Negative control of DNA replication by hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli

Tohru Mizushima, Satoshi Nishida, Kenji Kurokawa, Tsutomu Katayama, Takeyoshi Miki, Kazuhisa Sekimizu

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by ATP. ATP bound to DnaA protein is slowly hydrolyzed to ADP, but the physiological role of ATP hydrolysis is unclear. We constructed, by site-directed mutagenesis, mutated DnaA protein with lower ATPase activity, and we examined its function in vitro and in vivo. The ATPase activity of purified mutated DnaA protein (Glu204 → Gln) decreased to one-third that of the wild-type DnaA protein. The mutation did not significantly affect the affinity of DnaA protein for ATP or ADP. The mutant dnaA gene showed lethality in wild-type cells but not in cells growing independently of the function of oriC. Induction of the mutated DnaA protein in wild-type cells caused an overinitiation of DNA replication. Our results lead to the thesis that the intrinsic ATPase activity of DnaA protein negatively regulates chromosomal]DNA replication in E. coli cells.

Original languageEnglish
Pages (from-to)3724-3730
Number of pages7
JournalEMBO Journal
Volume16
Issue number12
DOIs
Publication statusPublished - 1997 Jun 16

Keywords

  • ATPase
  • DnaA
  • Escherichia coli
  • Negative regulation
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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