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

6 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
Externally publishedYes

Fingerprint

Origin Recognition Complex
Yeast
Yeasts
Cells
Degradation
Proteasome Endopeptidase Complex
Mutation
Temperature
G2 Phase Cell Cycle Checkpoints
Phenotype
Defects
Proteasome Inhibitors
Eukaryota
DNA Replication
Immunoprecipitation
Saccharomyces cerevisiae
Cell Cycle
Adenosine Triphosphate

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)

Cite this

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

In: Biochemical Journal, Vol. 402, No. 2, 01.03.2007, p. 397-403.

Research output: Contribution to journalArticle

Makise, Masaki ; Takahashi, Naoko ; Matsuda, Kazuya ; Yamairi, Fumiko ; Suzuki, Keitarou ; Tsuchiya, Tomofusa ; Mizushima, Tohru. / 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. In: Biochemical Journal. 2007 ; Vol. 402, No. 2. pp. 397-403.
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AU - Tsuchiya, Tomofusa

AU - Mizushima, Tohru

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