ADP-binding to origin recognition complex of Saccharomyces cerevisiae

Hitomi Takenaka, Masaki Makise, Wakako Kuwae, Naoko Takahashi, Tomofusa Tsuchiya, Tohru Mizushima

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

Abstract

The origin recognition complex (ORC), a possible initiator of chromosomal DNA replication in eukaryotes, binds to ATP through its subunits Orc1p and Orc5p. Orc1p possesses ATPase activity. As for DnaA, the Escherichia coli initiator, the ATP-DnaA complex is active but the ADP-DnaA complex is inactive for DNA replication and, therefore, the ATPase activity of DnaA inactivates the ATP-DnaA complex to suppress the re-initiation of chromosomal DNA replication. We investigated ADP-binding to ORC by a filter-binding assay. The Kd values for ADP-binding to wild-type ORC and to ORC-1A (ORC containing Orc1p with a defective Walker A motif) were less than 10nM, showing that Orc5p can bind to ADP with a high affinity, similar to ATP. ORC-5A (ORC containing Orc5p with a defective Walker A motif) did not bind to ADP, suggesting that the ADP-Orc1p complex is too unstable to be detected by the filter-binding assay. ADP dissociated more rapidly than ATP from wild-type ORC and ORC-1A. Origin DNA fragments did not stimulate ADP-binding to any type of ORC. In the presence of ADP, ORC could not bind to origin DNA in a sequence-specific manner. Thus, in eukaryotes, the ADP-ORC complex may be unable to initiate chromosomal DNA replication, and in this it resembles the ADP-DnaA complex in prokaryotes. However, overall control may be different. In eukaryotes, the ADP-ORC complex is unstable, suggesting that the ADP-ORC complex might rapidly become an ATP-ORC complex; whereas in prokaryotes, ADP remains bound to DnaA, keeping DnaA inactive, and preventing re-initiation for some periods.

Original languageEnglish
Pages (from-to)29-37
Number of pages9
JournalJournal of Molecular Biology
Volume340
Issue number1
DOIs
Publication statusPublished - 2004 Jun 25

Keywords

  • ADP
  • ATP
  • DNA replication
  • MCM, mini-chromosome maintenance proteins
  • ORC
  • ORC, origin recognition complex
  • Saccharomyces cerevisiae

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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