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

描述(申请人提供)：我们建议研究DNADSB(双链断裂)修复基因功能丧失突变对人类细胞末端连接过程的影响。我们将重点关注异二聚体(Ku70和Ku86亚基)Ku DNA末端结合复合体和LIGIII(DNA连接酶III)。Ku有着悠久的历史，几十年来人们已经知道，它是发生在体细胞中的大多数广义DSB修复所必需的，也是脊椎动物免疫系统正常发育的关键。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作为其他竞争的DSB修复途径，即A-NHEJ(替代-NHEJ)和HR(同源重组)的抑制者，具有到目前为止被低估的功能。因此，当Ku水平降低时，A-NHEJ和HR变得更加活跃，它们通常只占人类细胞末端连接活性的一小部分。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要么接受放射治疗，要么接受化疗(或两者兼而有之)，而这些抗肿瘤方式的有效性取决于他们产生DNA DSB的能力。因此，由于A-NHEJ和/或HR的上调，旨在通过放化疗提高肿瘤细胞杀伤力的策略可能会失败，这些策略涉及降低Ku水平。这是一个假设，我们将在这次拨款申请中直接测试。此外，Ku介导这种抑制的分子机制尚不清楚，我们建议进行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。已有多个基因与A-NHEJ有关，但重要的基因仍不明确。最引人注目的A-NHEJ候选之一是LIGIII(DNA连接酶III)。为了实验测试LIGIII的重要性，我们构建了一个可行的LIGIII缺失的人类细胞系，并在此建议表征LIGIII功能丧失对A-NHEJ的影响。特别是，一个特征涉及一个新的和强大的检测系统，通过它我们可以测量缺乏LIGIII(或Ku86)的人细胞中电离辐射诱导的总染色体重排。据我们所知，我们是世界上少数几个使用遗传、功能丧失方法来研究人类细胞中DNADSB的实验室之一，因此我们处于有利地位，能够获得其他地方无法获得的DSB修复机制的见解。

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.