Timely binding of IHF and Fis to DARS2 regulates ATP-DnaA production and replication initiation

Kazutoshi Kasho, Kazuyuki Fujimitsu, Toshihiro Matoba, Taku Oshima, Tsutomu Katayama

研究成果: ジャーナルへの寄稿記事

31 引用 (Scopus)

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In Escherichia coli, the ATP-bound form of DnaA (ATP-DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP-DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP-DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP-DnaA was fully active in replication initiation and underwent DnaA-ATP hydrolysis. ADP-DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP-DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP-DnaA production, thereby promoting timely initiation. Moreover, we show that IHF-DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP-DnaA and replication initiation in coordination with the cell cycle and growth phase.

元の言語英語
ページ(範囲)13134-13149
ページ数16
ジャーナルNucleic acids research
42
発行部数21
DOI
出版物ステータス出版済み - 12 1 2014

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All Science Journal Classification (ASJC) codes

  • Genetics

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