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。各种动物的衰老速度差别很大。因此，比较方法是获得衰老机制新见解的有力工具。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。考虑到最近对DNA DSB修复[和DSB反应机制]的理解进展，我们认为进行DNA DSB修复[和DSB反应]的种间比较研究是及时的。在这个应用程序中，我们建议使用一组啮齿动物来测试基因组维持机制与寿命共同进化的假设。啮齿类动物是这项研究的理想物种，因为它包括了不同寿命的系统发育相关物种，其中缓慢衰老已经独立进化了几次。我们的实验室收集了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、细胞生长特征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，我们将采用啮齿动物细胞和组织的收集：(1)测试基因组稳定性与长寿共同进化的假设。我们将通过流式细胞术检测非整倍体率，并通过核型分析特定的染色体畸变。(2)验证DSB修复效率和保真度随寿命进化的假设。我们最近开发了灵敏的荧光分析方法来分析DSB修复的两种途径，同源重组（HR）和非同源末端连接（NHEJ）的效率和保真度。这些试验将使多物种DSB修复的定量分析成为可能。(3)验证DSB特异性反应如凋亡和衰老随寿命共同进化的假设。我们将测量y照射后的细胞存活、诱导DSB修复病灶、凋亡、坏死和衰老。我们还将通过彗星试验分析DSB的修复率。这些数据将根据系统发育的不独立性进行校正，并对诸如体型等混杂变量进行控制。我们的实验室有独特的设备来进行拟议的研究。我们收集了一个精心构建的具有不同寿命的系统发育相关物种的集合，并开发了新的定量分析HR和NHEJ的方法，以及分析细胞凋亡和坏死的敏感方法。该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随寿命共同进化。它还将确定短寿和长寿啮齿动物之间基因组维持差异的分子机制。公共卫生相关性：造成动物物种之间寿命巨大差异的机制尚不清楚。该项目旨在研究基因组维持机制是否对物种间寿命差异有贡献，研究对象是短寿和长寿的啮齿类动物。这项研究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.