Mechanism for the degradation of origin recognition complex containing Orc5p with a defective Walker A motif and its suppression by over-production of Orc4p in yeast cells

Masaki Makise, Naoko Takahashi, Kazuya Matsuda, Fumiko Yamairi, Keitarou Suzuki, Tomofusa Tsuchiya, Tohru Mizushima

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Orc5p is one of six subunits constituting the ORC (origin recognition complex), a possible initiator of chromosomal DNA replication in eukaryotes. Orc5p contains a Walker A motif. We recently reported that a strain of Saccharomyces cerevisiae having a mutation in Orc5p's Walker A motif (orc5-A), showed cell-cycle arrest at G2/M and degradation of ORC at high temperatures (37°C). Over-production of Orc4p, another subunit of ORC, specifically suppressed these phenotypes [Takahashi, Yamaguchi, Yamairi, Makise, Takenaka, Tsuchiya and Mizushima (2004) J. Biol. Chem. 279, 8469-8477]. In the present study, we examined the mechanisms of ORC degradation and of its suppression by Orc4p over-production. In orc5-A, at high temperatures, ORC is degraded by proteasomes; either addition of a proteasome inhibitor, or introduction of a mutation of either tan1-1 or nob1-4 that inhibits proteasomes, prevented ORC degradation. Introduction of the tan1-1 mutation restored cell cycle progression, suggesting that the defect was due to ORC degradation by proteasomes. Yeast two-hybrid and co-immunoprecipitation analyses suggested that Orc5p interacts preferentially with Orc4p and that the orc5-A mutation diminishes this interaction. We suggest that this interaction is mediated by the C-terminal region of Orc4p, and the N-terminal region of Orc5p. Based on these observations, we consider that ATP binding to Orc5p is required for efficient interaction with Orc4p and that, in orc5-A, loss of this interaction at higher temperatures allows proteasomes to degrade ORC, causing growth defects. This model could also explain why overproduction of Orc4p suppresses the orc5-A strain's phenotype.

Original languageEnglish
Pages (from-to)397-403
Number of pages7
JournalBiochemical Journal
Volume402
Issue number2
DOIs
Publication statusPublished - 2007 Mar 1

Keywords

  • DNA replication
  • Orc4p
  • Orc5-ap
  • Origin recognition complex (ORC)
  • Walker A motif
  • Yeast

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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