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

酿酒酵母的各种温度敏感 DNA 聚合酶 II 突变体 已被表征。 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 DNA 聚合酶 II 中产生单个氨基酸变化的变化 基因。 这些结果证明酿酒酵母 DNA 聚合酶 II 参与其染色体DNA复制。 通过使用PCR 技术上，我们克隆了具有高度同源性的DNA聚合酶基因 转化为来自裂殖酵母 S. pombe 的酿酒酵母 DNA 聚合酶 II 以及来自 黑腹果蝇及其部分核苷酸序列已被 确定。 CDC7基因产物是启动前最后一步所必需的 酿酒酵母中染色体 DNA 的复制。 在过去的一年里，我们 已从遗传学角度证明 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 复制的起始并表明 DBF4 蛋白质激活启动所需的蛋白质 染色体DNA复制。 今年我们获得了生化 DBF4 蛋白和 CDC7 之间直接相互作用的证据 蛋白质。 此外，CDC7蛋白激酶的活性形式包括 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) 细胞周期蛋白并调节的提议 CDC7 蛋白激酶的活性。 最后，我们分离、测序、 并表征了与 DBF4、CDC15 和 DBF2 相互作用的 CDC5 基因 基因产物。

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.