期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 292, 期 4, 页码 1251-1266出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M116.762815
关键词
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资金
- Ministry of Education, Culture, Sports, Science, and Technology of Japan [KAKENHI 15K18479, 26291004, 26650127, 16H00775]
- Japan Society for the Promotion of Science
- Kyushu University Qdaijump Research Program (Former Interdisciplinary Programs in Education and Projects in Research Development) [26122, 28247]
- Grants-in-Aid for Scientific Research [26650127, 15K18479, 26291004, 16H00775] Funding Source: KAKEN
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.
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