Role of RPA Hyperphosphorylation in DNA Damage Responses

描述(申请人提供)：环境危害，包括紫外线辐射和遗传毒性化学物质，是DNA损伤的一些主要来源，被认为是人类癌症的主要原因，也是许多其他疾病的原因，如过早衰老和神经退化。DNA修复和DNA损伤检查点是防止细胞DNA损伤的两个主要生物防御系统。这两个机构之间的协调对于及时移除DNA损伤至关重要，防止DNA损伤因复制不忠而转变为永久性突变。然而，这种配位的分子细节在很大程度上仍不清楚。作为真核生物中主要的单链DNA结合蛋白，复制蛋白A参与了几乎所有的DNA代谢途径，如复制、重组、DNA损伤检查点和所有类型的DNA修复途径。尽管RPA在所有这些途径中都具有关键活性，但RPA是否以及如何在DNA修复和检查点之间的精确协调中发挥作用的问题仍有待阐明。我们实验室的最新发现表明，RPA在DNA损伤时经历了过度磷酸化。在这个项目中，我们将检验这一假说，即过度磷酸化可能改变RPA与单链DNA和蛋白质相互作用的生化活性，从而可能构成一个重要的调节机制，通过该机制，一些DNA损伤反应相互作用被抑制，而另一些由于与RPA的分子相互作用的失败或成功而被激活。为了验证这些假说，我们将具体确定：1)过度磷酸化的RPA在抑制起始放电中的作用；2)RAD51/Rad52与RPA之间的相互作用及其被RPA过度磷酸化的影响；以及3)RPA过度磷酸化刺激和/或破坏的细胞分子相互作用。该项目的长期目标是阐明人类细胞DNA损伤反应的分子机制，并更好地了解损伤诱导的突变和相关的人类疾病。

DESCRIPTION (provided by applicant): Environmental hazards, including UV irradiation and genotoxic chemicals, are some of the major sources for DNA damage and believed to be the major cause to human cancers and also a cause to many other diseases such as premature aging and neurodegeneration. DNA repair and DNA damage checkpoints are the two major biological defense systems against DNA damage in cells. Coordination between these two machineries is crucial for timely removal of DNA damage, preventing the conversion of DNA lesions to permanent mutations due to replication infidelity. However, the molecular details of the coordination remain largely unknown. As the major single-stranded DNA (ssDNA) binding protein in eukaryotes, replication protein A (RPA) is involved in almost all DNA metabolic pathways such as replication, recombination, DNA damage checkpoints, and all types of DNA repair pathways. Despite its critical activity in all these pathways the question of whether and how RPA plays a role in the precise coordination between DNA repair and checkpoints remain to be elucidated. Recent findings from our laboratory have shown that RPA undergoes hyperphosphorylatlon in response to DNA damage. In this project we will test the hypothesis that the hyperphosphorylatlon may alter the biochemical activity of RPA for interaction with ssDNA and proteins, and thus may constitute an important regulatory mechanism by which some DNA damage response interactions are inhibited while others are activated owing to the failure or success of the molecular interactions with RPA. To test these hypotheses, we will specifically determine: 1) the role of hyperphosphorylated RPA in the suppression of origin firing; 2) the interaction between Rad51/Rad52 and RPA and its effect by RPA hyperphosphorylatlon; and 3) the cellular molecular interactions stimulated and/or disrupted by the hyperphosphorylation of RPA. The long-term goal of this project is to elucidate the molecular mechanism of DNA damage responses in human cells, and to better understand the damage-induced mutagenesis and related human diseases.