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: 3289583
• 负责人: SUBHASIS B BISWAS
• 金额: $ 8.77万
• 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/3289583
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA directed RNA polymerase; Saccharomyces; affinity chromatography; binding proteins; chemical binding; cofactor; crosslink; enzyme complex; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; gel filtration chromatography; high performance liquid chromatography; immunochemistry; laboratory mouse; laboratory rabbit; 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。 我们的近期目标是描述这些机制 DNA复制的启动和延伸阶段的酶。 到目前为止，我们已经纯化了无聚合酶的酵母DNA引物酶 并开发了 DNA 聚合酶全酶的检测方法 酵母。 高分子量的部分纯化 (500,000Da)DNA pol I全酶已实现。 我们计划 完成全酶的纯化至均质。 我们将表征共纯化的每种多肽 通过免疫学和结构研究使用全酶。 我们将确定各种全酶相关活性， 这些活性位点在多肽组装中的位置，以及 它们在整体作用机制中的作用 全酶。 我们将测定DNA的体外率 全酶体外合成、DNA合成保真度 这种合成的持续进行性。 为了得出结论 关于全酶的结构和功能，我们会 确定各种亚基和辅因子的影响，例如 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-的机制 蛋白质相互作用，参与作用机制 通过紫外线光交联的全酶。 这可能会 提供对其行动模式的见解并补充我们的 如上所述的机理研究。 我们已启动隔离 酵母引物酶基因，我们计划完成引物酶基因 分离并深入了解其体内遗传需求。