title: Analysis of origin recognition complex in saccharomyces cerevisiae by use of Degron mutants.
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.
authors.affiliation: Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Makise
authors.forename: Nanako
authors.initials: N
authors.lastname: Matsui
authors.forename: Fumiko
authors.initials: F
authors.lastname: Yamairi
authors.forename: Naoko
authors.initials: N
authors.lastname: Takahashi
authors.forename: Masaya
authors.initials: M
authors.lastname: Takehara
authors.forename: Teita
authors.initials: T
authors.lastname: Asano
authors.forename: Tohru
authors.initials: T
authors.lastname: Mizushima
mesh_terms: D020816:Amino Acid Motifs; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis; D009154:Mutation; D051738:Origin Recognition Complex / Q000378:metabolism* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D004271:DNA, Fungal; D051738:Origin Recognition Complex