Modification of DNA Polymerase Delta by a Novel Mechanism During Replication Stre

描述(申请人提供)：DNA聚合酶Delta(POLD)是真核细胞染色体DNA复制所必需的关键酶。哺乳动物的Pol d由四个亚基组成，所有这些亚基都是其在体外发挥全部功能所必需的。这一建议是基于一个新的发现，即在紫外线和其他遗传毒性物质激活ATR/Chk1介导的S阶段检查点的DNA损伤后，人类Pold p12亚单位的水平显著下降。我们已经证明，这会导致POL d从四聚体转变为三聚体POL d3。将Pold3的生化性质与亲本酶的生化性质进行比较。我们将检测它的动力学特性，它绕过模板损伤的能力，以及它作为校对酶的能力。我们的工作假设是，向Pol d3的转换阻止了Pol d绕过模板损伤，从而允许修复过程发生。在紫外线损伤后，将用免疫荧光显微镜和激光扫描细胞术研究Pol d3定位于DNA损伤灶的时空方面，并与其他DNA损伤蛋白的募集进行比较。泛素化在Pold耗竭中的作用将被研究。泛素化系统的同一性将使用几种不同的方法来确定，包括候选泛素化蛋白的siRNA敲除，p12结合蛋白的鉴定，以及使用体外试验分离作用于泛素化p12的E3连接酶。

DESCRIPTION (provided by applicant): DNA polymerase delta (Pol d) is a key enzyme that is essential for eukaryotic chromosomal DNA replication. Mammalian Pol d consists of four subunits, all of which are required for its full function in vitro. This proposal is based on the novel discovery that levels of the human Pol d p12 subunit are dramatically depleted after DNA damage by UV and other genotoxic agents that activate the ATR/Chk1 mediated S-phase checkpoint. We have shown that this results in the conversion of Pol d from a tetramer into a trimer, Pol d3. The biochemical properties of pol d3 will be compared to those of the parent enzyme. We will examine its kinetic properties, its abilities to bypass template lesions, and its abilities to act as a proof reading enzyme. Our working hypothesis is that the conversion to Pol d3 prevents Pol d from bypassing template lesions, thereby allowing repair processes to take place. The spatiotemporal aspects of the localization of Pol d3 to DNA damage foci will be studied by immunofluorescence microscopy and laser scanning cytometry after UV damage and compared to the recruitment of other DNA damage proteins. The role of ubiquitination in the depletion of pol d will be studied. The identity of the ubiquitination system will be determined using several different approaches, including siRNA knockdown of candidate ubiquitination proteins, identification of p12 binding proteins, and isolation of E3 ligases that act to ubiquitinate p12 using in vitro assays.