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

我实验室以前的研究表明，Ku 80缺失的小鼠加速衰老的多种症状，已知Ku 80在各种染色质相关的细胞过程中起作用，最显著的是通过非同源末端连接修复双链断裂。项目4的长期目标是进一步研究Ku 80在长寿保证中的作用，基于其基因组维护功能及其与各种最近出现的潜在细胞防御系统的潜在遗传相互作用。拟议的研究符合方案项目的中心主题：即，氧化应激诱导的DNA损伤是衰老相关细胞变性和死亡的主要驱动力。这一长期目标将通过沿着四个具体目标来实现，这些目标将与本项目规划小组的其他项目密切合作，并广泛利用其核心设施。对于目标1，我们将分析ku 80-/-小鼠杂交到 染色质代谢（Rad 54、DNA-PKcs、Ku 70）或细胞周期反应（p53和p27/Kipl）受损的各种背景（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证基因Foxo 3a，Sir 2alpha，Sir 2L 2和Sir 2L 3，我们将筛选对调节氧化应激反应的信号转导途径重要的基因，p66/shc。我们将产生和分析这些基因的遗传改变细胞，并研究基因产物对多种DNA代谢途径的潜在影响，使用细胞培养系统和实验室最近开发的遗传毒性筛选（与项目1-3和核心C合作）。对于目标3，我们将测试以下假设：目标2中测试的这些基因或改变的基因，单独或组合，可用于改善ku 80-/-细胞中的加速老化症状，以及我们项目1的合作者发现的加速老化突变体。目标2的结果将决定我们如何实现目标3。项目的这一部分与项目1和项目3具有广泛的协同作用。对于目标4，我们将开发遗传工具，以促进对小鼠和细胞的分析。这包括两个部分：（1）开发能够在同一基因座过表达多个抗氧化防御基因的工具（这将避免现在获得这样的小鼠所必需的广泛育种）;和（2）开发携带一个或多个衰老报告基因的小鼠模型。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.