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: 8298501
• 负责人: ERIC A HENDRICKSON
• 金额: $ 30.73万
• 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/8298501
• 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）：我们建议研究 DNA DSB（双链断裂）修复基因的功能丧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（Ku70 和 Ku86 亚基）Ku DNA 末端结合复合物和 LIGIII（DNA 连接酶 III）。 Ku 有着悠久的历史，几十年来人们就知道它是体细胞中发生的大多数 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 作为称为 C-NHEJ（经典非同源末端连接）的修复途径的一部分履行这些职责。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））的抑制子，具有迄今为止未被充分认识的功能。因此，当 Ku 水平降低时，通常只占人类细胞末端连接活性一小部分的 A-NHEJ 和 HR 会变得更加活跃。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接受放疗或化疗（或两者兼而有之）治疗，这些抗肿瘤方式的功效在于它们产生 DNA DSB 的能力。因此，旨在通过放疗或化疗提高肿瘤细胞杀伤效力（涉及降低 Ku 水平）的策略可能会因 A-NHEJ 和/或 HR 的上调而失败。这是我们将在此拨款申请中直接测试的假设。此外，Ku介导这种抑制的分子机制尚不清楚，我们建议通过遗传和反向遗传实验来阐明这一过程。最后，我们开始从基因角度解决 A-NHEJ 通路问题。许多基因与 A-NHEJ 相关，但重要的基因仍不清楚。更引人注目的 A-NHEJ 候选者之一是 LIGIII（DNA 连接酶 III）。为了通过实验测试 LIGIII 的重要性，我们构建了可行的 LIGIII 缺失人类细胞系，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颖且强大的检测系统，通过该系统我们可以测量缺乏 LIGIII（或 Ku86）的人类细胞中电离辐射引起的总染色体重排。据我们所知，我们是世界上仅有的少数几个利用遗传、功能丧失方法来研究人类细胞中 DNA DSB 的实验室之一，因此我们有能力深入了解 DSB 修复机制，而这是其他地方无法获得的。