KU, TELOMERE MAINTENANCE AND CELLULAR SENESCENCE

这项建议的目标是研究Ku杂二聚体(Ku70/80) 在哺乳动物端粒维持和细胞衰老中的作用。 对Ku参与端粒维持的支持由最近的 证明端粒需要酵母的Ku同系物 维修。此外，研究表明，酵母菌Ku与 体内端粒DNA，提示Ku在端粒长度中起直接作用 监管。引人注目的是，尽管序列保守性相对较低 酵母和人Ku亚基之间的氨基酸水平上，外源 人Ku亚基在酵母中的表达挽救了Ku零突变。在这 建议书，申请人提出最近获得的新的初步证据 Ku与人类细胞中端粒DNA的关联。端粒 维护是衰老的关键，因为端粒酶的激活， 负责合成端粒DNA的酶，已被证明 直接控制原代人类细胞的复制衰老。申请人 对已知元素之间的相互作用提出了详细的调查 确定这些蛋白质在端粒和Ku中所起的作用 端粒维持和复制性衰老。申请者的具体目标 1)Ku/端粒DNA在哺乳动物端粒上的相互作用 使用体内和体外实验方法；2)是否已知哺乳动物 端粒结合蛋白介导Ku与端粒之间的相互作用； 3)胚胎干细胞端粒维持与细胞衰老 小鼠胚胎成纤维细胞Ku70、Ku 80、DNA-PKcs和DNA-PKcs基因突变的研究 端粒酶的RNA部分。这项建议将两个小组结合在一起， 互补专业知识：伊利莎白·布莱克本博士的团队 加州、旧金山拥有端粒和端粒酶方面的专业知识，以及 David Chen博士在洛斯阿拉莫斯国家实验室拥有Ku专业知识的团队 异源二聚体和DNA-PKcs。这项工作的目标是形成对 这些关键成分端粒和Ku异源二聚体的相互作用， 在哺乳动物细胞中。这些研究对于进一步了解 端粒维持，这将提供更好的理解细胞 衰老。

The goal of this proposal is to study the Ku heterodimer's (Ku70/80) role in telomere maintenance and cellular senescence in mammals. Support for Ku's involvement in telomere maintenance is provided by the recent demonstration that the Ku homologs of yeast are required for telomere maintenance. Moreover, it has been shown that yeast Ku is in close proximity to telomeric DNA in vivo, suggesting a direct role for Ku in telomere length regulation. Strikingly, despite the relatively low sequence conservation between yeast and human Ku subunits at the amino acid level, the exogenous expression of human Ku subunits in yeast rescues a Ku null mutation. In this proposal, the applicant presents recently obtained novel preliminary evidence for an association of Ku with telomeric DNA in human cells. Telomere maintenance is critical for senescence, since activation of telomerase, the enzyme responsible for the synthesis of telomeric DNA, has been shown to directly control replicative senescence in primary human cell. The applicant proposes a detailed investigation into the interactions between known elements of the telomere and Ku to determine the role played by these proteins in telomere maintenance and replicative senescence. The applicant's specific aims are to investigate: 1) Ku/telomeric DNA interactions at the mammalian telomere using in vivo and in vitro experimental approaches; 2) whether known mammalian telomere binding proteins mediate an interaction between Ku and the telomere; and 3) telomere maintenance in embryonic stem cells and cellular senescence in mouse embryonic fibroblasts with various mutations in Ku70, Ku 80, DNA-PKcs and the RNA moiety of telomerase. This proposal brings together two groups with complementary expertise: Dr. Elizabeth Blackburn's group at the University of California, San Francisco with expertise in the telomere and telomerase, and Dr. David Chen's group at Los Alamos National Lab with expertise in the Ku heterodimer and DNA-PKcs. The goal of this work is to form an understanding of the interaction of these key components, the telomere and the Ku heterodimer, in mammalian cells. These studies are critical for a further understanding of telomere maintenance, which will provide a better understanding of cellular senescence.