MECHANISM OF DNA-PROTEIN INTERACTION IN DNA REPLICATION

DNA复制中DNA-蛋白质相互作用的机制

• 批准号: 3289585
• 负责人: SUBHASIS B BISWAS
• 金额: $ 16.89万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289585
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA primase; DNA replication; Saccharomyces; chemical binding; enzyme complex; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; high performance liquid chromatography; laboratory mouse; laboratory rabbit; molecular cloning; protein purification; protein structure function; site directed mutagenesis

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.

我们研究的总体目标仍然是阐明其机制。 以酵母为模型在真核细胞中复制染色体的方式 系统。该项目在过去两年中取得的进展 明确了我们的目标，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。这个项目已经变成了 逐步地利用我们在分子方面的进展 一种高度进行性多聚体DNA聚合酶I的纯化 以及克隆DNA启动酶，以及在克隆ARS结合因子I时， 酵母DNA复制启动的推定因子。 关于多聚合酶I，我们的目标是：完成 多聚体聚合酶的纯化及性质研究 复合体，(2)完成启动酶基因的鉴定，(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)多聚合酶的特性 相关的酶活性、单个亚基和重组 多聚合酶活性，(5)核酸外切酶的性质 与多肽的络合和纯化有关的活性。 ARS结合因子I是一种独特的DNA结合蛋白，与高密度脂蛋白结合 在不同的ARS上对同源序列的特异性，似乎是 参与了DNA复制的启动。因此，对 这一因素在ARS中的作用对于全面理解 真核细胞DNA复制的机制。我们已经分离出一份 该因子的基因及进一步阐明其功能的目标 主要内容如下：(1)用DNA方法测定ABFI的一级结构 测序，(2)突变体的分离和ABFI的功能分析 在体内和体外。