Mechanisms of Telomeric DNA Loss and Repair

描述(由申请人提供)： 这项拟议研究的长期目标是了解与衰老和衰老相关疾病相关的基因组不稳定的分子机制。在衰老相关疾病和暴露于一些环境DNA损伤剂后，会发生加速的端粒DNA丢失。缺乏DNA修复蛋白WRN会加速端粒丢失，并导致人类程序性沃纳综合征(WS)，患者过早地发展为多种与衰老相关的病理。该建议的具体目标是1)确定与DNA损伤剂和WS相关的端粒DNA丢失的分子机制，以及2)确定修复蛋白WRN在端粒保存中的作用。需要检验的假设是，端粒DNA直接损伤导致端粒磨损，WRN蛋白通过防止和/或修复端粒DNA断裂来保护端粒。一种测量特定靶点突变的穿梭载体突变试验将用于检测WRN蛋白在防止复制诱导的端粒DNA缺失方面的作用，端粒DNA缺失可能是自发发生的，也可能是在暴露于环境诱变剂铬(VI)后发生的。接触铬(VI)与呼吸系统癌症有关，并诱导端粒中普遍存在的DNA序列中复制阻断加合物和断裂。因此，铬(VI)是研究环境DNA损伤对端粒DNA复制影响的一个很好的模型。穿梭载体方法允许分析独立的和罕见的突变事件，并消除了针对端粒重复序列丢失的选择性压力。为了研究WRN在直接修复端粒断裂中的作用，我们将使用激光共聚焦显微镜在活细胞中选择性地诱导端粒DNA双链断裂。这项建议的主要目标是确定DNA损伤和环境诱变剂对染色体末端端粒DNA完整性的影响，并检查修复端粒的细胞途径。鉴于端粒在衰老和癌症中的关键作用，从机制上理解加速端粒DNA丢失的遗传和环境因素应该有助于识别过早衰老的危险因素。识别端粒丢失的机制和保存端粒DNA的细胞过程对于设计干预疗法以预防或延缓与衰老和癌症相关的疾病的发生至关重要。

DESCRIPTION (provided by applicant): The long term objective of the proposed research is to understand the molecular mechanisms of genomic instability associated with aging and aging-related diseases. Accelerated telomeric DNA loss occurs in aging-related diseases and after exposure to some environmental DNA damaging agents. Lack of the DNA repair protein WRN accelerates telomere loss and causes the human progeroid Werner syndrome (WS) in which patients prematurely develop multiple aging-related pathologies. The specific goals of this proposal are 1) to determine the molecular mechanisms of telomeric DNA loss associated with DNA damaging agents and WS, and 2) to define the roles for the repair protein WRN in telomere preservation. The hypothesis to be tested is that damage directly to telomeric DNA contributes to telomere attrition, and that WRN protein preserves telomeres by preventing and/or repairing breaks in telomeric DNA. A shuttle vector mutagenesis assay that measures mutations in a defined target will be used to examine the role of WRN protein in preventing replication-induced telomeric DNA deletions that may occur either spontaneously or after exposure to the environmental mutagen chromium (VI). Exposure to Cr(VI) is associated with respiratory cancers, and induces replication-blocking adducts and breaks in DNA sequences that are prevalent in telomeres. Thus, Cr(VI) is an excellent model for investigating the consequences of environmental DNA damage on telomeric DNA replication. The shuttle vector approach allows for analysis of independent and rare mutation events and eliminates selective pressure against the loss of telomeric repeats. To investigate a role for WRN in repairing breaks at telomeres directly, confocal microscopy will be used to induce DNA double strand breaks selectively at telomeres with a UVA laser in live cells. The broad goals of this proposal are to determine the impact of DNA damage and environmental mutagens on the integrity of telomeric DNA in chromosome ends, and to examine cellular pathways for repairing telomeres. Given the critical role for telomeres in aging and cancer, a mechanistic understanding of the genetic and environmental factors that accelerate telomeric DNA loss should aid in the identification of risk factors for premature aging. Identifying mechanisms of telomere loss and cellular processes that preserve telomeric DNA is crucial for the design of intervention therapies that prevent or delay the onset of diseases associated with aging and cancer.