Comparative Analysis of DNA Break Repair in Short- and Long-Lived Rodent Species

描述(由申请人提供)：拟议研究的长期目标是了解决定寿命的机制。动物种类在衰老速度上有很大差异。因此，比较方法是获得对衰老机制的新见解的有力工具。体细胞突变和基因组重排的积累被认为是导致衰老的原因之一。从小鼠研究中收集的数据表明，DNA双链断裂(DSB)修复是一种重要的长寿保证机制。考虑到最近在了解DNA DSB修复[和DSB响应机制]方面的进展，我们认为现在是时候对DNA DSB修复[和DSB响应]进行物种间比较研究了。在这个应用中，我们建议使用一组啮齿动物物种来检验基因组维护机制与寿命共同进化的假设。啮齿动物是这项研究的理想物种，因为它包括具有不同寿命的系统发育相关物种，在这些物种中，缓慢衰老已经独立进化了几次。我们实验室收集了17种啮齿动物的原代细胞和组织，并对这些细胞的端粒酶活性、细胞生长特性和基因组稳定性进行了初步分析。我们的初步数据表明，长寿物种具有更高的基因组稳定性和更有效的DSB修复。在这个应用中，我们将使用原代啮齿动物细胞和组织的收集来：(1)测试基因组稳定性与长寿命共同进化的假设。我们将通过流式细胞术检测非整倍体率，并通过核型分析来分析特定的染色体异常。(2)检验DSB修复的效率和保真度与寿命同步演化的假设。我们最近开发了灵敏的荧光分析方法来分析DSB修复的两条途径-同源重组(HR)和非同源末端连接(NHEJ)-的效率和保真度。这些检测方法可以对多种物种的DSB修复进行定量分析。(3)检验特定的DSB反应(如细胞凋亡和衰老)与寿命共同进化的假设。我们将测量辐射后细胞存活率、DSB修复灶的诱导、细胞凋亡、坏死和衰老。我们还将通过彗星试验分析DSB修复率。这些数据将根据系统发育的非独立性进行校正，并对身体大小等混杂变量进行控制。我们的实验室装备独特，可以进行拟议中的研究。我们精心构建了具有不同寿命的系统发育相关物种的集合，并开发了用于HR和NHEJ分析的新的定量方法，以及用于分析细胞凋亡和坏死的灵敏方法。这项拟议的研究将确定诸如诱导细胞凋亡、衰老或DSB修复的效率和保真度等基因组维持机制是否与寿命共同进化。它还将确定导致短寿命和长寿命啮齿动物基因组维护差异的分子机制。与公共卫生相关：造成动物物种之间寿命巨大差异的机制尚不清楚。这项拟议的项目试图利用一组短寿命和长寿命的啮齿动物物种来研究基因组维护机制是否有助于物种间寿命的差异。这项研究中获得的信息将有助于制定预防癌症和延长人类寿命的策略。

DESCRIPTION (provided by applicant): The long-term goal of the proposed studies is to understand the mechanisms that determine longevity. Animal species differ enormously in their aging rates. Therefore, a comparative approach is a powerful tool to obtain new insights into the mechanisms of aging. Accumulation of somatic mutations and genomic rearrangements is believed to be a contributing cause of aging. Data collected from mouse studies suggest that DNA double-strand break (DSB) repair is an essential longevity assurance mechanism. Considering recent advances in understanding DNA DSB repair [and DSB response mechanisms], we believe that it is timely to undertake an interspecies comparative study of DNA DSB repair [and DSB response]. In this application we propose to test the hypothesis that genome maintenance mechanisms co evolve with lifespan, using a panel of rodent species. Rodents are an ideal species for this study as it includes phylogenetically related species with diverse lifespans, in which slow aging has evolved independently several times. Our laboratory has assembled a collection of primary cells and tissues from 17 rodent species, and performed preliminary analysis of telomerase activity, cell growth characteristics, and genome stability in these cells. Our preliminary data suggest that long-lived species have higher genome stability and more efficient DSB repair. In this application, we will employ the collection of primary rodent cells and tissues to: (1) Test the hypothesis that genome stability coevolves with long lifespan. We will examine aneuploidy rate by flow cytometry and analyze specific chromosomal aberrations by karyotyping. (2) Test the hypothesis that the efficiency and fidelity of DSB repair co evolve with lifespan. We have recently developed sensitive fluorescent assays for analysis of the efficiency and fidelity of the two pathways of DSB repair, homologous recombination (HR) and nonhomologous end joining (NHEJ). These assays will enable quantitative analysis of DSB repair in multiple species. (3) Test the hypothesis that specific DSB responses such as apoptosis and senescence co evolve with lifespan. We will measure cell survival, induction of DSB repair foci, apoptosis, necrosis, and senescence following y-irradiation. We will also analyze the rate DSB repair by comet assays. The data will be corrected for phylogenetic nonindependence, and controlled for confounding variables such as body size. Our laboratory is uniquely equipped to carry out the proposed research. We assembled a carefully constructed collection of phylogenetically related species with diverse lifespans, and developed novel quantitative assays for the analysis of HR and NHEJ, and sensitive assays for analysis of apoptosis and necrosis. The proposed study will determine whether such genome maintenance mechanisms as induction of apoptosis, senescence, or the efficiency and fidelity of DSB repair co evolve with lifespan. It will also identify molecular mechanisms responsible for the differences in genome maintenance between short- and long-lived rodents. PUBLIC HEALTH RELEVANCE: The mechanisms responsible for the vast differences in lifespan between animal species are unknown. The proposed project seeks to examine whether genome maintenance mechanisms contribute to interspecies differences in longevity using a collection of short- and long-lived rodent species. The information obtained in this study will help to develop strategies to prevent cancer and extend lifespan in humans.