Analysis of mutant origin recognition complex with reduced ATPase activity in vivo and in vitro

Masaya Takehara, Masaki Makise, Hitomi Takenaka, Teita Asano, Tohru Mizushima

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

In eukaryotes, ORC (origin recognition complex), a six-protein complex, is the most likely initiator of chromosomal DNA replication. ORC belongs to the AAA+ (ATPases associated with a variety of cellular activities) family of proteins and has intrinsic ATPase activity derived from Ore1p, one of its subunits. To reveal the role of this ATPase activity in Saccharomyces cerevisiae (baker's yeast) ORC, we mutated the Orc1p sensor 1 and sensor 2 regions, which are important for ATPase activity in AAA+ proteins. Plasmid-shuffling analysis revealed that Asn600, Arg694 and Arg704 are essential for the function of Orc1p. In yeast cells, overexpression of Orc1R694Ep inhibited growth, caused inefficient loading of MCM (mini-chromosome maintenance complex of proteins) and slowed the progression of S phase. In vitro, purified ORC-1R [ORC with Orc1R694Ep (Orc1p Arg 694-→ Glu mutant)] has decreased ATPase activity in the presence or absence of origin DNA. However, other activities (ATP binding and origin DNA binding) were indistinguishable from those of wild-type ORC. The present study showed that Arg694 of the Orc1p subunit is important for the ATPase activity of ORC and suggests that this ATPase activity is required for efficient MCM loading on to origin DNA and for progression of S phase.

Original languageEnglish
Pages (from-to)535-543
Number of pages9
JournalBiochemical Journal
Volume413
Issue number3
DOIs
Publication statusPublished - 2008 Aug 1

Keywords

  • ATPase
  • ATPases associated with a variety of cellular activities family of proteins (AAA family of proteins)
  • DNA replication
  • Origin recognition complex (Orc)
  • Origin recognition complex subunit 1p (orc1p)
  • Sensor 1 and sensor 2 regions

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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