title: Cooperative DnaA Binding to the Negatively Supercoiled datA Locus Stimulates DnaA-ATP Hydrolysis.
abstract: Timely initiation of replication in Escherichia coli requires functional regulation of the replication initiator, ATP-DnaA. The cellular level of ATP-DnaA increases just before initiation, after which its level decreases through hydrolysis of DnaA-bound ATP, yielding initiation-inactive ADP-DnaA. Previously, we reported a novel DnaA-ATP hydrolysis system involving the chromosomal locus datA and named it datA-dependent DnaA-ATP hydrolysis (DDAH). The datA locus contains a binding site for a nucleoid-associating factor integration host factor (IHF) and a cluster of three known DnaA-binding sites, which are important for DDAH. However, the mechanisms underlying the formation and regulation of the datA-IHF·DnaA complex remain unclear. We now demonstrate that a novel DnaA box within datA is essential for ATP-DnaA complex formation and DnaA-ATP hydrolysis. Specific DnaA residues, which are important for interaction with bound ATP and for head-to-tail inter-DnaA interaction, were also required for ATP-DnaA-specific oligomer formation on datA Furthermore, we show that negative DNA supercoiling of datA stabilizes ATP-DnaA oligomers, and stimulates datA-IHF interaction and DnaA-ATP hydrolysis. Relaxation of DNA supercoiling by the addition of novobiocin, a DNA gyrase inhibitor, inhibits datA function in cells. On the basis of these results, we propose a mechanistic model of datA-IHF·DnaA complex formation and DNA supercoiling-dependent regulation for DDAH.
authors.affiliation: From the Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Kasho
authors.affiliation: From the Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.affiliation: From the Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Ryuji
authors.initials: R
authors.lastname: Sakai
authors.affiliation: From the Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan katayama@phar.kyushu-u.ac.jp.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Katayama
mesh_terms: D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D056426:Genetic Loci / Q000502:physiology*; D006868:Hydrolysis; D034982:Integration Host Factors / Q000235:genetics / Q000378:metabolism; D009675:Novobiocin / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D034982:Integration Host Factors; C440502:integration host factor, E coli; D009675:Novobiocin; D000255:Adenosine Triphosphate; D027081:DNA Gyrase
keywords: ATPases associated with diverse cellular activities (AAA); DNA binding protein; DNA replication; DNA topology; DNA-protein interaction; Escherichia coli (E. coli); cell cycle; flow cytometry; nucleic acid; protein complex