title: Analysis on origin recognition complex containing Orc5p with defective Walker A motif.
abstract: Orc5p is one of six proteins that make up the origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes. To investigate the role of ATP binding to Orc5p in cells, we constructed orc5-A, a strain of Saccharomyces cerevisiae having a mutation in the Walker A motif of Orc5p (K43E). The strain showed temperature-sensitive growth. Incubation at a nonpermissive temperature (37 degrees C) caused accumulation of cells with nearly 2C DNA content. Overproduction of Orc4p, another subunit of ORC, suppresses this temperature sensitivity, but overproduction of other subunits did not. Overproduction of Orc4p did not suppress the temperature sensitivity of another orc5 mutant, orc5-1, whose mutation, L331P, is outside the ATP-binding motif. These results suggest that Orc4p is specifically involved in ATP binding to Orc5p itself or its function in DNA replication. Immunoblotting experiments revealed that in the orc5-A strain at a nonpermissive temperature, all ORC subunits gradually disappeared, suggesting that ORC5-A becomes degraded at nonpermissive temperatures. We therefore consider that ATP binding to Orc5p is involved in efficient ORC formation and that Orc4p is involved in this process.
authors: Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
authors: Naoko
authors: N
authors: Takahashi
authors: Yoshihiro
authors: Y
authors: Yamaguchi
authors: Fumiko
authors: F
authors: Yamairi
authors: Masaki
authors: M
authors: Makise
authors: Hitomi
authors: H
authors: Takenaka
authors: Tomofusa
authors: T
authors: Tsuchiya
authors: Tohru
authors: T
authors: Mizushima
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002453:Cell Cycle; D004261:DNA Replication; D004271:DNA, Fungal / Q000032:analysis / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004355:Drug Stability; D005434:Flow Cytometry; D015723:Gene Library; D016153:Genes, Suppressor; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D051738:Origin Recognition Complex; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature
chemical_list: D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C497220:ORC5 protein, S cerevisiae; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate