Ku86 Controls DNA Repair, Telomeres & Genomic Stability

描述(由申请人提供)：这笔赠款的具体目标是对人体细胞中的Ku86：Karp-1基因座进行体细胞遗传学分析。在小鼠中，Ku86和Ku70、DNA依赖的蛋白激酶催化亚单位(DNA-PK[CS])、XRCC-4、DNA连接酶IV和Artemis是DNA双链断裂(DSB)修复的关键形式，称为非同源末端连接(NHEJ)。最近，我们已经证明Ku86：Karp-1在人类体细胞中发挥着额外的、必不可少的作用。在这项资助申请中，我们提供了初步的数据来证明这一重要作用可能与Ku86：Karp-1调节端粒长度和基因组稳定性的能力有关。这是首次证明NHEJ因子之一在人类细胞中调节这两个过程。我们描述了下面的实验，这些实验阐明了KU86和KARP-1在DNA DSB修复、端粒长度调节和基因组稳定性中的遗传和分子作用(S)。大量癌症易感综合征的存在突显了识别和了解控制人类DNA修复的基因的重要性，如共济失调毛细血管扩张、共济失调性毛细血管扩张样障碍、奈梅根断裂综合征、范可尼贫血、Li-Fraumeni综合征、着色性干皮病以及乳腺癌和结肠癌，其中潜在的分子缺陷似乎存在于DNA修复基因中。我们打算通过四个实验来描述这些重要的通路：1.条件人Ku86：Karp-1-空细胞系的构建。2.Ku86：Karp-1缺失的人组织培养细胞为什么会发生凋亡？3.DNA-PK活性的丧失是否与Ku86：Karp-1缺失的表型有关？4.Ku86：Karp-1突变的细胞中端粒酶的生物合成或功能是否异常？这些研究的最终目的是利用Ku86和Karp-1突变细胞系作为工具来了解人类DNA DSB修复的分子机制。

DESCRIPTION (provided by applicant): The specific aims of this grant are directed towards a somatic cell genetic analysis of the Ku86:KARP-1 locus in human cells. In the mouse, Ku86, along with Ku70, the DNA-dependent protein kinase catalytic subunit (DNA-PK[CS]), XRCC-4, DNA ligase IV and Artemis, are required for a critical form of DNA double strand break (DSB) repair known as nonhomologous end joining (NHEJ). Recently, we have shown that Ku86:KARP-1 plays an additional, essential role in human somatic cells. In this grant application we provide preliminary data to demonstrate that this essential role is probably related to the ability of Ku86:KARP-1 to regulate telomere length and genomic stability. This is the first demonstration that one of the NHEJ factors regulates these two processes in human cells. We describe below experiments that elucidate the genetic and molecular role(s) of Ku86 and KARP-1 in DNA DSB repair, telomere length regulation and genomic stability in human cells. The importance of identifying and understanding the genes that control human DNA repair is underscored by the existence of a large number of cancer predisposition syndromes such as ataxia telangiectasia, ataxia telangiectasia-like disorder, Nijmegen Breakage syndrome, Fanconi's anemia, Li-Fraumeni syndrome, xeroderma pigmentosum and breast and colon cancer where it appears that the underlying molecular defects reside in DNA repair genes. We intend to characterize these important pathways using four lines of experimentation: 1. Construction of conditional human Ku86:KARP-1-null cell lines. 2. Why do human Ku86:KARP-1-null tissue culture human cells undergo apoptosis? 3. Is the loss of DNA-PK activity responsible for the Ku86:KARP-1-null phenotypes? 4. Is telomerase biogenesis or function aberrant in Ku86:KARP-1 mutant cells? The ultimate goal of these studies is to use Ku86 and KARP-1 mutant cell lines as tools to understand the molecular mechanisms of DNA DSB repair in humans.