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）： 拟议的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。与衰老有关的疾病和暴露于某些环境DNA破坏药物后，加速的端粒DNA损失发生。缺乏DNA修复蛋白WRN会加速端粒损失，并导致人类后代werner综合征（WS），其中患者过早发展了多种与衰老相关的病理。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损失的分子机制，以及2）定义修复蛋白WRN在端粒保存中的作用。要测试的假设是，直接对端粒DNA的损害会导致端粒损耗，并且WRN蛋白通过防止和/或修复端粒DNA中的休息来保留端粒。一种途径诱变测定法测量定义靶标的突变的方法将用于检查WRN蛋白在防止复制诱导的端粒DNA缺失中可能自发发生或暴露于环境诱变型棉布铬（VI）之后的作用。暴露于CR（VI）与呼吸癌有关，并在端粒中普遍存在的DNA序列中诱导复制阻滞性加合物和断裂。因此，CR（VI）是研究环境DNA损伤对端粒DNA复制的后果的绝佳模型。穿梭矢量方法可以分析独立和罕见的突变事件，并消除端粒重复损失的选择性压力。为了调查WRN在端粒的修复中断中的作用，共聚焦显微镜将用于在端粒上选择性地诱导DNA双链断裂，并在活细胞中使用UVA激光诱导。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，并检查用于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损失的遗传和环境因素的机械理解应有助于确定过早衰老的危险因素。识别端粒丢失和保留端粒DNA的细胞过程的机制对于预防或延迟与衰老和癌症相关的疾病发作的干预疗法的设计至关重要。