Regulation of DNA Double-Strand Break Repair

描述(申请人提供)：暴露于环境来源的电离辐射是普遍现象。此外，大多数癌症患者都会接受放射治疗。电离辐射通过诱导DNA双链断裂(DSB)部分地杀死细胞。DNA依赖蛋白激酶(DNA-PK)在DSB修复的调控中起着至关重要的作用。DNA-PK通过与断裂的DNA末端结合而激活，使自身、其他修复蛋白和信号分子(如p53)磷酸化。需要检验的假设是，DNA-PK具有决策或“检查点”功能。有人认为，DNA-PK最初与DNA末端结合形成滞留复合体。一个DNA末端与一个相容的、相反的DNA末端的突触可能通过DNA-PK催化亚基的自动磷酸化而导致激酶激活和检查点释放。有人认为，DNA-PK还可以感知DSB在不进行进一步处理的情况下无法修复的情况，并通过招募并潜在地磷酸化处理末端连接所需的酶来做出反应。或者，如果复合体仍然不可修复，DNA-PK可能会磷酸化复合体或抑癌基因P53中的负调控位点。三个具体的目标是：(1)表征当DNA-PK与分离的DNA末端结合时形成的初始复合体。将描述DNA-蛋白质的交联点以及蛋白质和DNA对化学和酶探针的可及性。(2)描述检查点释放所涉及的机制。相对于最初的复合体，突触复合体中蛋白质-DNA接触和蛋白质构象的变化将被识别。DNA-PKcs在突触复合体中的磷酸化位置将被确定，并对其功能进行研究。(3)鉴定在DNA末端形成的DNA-PK复合体，该复合体需要在修复之前进行处理。复合体将在有发夹末端的寡核苷酸上形成，因此如果不进行核溶解处理就无法修复。这个复合体招募和磷酸化发夹核酸内切酶Artemis的能力将被表征。将调查在没有Artemis的情况下使用替代底物，如P53。一个总体目标是获得对DSB修复调节的基本见解，这将导致新的、基于机制的策略来提高放射治疗的疗效。

DESCRIPTION (provided by applicant): Exposure to ionizing radiation from environmental sources is universal. In addition, most cancer patients receive radiation therapy. Ionizing radiation kills cells, in part, by inducing DNA double-strand breaks (DSBs). The DNA-dependent protein kinase (DNA-PK) is crucial to regulation of DSB repair. DNA-PK, which is activated by binding to broken DNA ends, phosphorylates itself, other repair proteins, and signaling molecules such as p53. The hypothesis to be tested is that DNA-PK has a decision-making, or "checkpoint" function. It is proposed that DNA-PK binds initially to DNA ends to form an arrested complex. Synapsis of one DNA end with a compatible, opposing, DNA end leads to kinase activation and checkpoint release, probably via DNA-PK catalytic subunit autophosphorylation. It is proposed that DNA-PK also senses when a DSB cannot be repaired without further processing and responds by recruiting, and potentially phosphorylating, enzymes that are required to process the ends for ligation. Alternatively, if the complex remains unrepairable, DNA-PK may phosphorylate negative regulatory sites in the complex or in tumor suppressor p53. Three specific aims are: (1) To characterize the initial complex formed when DNA-PK binds to an isolated DNA end. Points of DNA-protein crosslinking and the pattern of accessibility of protein and DNA to chemical and enzymatic probes will be characterized. (2) To characterize mechanisms involved in checkpoint release. Changes in protein-DNA contacts and protein conformation in the synaptic complex, relative to the initial complex, will be identified. Sites of DNA-PKcs phosphorylation in the synaptic complex will be identified and their function investigated. (3) To characterize the DNA-PK complex formed on a DNA end that requires processing prior to repair. Complexes will be formed on an oligonucleotide that has a hairpin terminus and is thus unrepairable without nucleolytic processing. The ability of this complex to recruit and phosphorylate the hairpin endonuclease, Artemis, will be characterized. The use of alternative substrates in the absence of Artemis, such as p53, will be investigated. An overall goal is to obtain basic insights into the regulation of DSB repair that will lead to new, mechanism-based strategies for increasing the efficacy of radiation therapy.