Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: Interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex

Masayuki Su'etsugu, Makoto Takata, Toshio Kubota, Yusaku Matsuda, Tsutomu Katayama

Research output: Contribution to journalArticlepeer-review

50 Citations (Scopus)

Abstract

In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding.

Original languageEnglish
Pages (from-to)509-522
Number of pages14
JournalGenes to Cells
Volume9
Issue number6
DOIs
Publication statusPublished - Jun 1 2004

All Science Journal Classification (ASJC) codes

  • Genetics
  • Cell Biology

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