Cooperative DnaA binding to the negatively supercoiled datA locus stimulates DnaA-ATP hydrolysis

Kazutoshi Kasho, Hiroyuki Tanaka, Ryuji Sakai, Tsutomu Katayama

Research output: Contribution to journalArticlepeer-review

17 Citations (Scopus)

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 DnaAbound 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 DnaAATP 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·Dna A 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·Dna A complex formation and DNA supercoiling-dependent regulation for DDAH.

Original languageEnglish
Pages (from-to)1251-1266
Number of pages16
JournalJournal of Biological Chemistry
Volume292
Issue number4
DOIs
Publication statusPublished - Jan 27 2017

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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