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

酿酒酵母不同温度敏感型DNA聚合酶II突变体 已经被刻画出来。从突变体中纯化的DNA聚合酶II 在体内表现出对温度敏感的DNA聚合酶活性 在限制性条件下，染色体DNA合成大大减少。 温度。此外，这些突变细胞有一个单碱基对 在DNA聚合酶II中产生单一氨基酸变化的变化 吉恩。这些结果证明了酿酒酵母DNA聚合酶II 参与其染色体DNA复制。利用聚合酶链式反应 技术，我们克隆了具有高度同源性的DNA聚合酶基因 从裂解酵母菌S.pombe和从 果蝇及其部分核苷酸序列已被测得 下定决心。 CDC7基因产物是启动前的最后一步所必需的 染色体DNA在酿酒酵母中的复制在前一年，我们 已经从基因上证明了CDC7基因产物(一种蛋白激酶) 直接与DBF4基因产物相互作用，这也是 染色体DNA复制的启动，并提示DBF4 蛋白质激活启动所需的蛋白质(S) 染色体DNA复制。在这一年里，我们获得了生化 DBF4蛋白与CDC7直接相互作用的证据 蛋白。此外，CDC7蛋白激酶的一种活性形式包括 CDC7和DBF4多肽。这些数据进一步证实了我们的 DBF4蛋白作为CDC28(CDC2)细胞周期蛋白发挥作用并调节 CDC7蛋白激酶的活性。最后，我们分离、测序、 并鉴定了与DBF4、CDC15和DBF2相互作用的CDC5基因 基因产品。

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.