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: 3289578
• 负责人: SUBHASIS B BISWAS
• 金额: $ 10.31万
• 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 1990-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289578
• 关键词: DNA directed DNA polymerase; DNA directed RNA polymerase; Saccharomyces; affinity chromatography; chemical binding; cofactor; crosslink; enzyme complex; enzyme reconstitution; enzyme structure; enzyme substrate; gel filtration chromatography; high performance liquid chromatography; immunochemistry; molecular cloning; nucleotides; photochemistry; radiobiology; ultraviolet radiation

DNA polymerase I and primase are the two major chromosomal DNA replication enzymes in the yeast Saccharomyces cerevisiae. Our immediate goal is to delineate the mechanism of these enzymes in the priming and elongation stages of DNA replication. Thus far, we have purified polymerase-free yeast DNA primase and developed assays for the DNA polymerase holoenzyme of yeast. Partial purification of a high molecular weight ( 500,000Da) DNA pol I holoenzyme has been achieved. We plan to complete the purification of the holoenzyme to homogeneity. We would characterize each of the polypeptides that copurify with holoenzyme through immunological and structural studies. We shall determine various holoenzyme-associated activities, location of these activity sites in the polypeptide assembly, and their function in the overall mechanism of action of the holoenzyme. We will determine the in vitro rate of DNA synthesis, fidelity of DNA synthesis of holoenzyme in vitro and processivity of such synthesis. In order to derive conclusions regarding the structure and function of holoenzyme we would determine the influence of various subunits and cofactors such as single stranded DNA binding protein in its various properties. We plan to study the mechanism of protein-nucleotide and DNA- protein interactions, involved in the mechanism of action of holoenzyme through ultraviolet photocrosslinking. This will likely provide insights in its modes of action and complement our mechanistic studies as outline above. We have initiated isolation of yeast primase gene and we plan to complete primase gene isolation and gain insights into its genetic requirement in vivo.

DNA聚合酶I和引物酶是两种主要的染色体 酿酒酵母中的DNA复制酶。 我们的直接目标是描绘这些机制， 酶在DNA复制的引发和延伸阶段。 到目前为止，我们已经纯化了无聚合酶的酵母DNA引发酶 并开发了DNA聚合酶全酶的测定方法， 酵母 部分纯化高分子量 ( 500，000 Da）DNA聚合酶Ⅰ全酶。 我们计划 以完成全酶的纯化至均一。 我们将描述每一种多肽， 通过免疫学和结构研究发现了全酶。 我们将测定各种全酶相关活性， 这些活性位点在多肽组装体中的位置，以及 它们在药物的总体作用机制中的作用 全酶 我们将确定DNA的体外率 全酶体外合成、DNA合成保真度及 这种合成的持续性。 为了得出结论 关于全酶的结构和功能， 确定各种亚基和辅因子的影响， 单链DNA结合蛋白的各种特性。 我们计划研究蛋白质-核苷酸和DNA- 蛋白质相互作用，参与的作用机制， 全酶经紫外光交联。 这将可能 提供其行动模式的见解，并补充我们的 机械研究如上所述。 我们已经开始隔离 酵母引发酶基因，我们计划完成引发酶基因 分离并深入了解其体内遗传要求。