In Escherichia coli, the activity of ATP-bound DnaA protein in initiating chromosomal replication is negatively controlled in a replication-coordinated manner. The RIDA (regulatory inactivation of DnaA) system promotes DnaA-ATP hydrolysis to produce the inactivated form DnaA-ADP in a manner depending on the Hda protein and the DNA-loaded form of the β-sliding clamp, a subunit of the replicase holoenzyme. A highly functional form of Hda was purified and shown to form a homodimer in solution, and two Hda dimers were found to associate with a single clamp molecule. Purified mutant Hda proteins were used in a staged in vitro RIDA system followed by a pull-down assay to show that Hda-clamp binding is a prerequisite for DnaA-ATP hydrolysis and that binding is mediated by an Hda N-terminal motif. Arg168 in the AAA+ Box VII motif of Hda plays a role in stable homodimer formation and in DnaA-ATP hydrolysis, but not in clamp binding. Furthermore, the DnaA N-terminal domain is required for the functional interaction of DnaA with the Hda-clamp complex. Single cells contain ∼50 Hda dimers, consistent with the results of in vitro experiments. These findings and the features of AAA+ proteins, including DnaA, suggest the following model. DnaA-ATP is hydrolyzed at a binding interface between the AAA+ domains of DnaA and Hda; the DnaA N-terminal domain supports this interaction; and the interaction of DnaA-ATP with the Hda-clamp complex occurs in a catalytic mode. In Escherichia coli, the activity of ATP-bound DnaA protein in initiating chromosomal replication is negatively controlled in a replication-coordinated manner. The RIDA (regulatory inactivation of DnaA) system promotes DnaA-ATP hydrolysis to produce the inactivated form DnaA-ADP in a manner depending on the Hda protein and the DNA-loaded form of the β-sliding clamp, a subunit of the replicase holoenzyme. A highly functional form of Hda was purified and shown to form a homodimer in solution, and two Hda dimers were found to associate with a single clamp molecule. Purified mutant Hda proteins were used in a staged in vitro RIDA system followed by a pull-down assay to show that Hda-clamp binding is a prerequisite for DnaA-ATP hydrolysis and that binding is mediated by an Hda N-terminal motif. Arg168 in the AAA+ Box VII motif of Hda plays a role in stable homodimer formation and in DnaA-ATP hydrolysis, but not in clamp binding. Furthermore, the DnaA N-terminal domain is required for the functional interaction of DnaA with the Hda-clamp complex. Single cells contain ∼50 Hda dimers, consistent with the results of in vitro experiments. These findings and the features of AAA+ proteins, including DnaA, suggest the following model. DnaA-ATP is hydrolyzed at a binding interface between the AAA+ domains of DnaA and Hda; the DnaA N-terminal domain supports this interaction; and the interaction of DnaA-ATP with the Hda-clamp complex occurs in a catalytic mode. The initiation of chromosomal replication is highly regulated so as to take place once and only once during the cell cycle. In Escherichia coli, this control is ensured by at least three pathways that target the replication origin oriC or the protein DnaA, which binds directly to oriC to initiate replication (1Boye E. L øbner-Olesen A. Skarstad K. EMBO Rep. 2000; 1: 479-483Crossref PubMed Scopus (130) Google Scholar, 2Katayama T. Mol. Microbiol. 2001; 41: 9-17Crossref PubMed Scopus (66) Google Scholar, 3Messer W. FEMS Microbiol. Rev. 2002; 26: 355-374PubMed Google Scholar). First, newly replicated oriC is temporarily inactivated by the binding of the SeqA protein (4Lu M. Campbell J.L. Boye E. Kleckner N. 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Second, the number of DnaA molecules accessible to oriC is restricted by the datA locus, which binds this protein, and a single copy of the locus is speculated to absorb up to ∼300 DnaA molecules (9Kitagawa R. Mitsuki H. Okazaki T. Ogawa T. Mol. Microbiol. 1996; 19: 1137-1147Crossref PubMed Scopus (103) Google Scholar, 10Kitagawa R. Ozaki T. Moriya S. Ogawa T. Genes Dev. 1998; 12: 3032-3043Crossref PubMed Scopus (145) Google Scholar, 11Ogawa T. Yamada Y. Kuroda T. Kishi T. Moriya S. Mol. Microbiol. 2002; 44: 1367-1375Crossref PubMed Scopus (66) Google Scholar). As the datA locus is located only 470 kb from oriC, its DnaA-titrating function is enhanced because it is replicated several minutes after initiation at oriC. The third mechanism, regulatory inactivation of DnaA (RIDA), 1The abbreviations used are: RIDA, regulatory inactivation of DnaA; pol, DNA polymerase; Ni-NTA, nickel-nitrilotriacetic acid; DAD III–IV, DnaA domains III and IV. converts the ATP-bound form of DnaA, which is active for initiation, to the inactive ADP-bound form (12Katayama T. Kubota T. Kurokawa K. Crooke E. Sekimizu K. Cell. 1998; 94: 61-71Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 13Kurokawa K. Nishida S. Emoto A. Sekimizu K. Katayama T. EMBO J. PubMed Scopus Google Scholar, T. K. Ogawa T. 2001; PubMed Scopus Google Scholar, M. H. Kurokawa K. Kubota T. M. Katayama T. Mol. Microbiol. 2001; PubMed Scopus Google Scholar, J. Katayama T. EMBO J. 2001; PubMed Scopus Google Scholar). 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The DnaA N-terminal domain to and DnaA domain with DNA the These a functional between Hda and the DnaA AAA+ domain A is that the between the AAA+ domains of Hda and DnaA is and that DnaA domains and the proteins to functional AAA+ domain The of of the Hda to DnaA-ATP hydrolysis in a manner with DnaA DnaA-ATP hydrolysis, the DnaA-ADP is from the Hda-clamp complex by by a of DnaA that by the from The hydrolysis of to the is required for of the complex from the clamp the DNA M. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. Rev. Mol. 2002; PubMed Scopus (145) Google Scholar). to S. for the and to J. for the the
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