MECHANISM OF DNA-PROTEIN INTERACTION IN DNA REPLICATION

DNA聚合酶I和启动子酶是两条主要的染色体 酿酒酵母中的DNA复制酶。 我们的直接目标是描绘出这些 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。 并建立了DNA聚合酶全酶的检测方法 酵母。一种高分子量的部分提纯 (500,000 Da)DNA PolI全酶已实现。我们计划 完成全酶的纯化，使其达到均一。 我们将对每一种相互作用的多肽进行表征 通过免疫学和结构研究获得全酶。 我们将测定各种与全酶相关的活性， 这些活性部位在多肽组件中的位置，以及 它们在整个行动机制中的作用 全酶。我们将测定DNA的体外百分率 全酶体外合成及DNA合成的保真度 这种合成的过程性。为了得出结论 关于全酶的结构和功能，我们将 确定各种亚基和辅因子的影响，如 单链DNA结合蛋白具有多种性质。 我们计划研究蛋白质核苷酸和DNA的作用机制。 蛋白质相互作用，参与作用机制 紫外光交联法合成全酶。这很可能 提供对其行动模式的见解，并补充我们的 如上所述的机械论研究。我们已经开始隔离 酵母底物酶基因，我们计划完成底物酶基因 分离并深入了解其在体内的遗传需求。

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.