title: The DnaA N-terminal domain interacts with Hda to facilitate replicase clamp-mediated inactivation of DnaA.
abstract: DnaA activity for replication initiation of the Escherichia coli chromosome is negatively regulated by feedback from the DNA-loaded form of the replicase clamp. In this process, called RIDA (regulatory inactivation of DnaA), ATP-bound DnaA transiently assembles into a complex consisting of Hda and the DNA-clamp, which promotes inter-AAA+ domain association between Hda and DnaA and stimulates hydrolysis of DnaA-bound ATP, producing inactive ADP-DnaA. Using a truncated DnaA mutant, we previously demonstrated that the DnaA N-terminal domain is involved in RIDA. However, the precise role of the N-terminal domain in RIDA has remained largely unclear. Here, we used an in vitro reconstituted system to demonstrate that the Asn-44 residue in the N-terminal domain of DnaA is crucial for RIDA but not for replication initiation. Moreover, an assay termed PDAX (pull-down after cross-linking) revealed an unstable interaction between a DnaA-N44A mutant and Hda. In vivo, this mutant exhibited an increase in the cellular level of ATP-bound DnaA. These results establish a model in which interaction between DnaA Asn-44 and Hda stabilizes the association between the AAA+ domains of DnaA and Hda to facilitate DnaA-ATP hydrolysis during RIDA.
authors.affiliation: Department of Molecular Biology, Kyushu University Graduate School of Pharmaceutical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Su'etsugu
authors.forename: Yuji
authors.initials: Y
authors.lastname: Harada
authors.forename: Kenji
authors.initials: K
authors.lastname: Keyamura
authors.forename: Chika
authors.initials: C
authors.lastname: Matsunaga
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Kasho
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Abe
authors.forename: Tadashi
authors.initials: T
authors.lastname: Ueda
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Katayama
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D054730:Protein Interaction Domains and Motifs
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C523634:DiaA protein, E coli; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C435211:Hda protein, E coli