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

描述（由申请人提供）：人类DNA聚合酶δ （Pol¿）是人类染色体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具有重要作用。我们已经确定，在细胞紫外线照射后，Pol¿4全酶（Pol¿4）通过失去其p12亚基转化为3亚基酶Pol¿3。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。该项目的目标是：a)阐明Pol¿3和Pol¿4在冈崎片段成熟和促进翻译合成中的特性；b)通过E3泛素连接酶RNF8和CRL4Cdt2表征p12降解的细胞和分子基础，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探讨了Pol¿3和Pol¿4与Fen1合作进行冈崎片段加工的能力，以及PCNA泛素化对这一过程的影响。模型底物模板将用于测试Pol¿3很好地适应参与冈崎片段加工的假设，以及DNA损伤期间ub- PCNA的形成抑制了这一过程。目的2解决了p12降解在翻译合成中的机制和作用，并将测试一个关于ub-PCNA上Pol h和Pol¿之间切换机制的新假设。这一假设考虑了这样一个事实，即ub-PCNA是一个六价分子，其上的Pol¿和Pol h都表现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转换速率。

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.