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

为了建立酿酒酵母DNA的体内功能， 聚合酶II（哺乳动物DNA聚合酶II的可能同源物） 通过质粒分离了多种温度敏感II型突变体 洗牌方法 研究表明，温度敏感突变体 (ts9 18和31）不仅具有温度敏感的DNA聚合酶II， 活性，但也表现出温度敏感的染色体复制， vivo. 此外，3'-5 '核酸外切酶缺陷型DNA聚合酶II突变体 表现出高自发突变频率，虽然染色体 复制不受这些突变的影响。 这些结果 这与DNA聚合酶II参与 酵母中的染色体复制 这些结果有力地支持了我们 目前的三种DNA聚合酶模型，能够解释这三种聚合酶如何 DNA聚合酶参与酵母染色体复制。 CDC7基因是启动前的最后一步所必需的。 酵母中的染色体复制。 到 了解染色体起始的调节机制 复制，我们已经分离了多拷贝质粒上的基因， 抑制对温度敏感的CDC7 - 1。 该基因与DBF4相同 我们之前通过与细胞周期互补分离出的 突变体dbf 4.此外，我们还发现多拷贝质粒上的CDC7也 抑制对温度敏感的DBF 4 - 1和DBF 4 - 2。 根据这些结果，我们 结论DBF4基因产物与CDC7基因产物直接相互作用 （丝氨酸-苏氨酸蛋白激酶）。 最后，我们分离出了 测定了三个抑制温度敏感型dbf4 - 1的新基因的序列 突变细胞并对其进行了广泛的表征。

In order to establish an in vivo function of Saccharmoyces cerevisiae DNA polymerase II (a probable homologue of mammalian DNA polymerase epsilon) various temperature-sensitive II mutants were isolated by the plasmid shuffling method. It has been shown that the temperature-sensitive mutants (ts9, 18, and 31) have not only temperature-sensitive DNA polymerase II activity, but also exhibit temperature-sensitive chromosomal replication in vivo. Furthermore, 3'-5' exonuclease-deficient DNA polymerase II mutants exhibit high spontaneous mutation frequency, although chromosomal replication is not affected by these mutations. These results are consistent with the notion that DNA polymerase II participates in chromosomal replication in yeast. These results strongly support our current three DNA polymerase model that is able to explain how the three DNA polymerases participate in yeast chromosomal replication. The CDC7 gene is required for the last step before initiation of chromosomal replication in the yeast Saccharomyces cerevisiae. To understand the regulatory mechanism of initiation of chromosomal replication, we have isolated the gene on a multicopy plasmid which suppresses temperature-sensitive cdc7-1. This gene is identical to DBF4 which we have previously isolated by complementation with a cell cycle mutant dbf4. Furthermore, we find that CDC7 on a multicopy plasmid also suppresses temperature-sensitive dbf4-l and dbf4-2. From these results, we concluded that DBF4 gene product directly interacts with CDC7 gene product (a serine-threonine protein kinase). Finally, we have isolated and sequenced three new genes which suppress temperature-sensitive dbf4-1 mutant cells and characterized them extensively.