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

基本信息

批准号：
8527889
负责人：
ERIC A HENDRICKSON
金额：
$ 1.55万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
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/8527889
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

项目摘要

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）修复基因对人类细胞中最终连接过程的功能丧失突变的影响。我们将重点（但不是只专注于异二聚体（KU70和KU86亚基）KU DNA末端结合复合物和ligiii（DNA连接酶III）。 KU具有悠久的历史，数十年来，大多数在体细胞中发生的广义DSB修复需要，这对于脊椎动物免疫系统的适当发展至关重要。 KU执行这些职责，作为所谓的C-NHEJ（经典非同源结局）的维修途径的一部分。在今年发表的工作中，我们证明了KU迄今为止的功能不足，作为其他竞争性DSB修复途径的抑制器，即A-NHEJ（替代性NHEJ）和HR（同源重组）。因此，当Ku的水平降低时，通常仅包含在人类细胞中结合活性的一小部分的A-NHEJ和HR变得更加活跃。该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了放射性或化学疗法（或两者兼而代之）的治疗，而这些抗肿瘤模式的功效则取决于其产生DNA DSB的能力。因此，由于A-NHEJ和/或HR的上调，涉及降低KU水平的放射性或化学疗法杀死肿瘤细胞杀死的疗效的策略可能会失败。这是一个假设，我们将直接在此赠款应用程序中进行测试。此外，KU介导这种抑制的分子机制尚不清楚，我们提出了遗传和反向遗传实验以阐明这一过程。最后，我们开始从基因上解决A-NHEJ途径。许多基因与A-NHEJ有关，但重要基因的定义仍然很差。 Ligiii（DNA连接酶III）是最引人注目的A-NHEJ候选者之一。为了通过实验测试Ligiii的重要性，我们构建了一个可行的Ligiii-null人类CEL系，并在此提议表征Ligiii对A-NHEJ的功能丧失的影响。特别是，一种表征涉及一个新颖而强大的测定系统，通过该系统，我们可以在缺乏LiGIII（或KU86）的人类细胞中测量辐射诱导的总染色体重排。据我们所知，我们是世界上仅有的少数实验室之一，利用遗传，功能丧失方法来研究人类细胞中的DNA DSB，因此我们可以很好地了解无法获得其他地方无法获得的DSB修复机制。