Mechanisms of Telomeric DNA Loss and Repair

描述（由申请人提供）： 该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性的分子机制。端粒DNA的加速丢失发生在与衰老相关的疾病中，以及暴露于某些环境DNA损伤剂之后。缺乏DNA修复蛋白WRN加速端粒丢失，并导致人类早老性沃纳综合征（WS），其中患者过早地发展出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，和2）确定修复蛋白WRN在端粒保存中的作用。待检验的假设是，直接对端粒DNA的损伤有助于端粒磨损，并且WRN蛋白通过防止和/或修复端粒DNA中的断裂来保存端粒。将使用穿梭载体诱变试验（可测量确定靶标中的突变）来检查WRN蛋白在预防复制诱导的端粒DNA缺失中的作用，端粒DNA缺失可能自发发生或在暴露于环境诱变剂铬（VI）后发生。暴露于Cr（VI）与呼吸道癌症有关，并诱导端粒中普遍存在的DNA序列的复制阻断加合物和断裂。因此，铬（VI）是一个很好的模型，用于调查端粒DNA复制的环境DNA损伤的后果。穿梭载体方法允许分析独立和罕见的突变事件，并消除了对端粒重复序列丢失的选择压力。为了研究WRN在直接修复端粒断裂中的作用，将使用共聚焦显微镜在活细胞中用UVA激光选择性地诱导端粒处的DNA双链断裂。该提案的广泛目标是确定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，对加速端粒DNA丢失的遗传和环境因素的机械理解应该有助于识别过早衰老的风险因素。确定端粒丢失的机制和保留端粒DNA的细胞过程对于设计预防或延迟与衰老和癌症相关的疾病发作的干预疗法至关重要。

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.