Biological Role of XPG DNA Repair Protein Interactions with WRN and BLM Helicases

描述(申请人提供)：基因组完整性持续受到来自细胞新陈代谢的内源性损害和外源性环境来源的威胁，包括紫外线、电离辐射和致突变化学物质。为了应对这种损伤，细胞进化出复杂和相互关联的DNA修复途径，以协调损伤的检测和移除与其他重要的细胞过程，如DNA复制、转录和重组。任何一个环节的失效都可能导致基因组不稳定，而基因组不稳定是癌症和衰老的已知先兆。这一建议的广泛重点是了解参与防止基因组不稳定的三种蛋白质之间相互作用的生物学意义：XPG，以及人类RecQ家族成员BLM和WRN。XPG在氧化损伤的转录偶联修复和碱基切除修复(BER)中都具有重要的非酶功能，阻断XPG功能的突变会导致一种严重的神经和发育障碍，并伴有过早衰老的症状，Cockayne综合征(XP-G/CS)。此外，WRN和BLM在复制过程中维持基因组完整性方面都发挥着重要作用。BLM的缺失会导致以癌症高发为特征的Bloom综合征，而WRN的缺失会导致Werner综合征，这是一种癌症发病率增加的早衰障碍。初步数据表明，XPG具有一个新的复制相关角色。有待检验的假设是，XPG与WRN或BLM一起作用于DNA损伤后停滞或崩溃的复制叉子的同源重组修复(HRR)，和/或在正常的端粒复制期间。目的1利用HRR中间体的DNA模型底物，研究XPG影响BLM和WRN酶活性的机制。目标2将使用免疫荧光、细胞分离和免疫沉淀来验证XPG定位于带有WRN或BLM的停滞或坍塌的复制叉处的假设。AIM 3将检查原代XP-G/CS细胞端粒的完整性，并确定XPG定位于端粒的细胞周期和DNA损伤诱导的条件。这些研究将为高度多功能的蛋白质调节DNA复制和修复之间的串扰提供有价值的见解。这两种DNA交易在维持基因组稳定性以及抵御癌症和衰老方面都至关重要。

DESCRIPTION (provided by applicant): Genomic integrity is persistently threatened by endogenous damage from the cellular metabolism and exogenous environmental sources including UV, ionizing radiation, and mutagenic chemicals. To counter this damage, cells have evolved complex and interconnected DNA repair pathways to coordinate lesion detection and removal with other vital cellular processes such as DNA replication, transcription and recombination. Failure in any link can lead to genomic instability, a known precursor to both cancer and aging. The broad focus of this proposal is to understand the biological significance of the interaction between three proteins involved in preventing genomic instability: XPG, and the human RecQ family members BLM and WRN. XPG has important non-enzymatic functions in both transcription-coupled repair and in base-excision repair (BER) of oxidative damage, and mutations that block functions of XPG lead to a severe neurological and developmental disorder with symptoms of premature aging, Cockayne syndrome (XP-G/CS). Further, WRN and BLM both play important roles in the maintenance of genomic integrity during replication. The loss of BLM leads to Bloom's syndrome, characterized by a high incidence of cancer, and the loss of WRN leads to Werner's syndrome, a premature aging disorder with increased cancer incidence. Preliminary data suggests a novel replication-associated role for XPG. The hypotheses to be tested are that XPG functions with WRN or BLM in homologous recombination repair (HRR) of stalled or collapsed replication forks after DNA damage, and/or during normal telomere replication. Aim 1 will use model DNA substrates of HRR intermediates to investigate the mechanisms by which XPG affects the function of BLM and WRN enzymatic activities. Aim 2 will use immunofluorescence, cell fractionation, and immunoprecipitations to test the hypothesis that XPG localizes to sites of stalled or collapsed replication forks with WRN or BLM. Aim 3 will examine primary XP-G/CS cells for telomere integrity, and determine the cell cycle and DNA damage-induced conditions under which XPG localizes to the telomeres. These studies will provide valuable insights into the roles of highly multifunctional proteins to regulate crosstalk between DNA replication and repair. Both of these DNA transactions are critically important in the maintenance of genomic stability and defense against cancer and aging.