Enzymatic Mechanisms of DNA Replication

描述(由申请人提供)： 基因组学方法以及大肠杆菌UMUC和DinB、酵母RAD30和着色性干皮病变异体(XPV)编码DNA聚合酶的发现导致了已知DNA聚合酶的数量的爆炸性增长。我们最初通过双杂交筛选发现，酵母DNA聚合酶(Pol)epsilon的必要但非催化的C端域是DNA复制、修复和DNA复制检查点所需的聚合酶，它与最新的聚合酶之一polsigma在物理和功能上都相互作用。酵母菌Polsigma突变体在S相完成、DNA修复、染色体凝聚和姐妹染色单体凝聚力方面存在缺陷。这种相互作用的发现为我们继续探索盆栽epsilon在S期染色体动力学中的确切作用提供了一个新的维度。我们发现polepsilon突变体在凝聚力方面存在缺陷。我们研究的总体目标是确定polepsilon和polsigma在这些不同的细胞过程中如何相互作用。首先，为了确定相互作用在体内的作用，将利用定点突变和两种蛋白质之间相互作用的相关效应与所产生的突变的表型进行结构/功能分析，特别是关于姐妹染色单体凝聚力。其次，我们将从生物化学的角度描述polsigma--底物的定义、保真度、加工性和增殖细胞核抗原的刺激作用。我们研究的一个决定性方面是Polsigma和POT epsilon之间的相互作用。我们还将讨论Polepsilon与另一种凝聚蛋白Ctf18的相互作用，Ctf18是另一种聚合酶钳夹加载器的组成部分。新生的姐妹染色单体凝聚力领域的关键问题是，染色体之间联系的性质是什么，DNA复制有助于形成这些联系的机制是什么。考虑到这一点，我们将确定是否需要Polepsilon来建立凝聚力和/或维护。我们将研究体内凝聚位点的复制分叉的命运以及聚合酶与这些位点的关联。我们将使用染色质免疫沉淀，DNA复制中间产物的2D凝胶电泳，以及使用荧光显微镜和GFP阻遏/操作者标记策略对染色体动力学的微观分析，这是我们在前一次授予期间建立的技术。为了确保进展，已经进行了几次合作。

DESCRIPTION (provided by applicant): Genomic approaches and the discovery that E. coli UmuC and DinB, yeast RAD30 and Xeroderma pigmentosum variant (XPV) encode DNA polymerases have led to an explosion in the number of known DNA polymerases. We have discovered, originally via a two hybrid screen, that the essential but non-catalytic C terminal domain of yeast DNA polymerase (pol) epsilon, a polymerase required for DNA replication, repair, and the DNA replication checkpoint, interacts both physically and functionally with one of the most novel of the new polymerases, pol sigma. Yeast pol sigma mutants are defective in completion of S phase, in DNA repair, in chromosome condensation, and in sister chromatid cohesion. The discovery of this interaction lends a new dimension to our continuing search for the precise role of pot epsilon in chromosome dynamics during S phase. We find that pol epsilon mutants are defective in cohesion. The overall goat of our studies is to determine how pol epsilon and pol sigma interact in these various cellular processes. First, to define the in vivo role of the interaction, structure/function analysis will be carried out by using site directed mutations and correlating effects on interaction between the two proteins with phenotypes of resulting mutants, especially with respect to sister chromatid cohesion. Second, we will characterize pol sigma biochemically- definition of substrates, fidelity, processivity, and stimulation by PCNA. A defining aspect of our studies is focus on the interaction between pol sigma and pot epsilon. We will also address pol epsilon's interaction with another cohesion protein,Ctf18, a component of an alternative polymerase clamp loader. The key questions in the nascent field of sister chromatid cohesion are what is the nature of the links between the chromosomes and what is the mechanism by which DNA replication contributes to forming these links. With this in mind, we will determine if pol epsilon is required for establishing cohesion and/or maintenance. We will study the fate of replication forks at sites of cohesion in vivo and the association of polymerases with these sites. We will use chromatin immunoprecipitation, 2D gel electrophoresis of DNA replication intermediates, and microscopic analysis of chromosome dynamics using fluorescence microscopy and the GFP-repressor/operator tagging strategy, techniques set up by us during the previous granting period. Several collaborations are in place to insure progress.