Molecular mechanism of age-related decline of nonhomologous DNA end joining

非同源DNA末端连接与年龄相关的衰退的分子机制

• 批准号: 7147134
• 负责人: Vera Gorbunova
• 金额: $ 28.74万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7147134
• 关键词: DNA damage; DNA repair; age difference; aging; animal old age; biotechnology; cell senescence; disease /disorder model; fibroblasts; fluorescent in situ hybridization; gene expression; gene induction /repression; gene rearrangement; genetic regulatory element; genetic transcription; genetically modified animals; green fluorescent proteins; laboratory mouse; messenger RNA; model design /development; posttranslational modifications; protein localization; transfection /expression vector

DESCRIPTION (provided by applicant): Genomic instability is a hallmark of aging cells, and the major cause of tumorigenesis. The long-term goal of the proposed research is to understand the molecular mechanism of age-related genomic instability. Genomic rearrangements are thought to arise as a result of errors in the repair of DNA double-strand breaks (DSB). We have recently developed a sensitive fluorescent assay for analysis of DNA DSB repair via nonhomologous end joining (NHEJ) pathway in senescent cells. Using this assay we have demonstrated that NHEJ becomes less efficient and more error-prone during cellular senescence. Our preliminary data suggest that the protein levels of several key components of NHEJ pathway are reduced dramatically in senescent cells, consistent with the observed age-related decline of NHEJ. Therefore, the focus of this application is to further examine NHEJ during aging and to understand the mechanism responsible for the low levels of NHEJ proteins in senescent cells. Our Specific Aims are: (1) To test the hypothesis that NHEJ becomes less efficient and more error-prone during organismal aging. General decline of DNA repair has been observed in aged individuals, suggesting that NHEJ is likely to decline during aging. We will examine the efficiency and fidelity of NHEJ in fibroblasts from donors of different age from the Baltimore Longitudinal Study on Aging, and in aging mice. We will use the NHEJ assay we developed for analysis of end joining in senescent cells. (2) To test the hypothesis that the decline of NHEJ is caused by reduction in the amount of DSB repair proteins. Our preliminary experiments show that Ku70, Ku80 and NBS1 proteins are virtually absent from senescent cells. To expand this observation, we will examine mRNA and protein levels of the key components of NHEJ machinery in replicatively senescent human fibroblasts and in tissues of old mice. We will determine whether a transcriptional or post-translational mechanism is responsible for the reduction of NHEJ protein levels: (3) Generate a transgenic mouse model for analysis of age-related changes in NHEJ. Human genome becomes unstable with age leading to cancer and age-related diseases. Why genomes become unstable with age is unknown. The proposed study aims to understand the mechanism of age-related genomic instability, which will help develop ways to stabilize the aging genome and prevent cancer.

描述（由申请人提供）：基因组不稳定性是衰老细胞的标志，也是肿瘤发生的主要原因。拟议的研究的长期目标是了解与年龄相关的基因组不稳定性的分子机制。基因组重排被认为是由于DNA双链断裂修复（DSB）的误差而引起的。我们最近开发了一种敏感的荧光测定法，用于通过非同源末端连接（NHEJ）途径分析DNA DSB修复。使用此测定，我们证明了NHEJ在细胞衰老过程中的效率较低，易于错误。我们的初步数据表明，在衰老细胞中，NHEJ途径的几个关键成分的蛋白质水平大大降低，与观察到的NHEJ年龄相关的下降一致。因此，该应用的重点是在衰老过程中进一步检查NHEJ，并了解导致衰老细胞中NHEJ蛋白低水平的机制。我们的具体目的是：（1）检验以下假设：NHEJ在有机体衰老过程中效率较低，易于错误。在老年人中观察到DNA修复的总体下降，表明NHEJ在衰老期间可能会下降。我们将研究来自巴尔的摩衰老纵向研究和衰老小鼠的捐助者的成纤维细胞中NHEJ的效率和保真度。我们将使用我们开发的NHEJ分析来分析衰老细胞中的结合。 （2）测试了NHEJ的下降是由DSB修复蛋白量减少引起的假设。我们的初步实验表明，KU70，KU80和NBS1蛋白实际上没有衰老细胞。为了扩展这一观察结果，我们将检查复制性衰老的人成纤维细胞和旧小鼠组织中NHEJ机械关键成分的mRNA和蛋白质水平。我们将确定转录或翻译后机制是否负责NHEJ蛋白水平的降低：（3）生成一种转基因小鼠模型，用于分析NHEJ年龄相关的变化。人类基因组随着年龄的导致癌症和与年龄有关的疾病而变得不稳定。为什么基因组随着年龄而变得不稳定是未知的。拟议的研究旨在了解与年龄相关的基因组不稳定性的机制，这将有助于开发稳定衰老基因组和预防癌症的方法。