THE IMPACT OF CELLULAR DEFENSE ON THE ROLE OF Ku80 IN GENOME MAINTENANCE AND LONG

我的实验室以前的研究表明，Ku80基因缺失的小鼠出现了多种加速衰老的症状，Ku80基因已知在各种与染色质相关的细胞过程中发挥作用，最显著的是通过非同源末端连接修复双链断裂。项目4的长期目标是基于Ku80的基因组维持功能以及它与最近出现的各种潜在的细胞防御系统的潜在遗传相互作用，进一步研究Ku80在长寿保证中的作用。拟议的研究与该计划项目的中心主题一致：即，氧化应激诱导的DNA损伤是衰老相关细胞退化和死亡的主要驱动力。这一长期目标将沿着四个具体目标进行，与该项目小组的其他项目密切合作制定，并广泛利用其核心设施。对于目标1，我们将分析ku80-/-小鼠进入 在染色质代谢(RAD54、DNA-PKcs、Ku70)或细胞周期反应(P53和p27/KIPL)方面受到损害的各种背景(与项目1-3、核心B和核心C合作)。对于目标2，我们将筛选最近出现的候选长寿保障基因Foxo3a、Sir2pha、Sir2L2和Sir2L3，并筛选一个对调节氧化应激反应的信号转导途径重要的基因，p66/SHC。我们将利用细胞培养系统和实验室最近开发的遗传毒性筛查(与项目1-3和核心C合作)来生成和分析这些基因的转基因细胞，并研究基因产品对多种DNA代谢途径的潜在影响。对于目标3，我们将测试这样的假设，即这些基因或在目标2中测试的改变的基因，可以单独或组合用于改善ku80-/-细胞中的加速衰老症状，以及我们项目1的合作者发现的加速衰老突变体。目标2的结果将决定我们如何继续进行目标3。该项目的这一部分与项目1和3提供广泛的协同作用。对于目标4，我们将开发基因工具来促进对小鼠和细胞的分析。这包括两个部分：(1)开发工具，使多个抗氧化防御基因在同一基因座上过度表达(这将避免目前获得此类小鼠所必需的广泛繁殖)；(2)开发含有一个或多个衰老报告的小鼠模型。这两个鼠标建模工具将分别与项目1和项目3密切合作开发。

Previous studies in my laboratory have indicated multiple symptoms of accelerated aging in mice deleted for Ku80, which is known to act in a variety of chromatin-related cellular processes, most notably in the repair of double-strand breaks through non-homologous end joining. The long-term objective of Project 4 is to further investigate the role of Ku80 in longevity assurance, based on its genome maintenance function and its potential genetic interactions with a variety of recently emerged potential cellular defense systems. The proposed studies are in keeping with the central theme of the program project: i.e., oxidative stress-induced DNA damage as a major driving force of aging-related cellular degeneration and death. This long-term objective will be pursued along four specific aims, worked out in close collaboration with the other projects of this PPG, and extensively using its core facilities. For Aim 1, we will analyze ku80 -/- mice crossed into various backgrounds that are compromised for chromatin metabolism (Rad54, DNA-PKcs, Ku70) or cell cycle response, (p53 and p27/Kipl)(Collaboration with projects 1-3, Core B and Core C). For Aim 2 we will screen the recently emerged candidate longevity assurance genes Foxo3a, Sir2alpha, Sir2L2 and Sir2L3, and we will screen a gene important for a signal transduction pathway that regulates oxidative stress responses, p66/shc. We will generate and analyze genetically altered cells for these genes and investigate the potential influence of the gene products on a multitude of DNA metabolism pathways, using cell culture systems and a genotoxic screen recently developed in the laboratory (Collaboration with projects 1-3 and core C). For Aim 3, we will test the hypothesis that these genes or altered genes tested in aim 2, alone or in combination, can be used to ameliorate the accelerated aging symptoms in the ku80 -/- cells, and in the accelerated aging mutants discovered by our collaborators of project 1. The results from aim 2 will determine how we proceed for aim 3. This part of the project provides extensive synergy with projects 1 and 3. For Aim 4, we will develop genetic tools to facilitate analysis of mice and cells. This consists of two parts: (1) development of tools that enable over-expression of multiple anti-oxidant defense genes at the same locus (which will avoid the extensive breeding that is now necessary to obtain such mice); and (2) development of a mouse model harboring one or more senescence reporters. These two mouse-modeling tools will be developed in close collaboration with projects 1 and 3, respectively.