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

描述（由申请人提供）：DNA聚合酶delta （Pol d）是真核生物染色体DNA复制所必需的关键酶。哺乳动物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。这一建议是基于一项新的发现，即人类Pol d p12亚基的水平在DNA受到紫外线和其他基因毒性物质损伤后急剧减少，这些物质激活了ATR/Chk1介导的s期检查点。我们已经证明，这导致Pol d从四聚体转化为三聚体，Pol d3。将pol d3的生化特性与母体酶的生化特性进行比较。我们将研究它的动力学性质，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，转化为Pol d3可以防止Pol d绕过模板病变，从而允许修复过程发生。我们将通过免疫荧光显微镜和激光扫描细胞术研究Pol d3在紫外线损伤后对DNA损伤灶定位的时空方面，并与其他DNA损伤蛋白的募集进行比较。我们将研究泛素化在pol - d耗竭中的作用。泛素化系统的身份将通过几种不同的方法来确定，包括siRNA敲除候选泛素化蛋白，鉴定p12结合蛋白，以及使用体外实验分离作用于p12泛素化的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.