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epsilon的同源物) 去年鉴定和纯化，该基因已经被鉴定，克隆 纯化的DNA聚合酶II及其部分氨基的抗血清 测定了该病毒的酸序列及其核苷酸序列。突变者 已经产生了各种缺失和基因中断。vbl.使用 这些突变体，已经证明DNA聚合酶II是一种必不可少的 酵母菌染色体复制的酶，可以想象DNA 聚合酶epsilon可能是DNA复制的关键酶 哺乳动物细胞。基于这些结果，我们提出了一个新的模型 解释三种DNA聚合酶如何复制真核染色体 在细胞内。我们还克隆了编码DNA的基因并对其进行了测序 聚合酶II辅助蛋白(B、C和D亚基)。 众所周知，CDC7基因是启动染色体所必需的。 复制，但不是来源特定的结合蛋白。我们已经隔离了 并对一个能够抑制cdc7突变的基因进行了测序。这个基因 是一个已知的细胞分裂周期基因DBF4，我们已经 独立学习，独立完成上述任务。由此，我们有了 结论是DBF4蛋白与CDC7蛋白直接相互作用 (与蛋白激酶活性相关)以促进启动 酿酒酵母S期的染色体复制。 我们今年研究的另一种DNA复制蛋白是一种新的 一种单链DNA结合蛋白，由三个 不同的多肽。这个蛋白质复合体是人类RF-A的同源物 蛋白。这种蛋白质极大地刺激了DNA聚合酶I反应，但 对DNA聚合酶II或III反应几乎没有影响。大多数 蛋白质定位在酵母核基质中，表明它发挥着非常重要的 染色体复制的重要作用。

In order to establish an in vivo function of Saccharomyces cerevisiae DNA polymerase II (a homolog of mammalian DNA polymerase epsilon) which we have identified and purified last year, the gene has been identified, cloned by both antiserum against the purified DNA polymerase II and its partial amino acid sequence and its nucleotide sequence have been determined. Mutants having various deletions and disrupted gene have been generated. Using these mutants, it has been shown that DNA polymerase II is an essential enzyme for yeast chromosome replication and it is conceivable that DNA polymerase epsilon could be an essential enzyme for DNA replication in mammalian cells. Based upon these results, we have proposed a new model to explain how eucaryotic chromosome is replicated by three DNA polymerases inside the cells. We also have cloned and sequences the genes encoding DNA polymerase II accessory proteins (subunits B, C, and D). CDC7 gene is well known to be required for initiation of chromosome replication, but not an origin-specific binding protein. We have isolated and sequenced one gene which is able to suppress cdc7 mutations. This gene is an already known cell-division-cycle gene, DBF4, which we have been studying independently from the above mentioned. From this, we have concluded that the DBF4 protein interacts directly with the CDC7 protein (associated with a protein kinase activity) to facilitate initiation of chromosome replication during S-phase in S. cerevisiae. Another DNA replication protein that we have studied in this year is a new type of single-stranded DNA binding protein which consists of three different polypeptides. This protein complex is a homolog of human RF-A protein. This protein greatly stimulates DNA polymerase I reaction, but has little effect on either DNA polymerase II or III reaction. Most of the protein localizes in yeast nuclear matrix, suggesting that it plays a very important role for chromosome replication.