Ku regulates non-homologous end joining pathways in human somatic cells

Ku 调节人类体细胞中的非同源末端连接途径

• 批准号: 8649877
• 负责人: ERIC A HENDRICKSON
• 金额: $ 1.83万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-07 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8649877
• 关键词: Address; Affect; Applications Grants; Belief; Binding; Biological Assay; Cancer Patient; Cell Line; Cells; Chemicals; Chromosomal Breaks; Chromosomal Rearrangement; Clinical; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA ligase III; Defect; Development; Double Strand Break Repair; Ensure; Event; Fertility; Frequencies; G22P1 gene; Gene Mutation; Gene Targeting; Generations; Genes; Genetic; Genetic Recombination; Genome; Goals; Health; Homeostasis; Homologous Gene; Human; Human Cell Line; Human Genome; Immune; Immune system; Investigation; Ionizing radiation; Knowledge; Laboratories; Left; Lesion; Maintenance; Malignant Neoplasms; Measures; Mediating; Meiosis; Metabolism; Minor; Mission; Modality; Molecular; Nonhomologous DNA End Joining; Nucleotides; Null Lymphocytes; Oncogenic; Organism; Outcome; Pathway interactions; Patients; Positioning Attribute; Predisposition; Process; Publishing; Radiation; Radiation induced damage; Radiation induced double strand break; Radiation therapy; Recording of previous events; Reliance; Rodent; Signal Transduction; Sister Chromatid; Somatic Cell; Syndrome; System; Systems Development; Testing; Therapeutic; Tumor Tissue; United States National Institutes of Health; Up-Regulation; Work; cell killing; chemotherapy; cost; design; gene repair; genetic analysis; homologous recombination; human DNA; inhibitor/antagonist; insight; loss of function; loss of function mutation; mutant; neoplastic cell; novel; positional cloning; repaired; research study; response; telomere; tumor

DESCRIPTION (provided by applicant): We propose to study the impact of loss-of-function mutations of DNA DSB (double-strand break) repair genes on end joining processes in human cells. We will focus heavily, but not exclusively, on the heterodimeric (Ku70 and Ku86 subunits) Ku DNA end-binding complex and LIGIII (DNA ligase III). Ku has a storied history and it has been known for decades that it is required for most of the generalized DSB repair occurring in somatic cells as well as being critical for the proper development of the vertebrate immune system. Ku performs these duties as part of a repair pathway referred to as C-NHEJ (classic-non-homologous end joining). In work published this year, we demonstrated that Ku has a hitherto underappreciated function as a suppressor of the other competing DSB repair pathways, namely A-NHEJ (alternative-NHEJ) and HR (homologous recombination). Thus, when the levels of Ku are reduced, A-NHEJ and HR, which normally comprise only a small fraction of the end joining activity in human cells, become more active. This observation has potential clinical implications. Thus, the vast majority of human cancer patients are treated either with radio- or chemotherapy (or both) and the efficacy of these anti-tumor modalities resides in their ability to generate DNA DSBs. Thus, strategies designed to increase the efficacy of tumor cell killing by radio- or chemotherapy that involve reducing the levels of Ku are likely to fail due to the up-regulation of A-NHEJ and/or HR. This is a hypothesis that we will directly test in this grant application. Moreover, the molecular mechanism by which Ku mediates this suppression is unknown and we propose genetic and reverse genetic experiments to elucidate this process. Finally, we have begun to genetically tackle the A-NHEJ pathway. A number of genes have been implicated in A-NHEJ, but the important genes remain poorly defined. One of the more compelling A- NHEJ candidates is LIGIII (DNA ligase III). To experimentally test the importance of LIGIII we have constructed a viable LIGIII-null human cel line and herein propose to characterize the impact of the loss-of- function of LIGIII on A-NHEJ. In particular, one characterization involves a novel and powerful assay system by which we can measure ionizing radiation-induced gross chromosomal rearrangements in human cells lacking LIGIII (or Ku86). To our knowledge, we are one of only a few laboratories in the world utilizing genetic, loss-of-function approaches to study DNA DSB in human cells and thus we are well positioned to gain insights into the mechanism of DSB repair that cannot be obtained elsewhere.

描述（由申请人提供）：我们计划研究DNA DSB（双链断裂）修复基因功能缺失突变对人类细胞末端连接过程的影响。我们将重点关注，但不限于异二聚体（Ku 70和Ku 86亚基）Ku DNA末端结合复合物和LIGIII（DNA连接酶III）。Ku具有传奇的历史，几十年来人们已经知道它是体细胞中发生的大多数广义DSB修复所必需的，并且对于脊椎动物免疫系统的正常发育至关重要。Ku作为被称为C-NHEJ（经典-非同源末端连接）的修复途径的一部分执行这些职责。在今年发表的工作中，我们证明了Ku作为其他竞争性DSB修复途径（即A-NHEJ（替代NHEJ）和HR（同源重组））的抑制剂具有迄今未被充分认识的功能。因此，当Ku的水平降低时，通常仅包含人细胞中末端连接活性的一小部分的A-NHEJ和HR变得更活跃。这一观察结果具有潜在的临床意义。因此，绝大多数人类癌症患者用放疗或化疗（或两者）治疗，并且这些抗肿瘤方式的功效在于它们产生DNA DSB的能力。因此，旨在通过涉及降低Ku水平的放疗或化疗来增加肿瘤细胞杀伤功效的策略可能会由于A-NHEJ和/或HR的上调而失败。这是我们将在本资助申请中直接测试的假设。此外，Ku介导这种抑制的分子机制是未知的，我们建议遗传和反向遗传实验来阐明这一过程。最后，我们已经开始从基因上解决A-NHEJ途径。许多基因与A-NHEJ有关，但重要的基因仍然定义不清。其中一个更引人注目的A-NHEJ候选者是LIGIII（DNA连接酶III）。为了实验性地测试LIGIII的重要性，我们构建了一种可行的LIGIII无效的人细胞系，并在此提出表征LIGIII功能丧失对A-NHEJ的影响。特别是，一个表征涉及一种新的和强大的测定系统，通过该系统，我们可以测量电离辐射诱导的总染色体重排缺乏LIGIII（或Ku 86）的人类细胞。据我们所知，我们是世界上仅有的几个利用遗传，功能丧失方法研究人类细胞中DNA DSB的实验室之一，因此我们有能力深入了解其他地方无法获得的DSB修复机制。