previously for purification of DnaA (37Sekimizu K. Yung B.Y.-M. Kornberg A. J. Biol. Chem. 1988; 263: 7136-7140Abstract Full Text PDF PubMed Google Scholar, 38Takata M. Guo L. Katayama T. Hase M. Seyama Y. Miki T. Sekimizu K. Mol. Microbiol. 2000; 35: 454-462Crossref PubMed Scopus (13) Google Scholar). MC1061 cells bearing pBirA and pTKM13 or pTKM14 were similarly used. BioDnaA and bioDAD III–IV were purified fro