Novel Interactions of DNA Repair Processes in Replication Fork Maintenance

描述（由申请人提供）：细胞必须保持其基因组的完整性，以便繁殖和存活。然而，基因组不断受到内源性代谢物和环境来源的损害，这些损害会阻碍复制和转录。复制过程中会产生特殊风险。如果复制叉没有得到保护，复制体在病变处的失速最终会导致链断裂、遗传信息丢失和基因组不稳定。为了应对这些挑战，生物体进化出多种DNA修复和检查点途径，这些途径必须相互协调，也必须与DNA复制协调。这些过程中的任何一个或它们的协调发生故障，都可能破坏基因组的完整性，并导致从衰老到癌症等人类疾病的表型。本提案旨在了解两个意外发现的生化基础和生物学意义。首先，DNA修复蛋白XPG在s期上调，定位于含有修复受损复制叉蛋白的病灶。其次，XPG在物理和功能上与WRN相互作用，这对于维持s期基因组完整性很重要，并且还直接与RAD51重组酶相互作用。待验证的中心假设是，XPG在与WRN合作维持复制叉以及通过促进rad51介导的同源重组中具有新的作用。提出的方法利用两个已建立的实验室及其合作者的互补生化和细胞生物学专业知识。目的1将定义XPG在复制叉中的作用，并描述其丢失的生物学后果。目的2将验证在s期XPG与WRN在停滞复制分叉的一个子集上起作用的假设，并确定它们在细胞中相互作用的条件。目的3将研究XPG作为复制相关DNA双链断裂同源重组修复介质的作用。这些研究将确定XPG和WRN介导的DNA修复过程之间相互作用的分子基础，以及它们在防止S期基因组完整性丧失中的新作用，这对环境暴露的知情调节和新型癌症治疗的合理开发都具有重要意义。

DESCRIPTION (provided by applicant): Cells must maintain the integrity of their genomes in order to propagate and survive. However, genomes are constantly challenged by damage from endogenous metabolites and environmental sources that pose impediments to replication and transcription. Special risks arise during replication. If the replication fork is not protected, replisome stalling at lesions can ultimately lead to strand breakage, loss of genetic information, and genomic instability. To face these challenges, organisms evolved multiple DNA repair and checkpoint pathways that must be coordinated with each other, as well as with DNA replication. Breakdown either in any of these processes or their coordination can corrupt genome integrity and cause human disease phenotypes ranging from aging to cancer. This proposal aims to understand the biochemical basis and biological significance of two unexpected findings. First, the DNA repair protein XPG is up-regulated in S-phase and localizes to foci containing proteins that repair damaged replication forks. Second, XPG interacts physically and functionally with WRN, which is known to be important for maintaining genomic integrity during S-phase, and also interacts directly with the RAD51 recombinase. The central hypothesis to be tested is that XPG has novel roles in replication fork maintenance both cooperatively with WRN and through facilitation of RAD51-mediated homologous recombination. The proposed approaches harness the complementary biochemical and cell biological expertise of two established laboratories and their collaborators. Aim 1 will define the role of XPG at replication forks and characterize the biological consequences of its loss. Aim 2 will test the hypothesis that during S-phase XPG functions with WRN at a subset of stalled replication forks and determine the conditions under which they interact in cells. Aim 3 will investigate a proposed role for XPG as a mediator of homologous recombinational repair of replication-associated DNA double-strand breaks. The proposed studies will define the molecular basis for interactions among the DNA repair processes mediated by XPG and WRN and their novel role in preventing loss of genomic integrity during S phase, with implications both for informed regulation of environmental exposures and rational development of novel cancer therapies. PUBLIC HEALTH RELEVANCE: The proposed studies will determine the mechanisms and biological significance of a recently discovered role for the multi-functional DNA repair protein XPG in replication fork maintenance. This novel function for XPG is mediated in part through direct interactions both with the RAD51 recombinase and with another multi-functional DNA repair protein WRN, both of which are important in preserving the integrity and stability of the genome. Mutations in XPG or WRN give rise to different inherited diseases that include cancer predisposition and aging phenotypes, while the RAD51 pathway is essential for cell viability. The project results will provide new insights into interactions among DNA repair systems, and how they protect humans from neurodegeneration, premature aging and cancer as a result of DNA damage from environmental and endogenous sources. The proposed studies will also help provide a rational basis for regulating environmental exposure to DNA damaging agents and for administering DNA damaging anti-cancer therapies.