MECHANISM OF DNA REPLICATION IN EUCARYOTES--YEAST AS A MODEL SYSTEM

真核生物DNA复制机制——以酵母为模型系统

• 批准号: 3841119
• 负责人: A SUGINO
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3841119
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA replication; DNA replication origin; Saccharomyces cerevisiae; enzyme activity; fungal genetics; gene deletion mutation; gene induction /repression; gene interaction; genetic models; molecular cloning; nucleic acid sequence; polymerase chain reaction; protein kinase; temperature sensitive mutant

Various temperature-sensitive DNA polymerase II mutants of S. cerevisiae have been characterized. The purified DNA polymerase II from the mutants exhibited temperature-sensitive DNA polymerase activity, while in vivo chromosomal DNA synthesis was greatly reduced at the restrictive temperatures. Furthermore, these mutant cells had a single-base-pair change which generates a single amino acid change in the DNA polymerase II gene. These results proved that S. cerevisiae DNA polymerase II participates in its chromosomal DNA replication. By using the PCR technique, we have cloned the DNA polymerase gene which has a high homology to S. cerevisiae DNA polymerase II from a fission yeast S. pombe and from Drosophila melanogaster and their partial nucleotide sequences have been determined. The CDC7 gene product is required for the last step before initiation of chromosomal DNA replication in S. cerevisiae. In the previous year, we have shown genetically that the CDC7 gene product (a protein kinase) directly interacts with the DBF4 gene product which is also required for initiation of chromosomal DNA replication and suggested that the DBF4 protein activates the protein(s) which is needed for initiation of chromosomal DNA replication. In this year, we have obtained biochemical evidences for the direct interaction between the DBF4 protein and the CDC7 protein. Furthermore, an active form of the CDC7 protein kinase consisted of the CDC7 and DBF4 polypeptides. These data further substantiated our proposal that the DBF4 protein works as a CDC28 (CDC2) cyclin and modulates activity of the CDC7 protein kinase. Finally, we have isolated, sequenced, and characterized the CDC5 gene which interacts with DBF4, CDC15, and DBF2 gene products.

不同的温度敏感性DNA聚合酶II突变体的S。酿酒酵母 已经被定性。 从突变体中纯化的DNA聚合酶II 表现出温度敏感的DNA聚合酶活性，而在体内 染色体DNA合成在限制性条件下大大减少， 温度 此外，这些突变细胞具有单碱基对 在DNA聚合酶II中产生单个氨基酸变化的变化 基因 这些结果证明S.酿酒酵母DNA聚合酶Ⅱ 参与其染色体DNA复制。 用pcr 技术，克隆了具有高度同源性的DNA聚合酶基因 对鼠伤寒沙门氏酿酒酵母DNA聚合酶II从裂殖酵母S.粟酒和从 果蝇及其部分核苷酸序列已被 测定 CDC7基因产物是启动前的最后一步所需的。 染色体DNA复制。啤酒。 去年，我们 从遗传学上表明，CDC7基因产物（一种蛋白激酶） 直接与DBF4基因产物相互作用， 启动染色体DNA复制，并建议DBF4 蛋白质激活启动所需的蛋白质， 染色体DNA复制 在这一年里，我们已经获得了生化 DBF4蛋白和CDC7之间直接相互作用的证据 蛋白 此外，CDC7蛋白激酶的活性形式包括： 的CDC7和DBF4多肽。 这些数据进一步证实了我们 DBF4蛋白作为CDC28（CDC2）细胞周期蛋白发挥作用， CDC7蛋白激酶的活性。 最后，我们已经分离，测序， 并鉴定了与DBF4、CDC 15和DBF2相互作用的CDC 5基因 基因产物