Checkpoint Signaling and Repair of UV Damage to Human DNA

描述（由申请人提供）：该项目的长期目标是描述核苷酸切除修复（NER）和DNA损伤检查点之间通路相互作用的分子机制。DNA损伤是由紫外线照射和各种环境遗传毒性化学物质和致癌物引起的大体积损伤引起的，而细胞对DNA损伤的反应中，NER和损伤检查点是两个主要组成部分。这些通路的缺陷与人类癌症易感性死亡直接相关，如着色性干皮病（XP）和共济失调-毛细血管扩张症（AT）。尽管在理解细胞DNA损伤反应的整体机制方面已经做出了巨大的努力，但由于这两个系统在很大程度上是分开研究的，因此关于这两种途径之间关系的分子细节仍然难以捉摸。为了更系统和精确地了解细胞对DNA损伤的反应，研究人员开发了实验和策略来解决以下问题：DNA损伤时细胞中的DNA损伤检查点和DNA修复是如何协调的？哪些蛋白质因子参与了相互作用？相互作用的分子基础是什么？本项目采用生物化学、分子生物学和结构学相结合的方法，旨在(1)明确XPA在ATR调控NER中的作用及其对细胞周期的依赖性，以验证XPA是NER中ATR检查点调控NER功能的主要靶点的假设；(2)确定ATR-XPA蛋白-蛋白质相互作用对紫外线下DNA损伤反应的影响，并验证这种相互作用是紫外线照射下ATR调节的NER中XPA核积累的决定因素的基本假设；(3)研究XPA核易位对ATR检查点信号的依赖性。我们将验证ATR检查点信号是DNA损伤诱导的XPA细胞质到核运输所必需的假设；(4)评估ATR调控的XPA磷酸化在DNA损伤反应中的作用以及XPA磷酸化对核苷酸切除修复的影响。

DESCRIPTION (provided by applicant): The long-term objectives of this project are to delineate the molecular mechanisms of interpathway interactions between nucleotide excision repair (NER) and DNA damage checkpoints. NER and the damage checkpoints are two major components in cellular responses to DNA damage due to formation of bulky lesions induced by UV irradiation and various environmental genotoxic chemicals and carcinogens. Defects in these pathways have been directly correlated to human cancer prone deceases such as xeroderma pigmentosum (XP) and Ataxia-Telangiectasia (AT). Although great efforts have been made in understanding the overall mechanism of cellular DNA damage responses, the molecular details about the relationship between the two pathways remain elusive due to the fact that the two systems have been largely studied separately. To gain a more systematic and precise view of cellular responses to DNA damage, experiments and strategies are developed to address the following questions: How the DNA damage checkpoints and DNA repair are coordinated in cells upon DNA damage? What are the protein factors involved in the interactions? And what is the molecular basis of the interactions? Using a combined biochemical, molecular biology and structural approach, this project aims (1) to define the role XPA in ATR regulation of NER and the cell-cycle dependency of the regulation for testing the hypothesis that XPA is the main target in NER for regulation of NER functions by ATR checkpoint; (2) to determine the effects of ATR-XPA protein-protein interaction on DNA damage responses to UV and to test the underlying hypothesis that the interaction is a determinant for nuclear accumulation of XPA for NER regulated by ATR upon UV irradiation; (3) to examine the dependence of nuclear-translocation of XPA on ATR checkpoint signaling. We will test the hypothesis that ATR checkpoint signaling is required for the DNA damage-induced cytoplasm-to-nuclear trafficking of XPA; and (4) to assess the role of ATR- regulated XPA phosphorylation in DNA damage responses and the effects of XPA phosphorylation on nucleotide excision repair.