Mechanisms of Telomeric DNA Loss and Repair

端粒 DNA 丢失和修复的机制

• 批准号: 7900269
• 负责人: Patricia L Opresko
• 金额: $ 21.83万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7900269
• 关键词: Age; Age of Onset; Aging; Biological Assay; Biological Preservation; Cataract; Cell Line; Cell physiology; Cells; Chromosomes; Confocal Microscopy; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Sequence; DNA biosynthesis; DNA repair protein; DNA-Directed DNA Polymerase; Deletion Mutation; Disease; Environmental Risk Factor; Event; Exposure to; Frequencies; Functional disorder; Genetic; Genomic Instability; Genomics; Goals; Guanine; Heart Diseases; Human; In Vitro; Induced Mutation; Kinetics; Knowledge; Lasers; Lesion; Life; Longevity; Malignant Neoplasms; Measures; Metals; Modeling; Molecular; Monitor; Mutagenesis; Mutation; Normal Cell; Onset of illness; Pathology; Pathway interactions; Patients; Phosphorylation; Premature aging syndrome; Process; Proteins; Research; Research Personnel; Risk Factors; Role; Running; Shuttle Vectors; Somatic Cell; Technology; Telomerase; Telomere Shortening; Testing; Therapeutic Intervention; Werner Syndrome; Work; adduct; age related; base; chromium hexavalent ion; design; environmental mutagens; helicase; human WRN protein; in vivo; migration; pressure; prevent; programs; protein function; repaired; research study; respiratory; telomere; vector

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.

描述（由申请人提供）： 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性的分子机制。端粒 DNA 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 损伤剂后。 DNA 修复蛋白 WRN 的缺乏会加速端粒丢失，并导致人类早衰维尔纳综合征 (WS)，患者过早出现多种与衰老相关的病症。该提案的具体目标是 1) 确定与 DNA 损伤剂和 WS 相关的端粒 DNA 丢失的分子机制，2) 确定修复蛋白 WRN 在端粒保存中的作用。待测试的假设是端粒 DNA 的直接损伤会导致端粒磨损，而 WRN 蛋白通过防止和/或修复端粒 DNA 的断裂来保护端粒。穿梭载体诱变测定可测量特定靶标中的突变，用于检查 WRN 蛋白在防止复制诱导的端粒 DNA 缺失中的作用，这种缺失可能自发发生或暴露于环境诱变剂铬 (VI) 后发生。接触 Cr(VI) 与呼吸道癌有关，并会诱导端粒中普遍存在的复制阻断加合物和 DNA 序列断裂。因此，Cr(VI) 是研究环境 DNA 损伤对端粒 DNA 复制的影响的绝佳模型。穿梭载体方法允许分析独立和罕见的突变事件，并消除针对端粒重复丢失的选择性压力。为了研究 WRN 在直接修复端粒断裂中的作用，共聚焦显微镜将用于在活细胞中用 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，对加速端粒 DNA 丢失的遗传和环境因素的机制理解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预防或延缓与衰老和癌症相关的疾病发作的干预疗法至关重要。