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

描述(申请人提供)：人类DNA聚合酶增量(POL？)是人类染色体DNA复制和修复的中心酶。因此，POL？在维持基因组完整性方面具有重要作用。我们已经证实，POL？全酶(POL？4)在细胞紫外线照射后，通过失去其p12亚基而转化为3-亚基的POL？3。我们的中心假设是，Polo3已经改变了有助于细胞防御基因组损伤的属性。该项目的目标是a)阐明Pol？3和Pol？4在Okazaki片段成熟和促进跨损伤合成方面的特性，以及b)表征E3泛素连接酶RNF8和CRL4CDt2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研究Pol？3和Pol？4与Fen1协同进行Okazaki片段加工的能力，以及增殖细胞核抗原泛素化对这一过程的影响。模型底物模板将用于检验假设，即Pol？3很好地适应参与Okazaki片段处理，并且在DNA损伤过程中UB-PCNA的形成抑制了这一过程。目的2探讨p12降解在跨损伤合成中的机制和作用，并将验证一种新的假说，即在UB-PCNA上Pol h和Pol？之间的转换机制。这一假说考虑到UB-PCNA是一个六价分子，在该分子上Pol？和Pol h都显示出驱动招募和转换过程的多价相互作用。我们将使用严格的稳态前动力学分析来定量评估它们的切换率。 这些研究将包括其他表现出与Pol h相似结构的跨损伤聚合酶，以确定这一机制的一般性。目的3解决我们的主要发现，即p12是CRL4CDT2 E3连接酶的底物。在这里，我们将以一种严格的方式描述它在体内调节p12降解以应对紫外线损伤以及在细胞周期进展中的作用。P12的缺失或过度表达对紫外线敏感性和细胞生长的影响将被研究，以阐明p12的细胞功能。目的4探讨RNF8的作用，它以p12为靶点进行降解，此外，泛素化增殖细胞核抗原调节跨损伤合成。在这里，我们将确定RNF8途径中p12降解所需的其他成分，并检验RNF8将p12降解整合到几个DNA损伤信号网络中的假设。这将集中在目前尚不清楚的IR在触发p12降解中的作用及其对HR的影响。这个项目的目标是对紫外线或化学试剂造成的DNA损伤做出新的反应，这与NIEHS的目标直接相关。这种DNA损伤反应的损伤可能会增加基因组的不稳定性，这是致癌过程中的一个关键事件。

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.