Modification of DNA Polymerase d by a Novel Mechanism During Replication Stress

描述（由申请人提供）：人DNA聚合酶δ（Pol？）是人染色体DNA复制及其修复的中心酶。因此，Pol <$在维持基因组完整性方面发挥着重要作用。我们已经确定，Pol <$全酶（Pol <$4）转化为3-亚基酶，Pol <$3，其p12亚基的细胞紫外线照射后的损失。我们的中心假设是，Pol 3改变了有助于细胞防御基因组损伤的特性。本项目的目标是：a）阐明Pol <$3和Pol <$4在Okazaki片段成熟和促进跨损伤合成中的性质，b）表征E3泛素连接酶RNF8和CRL 4Cdt2降解p12的细胞和分子基础，以及它们如何将p12降解整合到DNA损伤反应的细胞网络中。目的1解决了Pol <$3和Pol <$4与Fen 1合作进行Okazaki片段加工的能力，以及PCNA的泛素化对该过程的影响。模型底物模板将被用来测试假设，即Pol <$3很好地适应于参与冈崎片段加工，并且在DNA损伤期间形成ub-PCNA抑制该过程。目的2阐明p12降解在跨损伤合成中的机制和作用，并将检验一种新的关于亚增殖细胞核抗原上Pol h和Pol <$之间转换机制的假说。这一假设考虑到了这样一个事实，即ub-PCNA是一种六价分子，Pol和Pol h都表现出多价相互作用，从而驱动招募和转换过程。我们将使用严格的前稳态动力学分析，定量评估他们的转换率。 这些研究将包括表现出与Pol h相似的结构域结构的其他跨损伤聚合酶，以确定该机制的一般性。目的3阐述了我们的主要发现，即p12是CRL 4Cdt2 E3连接酶的底物。在这里，我们将以严格的方式表征它在调节p12降解中的体内作用，以响应UV损伤，以及在细胞周期进程中。耗尽或过度表达p12对紫外线敏感性和细胞生长的后果将被调查，以阐明p12的细胞功能。目的4阐明RNF8的作用，其靶向p12降解，此外，泛素化PCNA以调节跨损伤合成。在这里，我们将确定p12降解所需的RNF8途径的其他组件，并测试RNF8将p12降解整合到几个DNA损伤信号网络中的假设。这将集中在目前未知的作用，IR在触发p12降解和其对HR.The的新的响应UV或化学试剂引起的DNA损伤的目标是直接相关的NIEHS的目标的影响。这种DNA损伤反应的损伤可能会增加基因组的不稳定性，这是癌发生的关键事件。

DESCRIPTION (provided by applicant): Human DNA polymerase delta (Pol ¿) is a central enzyme in the replication of human chromosomal DNA and its repair. Pol ¿ thus has important roles in maintaining genomic integrity. We have established that the Pol ¿ holoenzyme (Pol ¿4) is converted into a 3-subunit enzyme, Pol ¿3, by the loss of its p12 subunit after cellular UV irradiation. Our central hypothesis is that Pol ¿3 has altered properties which contribute to the cellular defense against genomic damage. The goals of this project are directed toward a) elucidating the properties of Pol ¿3 and Pol ¿4 in Okazaki fragment maturation and in facilitating translesion synthesis, and b) characterizing the cellular and molecular basis of p12 degradation by the E3 ubiquitin ligases RNF8 and CRL4Cdt2 and how they integrate p12 degradation into the cellular networks of DNA damage responses. Aim 1 addresses the abilities of Pol ¿3 and Pol ¿4 to perform Okazaki fragment processing in cooperation with Fen1, as well as the impact of ubiquitination of PCNA on this process. Model substrate templates will be used to test the hypotheses that Pol ¿3 is well adapted to participate in Okazaki fragment processing, and that formation of ub- PCNA during DNA damage inhibits this process. Aim 2 addresses the mechanism and role of p12 degradation in translesion synthesis, and will test a novel hypothesis for the mechanisms of switching between Pol h and Pol ¿ on ub-PCNA. This hypothesis takes into account the fact that ub-PCNA is a hexavalent molecule on which both Pol ¿ and Pol h exhibit multivalent interactions that drive the recruitment and switching process. We will use rigorous pre-steady state kinetic analysis to quantitatively assess their switching rates. These studies will include other translesion polymerases that exhibit similar domain structures as Pol h to determine the generality of this mechanism. Aim 3 addresses our major discovery that p12 is a substrate of the CRL4Cdt2 E3 ligase. Here we will characterize in a rigorous manner it's in vivo role in regulating p12 degradation in response to UV damage, as well as in cell cycle progression. The consequences of depletion or overexpression of p12 on UV sensitivity and cell growth will be investigated to elucidate the cellular functions of p12. Aim 4 addresses the role of RNF8 which targets p12 for degradation, and in addition, ubiquitinates PCNA to regulate translesion synthesis. Here we will determine the other components of RNF8 pathway that are needed for p12 degradation, and test the hypothesis that RNF8 integrates p12 degradation into several DNA damage signaling networks. This will focus on the currently unknown role of IR in triggering p12 degradation and its effect on HR. The goals of this project on the novel response to DNA damage caused by UV or chemical agents are directly related to the goals of the NIEHS. Impairments in this DNA damage response may increase genomic instability which is a key event in carcinogenesis.