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: 3289579
• 负责人: SUBHASIS B BISWAS
• 金额: $ 16.3万
• 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/3289579
• 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，染色体在真核细胞中复制 系统。该项目近两年来取得的进展 确定了我们的目标，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。该程序已成为 逐步分子化以利用我们在 高度持续进行的多聚体 DNA 聚合酶 I 的纯化，纯化 并克隆 DNA 引物酶，并在克隆 ARS 结合因子 I 时， 参与酵母 DNA 复制启动的假定因素。 关于多聚体聚合酶 I，我们的目标是：完成 多聚引物酶-聚合酶的纯化和表征 复杂，(2) 完成引物酶基因的表征，(3) 分析新发现的酵母单链 DNA 的作用 结合蛋白（酵母SSB）对多聚体DNA功能的影响 聚合酶 I，(4) 多聚引物酶-聚合酶的表征 相关的酶活性、单个亚基和重构 多聚体聚合酶活性，(5) 核酸外切酶的表征 与多肽的复合和纯化相关的活性。 ARS 结合因子 I 是一种独特的 DNA 结合蛋白，可与高 对不同 ARS 处的同源序列具有特异性，并且似乎是 参与 DNA 复制的起始。 因此，阐明 该因素在 ARS 中的作用对于全面理解 ARS 非常重要 真核生物DNA复制机制。 我们已经隔离了一个副本 该因子的基因以及我们进一步阐明其功能的目标 如下： (1) DNA测定ABFI的一级结构 测序，（2）突变体分离及ABFI功能分析 体内和体外。