Biochemical Studies of Human DNA Polymerase Delta

描述(由申请人提供)：基因组的完整和准确复制是生物学中的一个中心过程。这涉及多个过程以及复杂的调控机制，允许在每个细胞周期内进行染色体的临时协调复制。波尔？在DNA复制中起着至关重要的作用，但也是DNA修复过程中的主要参与者。因此，了解人类Pol？的特性、其调控以及通过蛋白质-蛋白质与复制体的其他成分以及DNA修复机制的相互作用整合其功能对于我们理解人类DNA的复制和修复是至关重要的。对人类波兰人的了解要少得多吗？这一点很重要，因为它们的亚基组成不同。我们的目标是剖析p12和p68亚基在POL？功能。我们的研究是由这样的假设驱动的，即p12直接影响POL？的聚合酶和核酸外切酶位点的催化功能，而p68则起到蛋白质-蛋白质相互作用的支架作用。在目标1中，我们利用了最近在表达重组POL？方面取得的进展。对核心酶(p125/p50)、核心酶+p12、核心酶+p68和全酶进行严格的生化比较。在目标2中，我们专注于通过确定p12与核心酶的p125和p50亚基结合的位置来确定其作用，并分析它们的单独作用。我们还将分析Pol？和增殖细胞核抗原。在目标3中，我们将重点放在p68的功能上，认为它是介导Pol？与其他蛋白质结合。我们将研究几个关于磷酸化-去磷酸化调节p68蛋白-蛋白相互作用的机制的假说，以及它作为靶向蛋白招募蛋白磷酸酶-1的作用。我们的研究可能有助于理解癌症病因学，因为POL？可能会导致基因组的不稳定。公共卫生相关性：人权？是参与染色体DNA复制和修复的关键酶之一。因此，它的功能在无错误地复制DNA以及修复错误方面非常重要，从而可以避免突变。我们的研究旨在了解该酶的不同亚基如何对催化核心的功能做出贡献。这些研究对于了解它如何能够准确地进行DNA复制，以及它如何与组成复制和修复机制的其他蛋白质相互作用至关重要。我们的工作还将提供关于如何Pol？职能受到规范。目前我们对人类极地的认识存在很大差距？酶，我们的工作对于理解它的功能变化如何导致人类细胞突变增加以及癌症的病因学是很重要的。

DESCRIPTION (provided by applicant): The complete and accurate replication of the genome is a central process in biology. This involves multiple processes as well as complex regulatory mechanisms that allow the temporally coordinated duplication of the chromosomes during each cell cycle. Pol ? plays a crucial role in DNA replication but in addition also is a major participant in DNA repair processes. A knowledge of the properties of human Pol ?, its regulation and the integration of its functions by protein-protein interactions with other components of the replisome and with DNA repair machinery is thus central to our understanding of human DNA replication and repair. Far less is known about human Pol ? than its more extensively studied counterpart in budding yeast, an important point because they differ in subunit composition. Our goals are directed toward dissecting the roles of the p12 and p68 subunits in Pol ? function. Our research is driven by the hypothesis that p12 directly affects the catalytic functions of the polymerase and exonuclease sites of Pol ?, while p68 plays a role as a scaffold for protein-protein interactions. In Aim 1 we take advantage of recent advances we have made in the expression of recombinant Pol ? and its subassemblies to make a rigorous biochemical comparisons of the core enzyme (p125/p50), core + p12, core + p68 and the holoenzyme. In Aim 2 we focus on determining the roles by identifying the sites of p12 that allow it to bind to the p125 and p50 subunits of the core enzyme, and the analysis of their individual effects We also will analyze the subunit interactions of Pol ? and PCNA. In Aim 3 we focus on the functions of p68, with the view that it acts a scaffold that mediates interactions of Pol ? with other proteins. We will investigate several hypotheses regarding the mechanisms by which phosphorylation-dephosphorylation regulates protein-protein interactions of p68, and also its role as a targeting protein that recruits protein phosphatase-1. Our studies could contribute to the understanding of cancer etiology, since it is more than likely that alterations in Pol ? could contribute to genomic instability. PUBLIC HEALTH RELEVANCE: Human Pol ? is one of the key enzymes that are involved in the duplication and repair of chromosomal DNA. Thus, its functions are highly important in replicating DNA without errors, as well as in repairing errors, so that mutations can be avoided. Our research is directed toward understanding how the different subunits of this enzyme contribute to the function of the catalytic core. These studies are crucial to understanding how it is able to perform DNA replication accurately, and how it interacts with other proteins that comprise the replication and repair machinery. Our work will also provide insights as to how Pol ? functions are regulated. There is currently a large gap in our knowledge of the human Pol ? enzyme, and our work is important to understanding how alterations in its functions could contribute to increased mutations in human cells, and to the etiology of cancer.