Analysis of origin recognition complex in Saccharomyces cerevisiae by use of degron mutants

Masaki Makise, Nanako Matsui, Fumiko Yamairi, Naoko Takahashi, Masaya Takehara, Teita Asano, Tohru Mizushima

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5 Citations (Scopus)

Abstract

Origin recognition complex (ORC), a six-protein complex (Orc1p-Orc6p), may deeply involve in initiation of chromosomal DNA replication. However, since most temperature-sensitive orc mutants of Saccharomyces cerevisiae show the accumulation of cells with nearly 2C DNA content, the exact stage at which ORC acts is not fully understood. In this study, we constructed a heat-inducible degron mutant for each ORC subunit. As well as each targeted subunit, other subunits of ORC were also rapidly degraded under non-permissive conditions. In the orc5 degron mutant, incubation under the non-permissive conditions caused accumulation of cells with nearly 2C DNA content, and phosphorylation of Rad53p. When Orc5p (ORC) is depleted, this inhibits G1/S transition and formation of a pre-replicative complex (pre-RC). For pre-RC to form, and G1/S transition to proceed, Orc5p (ORC) must be present in late G1, rather than early G1, or G2/M. Block and release experiments revealed that Orc5p (ORC) is not necessary for S and G2/M phase progression. We therefore propose that ORC is necessary for the G1/S transition and pre-RC formation, and accumulation of cells with nearly 2C DNA content seen in various orc mutants is due to inefficient pre-RC formation, and/or induction of checkpoint systems.

Original languageEnglish
Pages (from-to)455-465
Number of pages11
JournalJournal of biochemistry
Volume143
Issue number4
DOIs
Publication statusPublished - 2008 Apr 1

Keywords

  • DNA replication
  • Heat inducible degron mutant
  • ORC
  • Orc5p
  • Pre-replicative complex

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Makise, M., Matsui, N., Yamairi, F., Takahashi, N., Takehara, M., Asano, T., & Mizushima, T. (2008). Analysis of origin recognition complex in Saccharomyces cerevisiae by use of degron mutants. Journal of biochemistry, 143(4), 455-465. https://doi.org/10.1093/jb/mvn005