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(可能是哺乳动物DNA聚合酶epsilon的同源物) 通过该质粒分离得到了多种温度敏感型II突变体。 洗牌法。已有研究表明，温度敏感突变体 (TS9、18和31)不仅有对温度敏感的DNA聚合酶II 活性，但也表现出对温度敏感的染色体复制 活着。此外，3‘-5’核酸外切酶缺陷DNA聚合酶II突变体 表现出很高的自发突变频率，尽管染色体 复制不受这些突变的影响。这些结果是 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的 目前的三种DNA聚合酶模型能够解释三种 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 酿酒酵母中的染色体复制。至 了解染色体起始的调控机制 复制，我们已经分离了一个多拷贝质粒上的基因 抑制对温度敏感的CDC7-1。该基因与DBF4完全相同 我们之前已经通过与细胞周期互补来分离它 突变体DBF4。此外，我们还发现多拷贝质粒上的CDC7也 抑制对温度敏感的DBF4-L和DBF4-2。从这些结果来看，我们 结论DBF4基因产物与CDC7基因产物直接相互作用 (一种丝氨酸-苏氨酸蛋白激酶)。最后，我们分离出了 测序了三个新的抑制温度敏感的dbf4-1基因 突变细胞，并对它们进行了广泛的表征。

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.