MECHANISM OF DNA-PROTEIN INTERACTION IN DNA REPLICATION

我们研究的总体目标仍然是阐明其机制 以酵母为模型， 系统该项目过去两年取得的进展， 定义了我们的目标，我们的研究现在集中在多聚体DNA上， 聚合酶I和ARS结合因子。该计划已成为 逐步分子化，以利用我们在 纯化高度进行性多聚体DNA聚合酶I，纯化 以及克隆DNA引发酶，并且在克隆ARS结合因子I中， 参与酵母DNA复制起始的假定因子。 关于多聚体聚合酶I，我们的目标是： 多聚引物酶-聚合酶的纯化和表征 复合物，（2）完成引物酶基因的表征，（3） 一种新发现的酵母单链DNA的作用分析 结合蛋白（酵母SSB）对多聚体DNA功能的影响 聚合酶I，（4）多聚引物聚合酶的表征 相关的酶活性，单个亚基，以及 多聚体聚合酶活性，（5）外切核酸酶的表征 与多肽的复合物和纯化相关的活性。 ARS结合因子I是一种独特的DNA结合蛋白， 特异性同源序列在各种ARS，似乎是 参与DNA复制的起始。 因此， 这一因素在ARS中的作用对于全面理解 真核生物DNA复制机制。 我们分离出了一个 我们的目标是进一步阐明其功能 主要内容如下：（1）用DNA法测定ABFI的一级结构 测序;（2）ABFI突变体的分离和功能分析 在体内和体外。

The overall goal of our research remains the elucidation of the mechanism by which chromosomes replicate in eukaryotic cells using yeast as a model system. The progress made during the last two years of this project has defined our goals and our research is now centered on the multimeric DNA polymerase I and an ARS binding factor. The program has become progressively molecular to take advantage of our progress in the purification of a highly processive multimeric DNA polymerase I, purifying and cloning the DNA primase, and in cloning the ARS binding factor I, the putative factor involved in the initiation of yeast DNA replication. With regard to the multimeric polymerase I our goals are: completion of the purification and characterization of the multimeric primase-polymerase complex, (2) completion of the characterization of the primase gene, (3) analysis of the effect of a newly-discovered yeast single stranded DNA binding protein (yeast SSB) on the functions of the multimeric DNA polymerase I, (4) characterization of the multimeric primase-polymerase associated enzymatic activities, individual subunits, and reconstitution of the multimeric polymerase activity, (5) characterization of the exonuclease activity associated with the complex and purification of the polypeptide. ARS Binding Factor I is a unique DNA binding protein that binds with high specificity to homologous sequences at various ARSs and appears to be involved in the initiation of DNA replication. Thus, elucidation of the role of this factor in ARSs is important for overall understanding of the mechanism of eukaryotic DNA replication. We have isolated a single copy gene for this factor and our goals for further elucidation of its functions are as follows: (1) determination of the primary structure of ABFI by DNA sequencing, (2) isolation of mutants and analysis of the functions of ABFI in vivo and in vitro.