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的核心过程。这涉及多个过程以及复杂的调节机制，这些机制允许在每个细胞周期中染色体的临时协调复制。波尔吗?在DNA复制中起着至关重要的作用，同时也是DNA修复过程的主要参与者。人类Pol属性的知识？因此，通过与复制体的其他组成部分和DNA修复机制的蛋白质-蛋白质相互作用来调节和整合其功能是我们理解人类DNA复制和修复的核心。我们对人类Pol知之甚少？与在出芽酵母中被广泛研究的对应物相比，这一点很重要，因为它们的亚基组成不同。我们的目标是解剖p12和p68亚基在Pol ？函数。我们的研究是基于一个假设，即p12直接影响聚合酶和Pol ？而p68则作为蛋白质相互作用的支架发挥作用。在Aim 1中，我们利用了我们在重组Pol ？并对其子组装体进行了严格的生化比较，比较了核心酶（p125/p50）、核心+ p12、核心+ p68和全酶。在Aim 2中，我们将通过鉴定p12与核心酶的p125和p50亚基结合的位点来确定其作用，并分析它们的个体效应。我们还将分析Pol ？和PCNA。在Aim 3中，我们关注p68的功能，认为它是介导Pol ？和其他蛋白质。我们将研究关于磷酸化-去磷酸化调节p68蛋白-蛋白相互作用的机制的几种假设，以及它作为募集蛋白磷酸酶-1的靶向蛋白的作用。我们的研究可能有助于了解癌症的病因，因为Pol ？可能会导致基因组不稳定。公共卫生相关性：人类Pol ？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。我们的研究方向是了解这种酶的不同亚基如何促进催化核心的功能。这些研究对于理解它如何能够准确地进行DNA复制，以及它如何与构成复制和修复机制的其他蛋白质相互作用至关重要。我们的工作也将提供关于波尔？功能被调节。目前我们对人类Pol ？我们的工作对于理解其功能的改变如何导致人类细胞中突变的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.