Regulation of DNA Double-Strand Break Repair

描述（由申请人提供）：暴露于环境源的电离辐射是普遍的。此外，大多数癌症患者接受放射治疗。电离辐射杀死细胞，部分原因是通过诱导DNA双链断裂（DSBs）。dna依赖性蛋白激酶（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p53等信号分子磷酸化。需要验证的假设是，DNA-PK具有决策或“检查点”功能。有人提出，DNA- pk最初结合到DNA末端形成一个阻滞复合物。一个DNA末端与另一个兼容的、相反的DNA末端的突触可能通过DNA- pk催化亚基自磷酸化导致激酶激活和检查点释放。有人提出，DNA-PK也能感知到DSB在没有进一步加工的情况下无法修复，并通过招募和潜在的磷酸化酶来响应，这些酶是处理末端进行结扎所需的。或者，如果复合体仍然无法修复，DNA-PK可能使复合体或肿瘤抑制因子p53中的负调控位点磷酸化。三个具体目的是：(1)表征DNA- pk与分离的DNA末端结合时形成的初始复合物。DNA-蛋白质交联点以及蛋白质和DNA对化学和酶探针的可及性模式将被表征。(2)描述检查点释放的机制。相对于初始复合物，将鉴定突触复合物中蛋白质- dna接触和蛋白质构象的变化。将鉴定突触复合体中DNA-PKcs磷酸化位点并研究其功能。(3)表征DNA末端形成的DNA- pk复合物，该复合物在修复前需要加工。配合物将在具有发夹末端的寡核苷酸上形成，因此不进行核分解处理是无法修复的。这种复合物招募和磷酸化发夹核酸内切酶Artemis的能力将被表征。在没有Artemis的情况下使用替代底物，如p53，将进行研究。总体目标是获得DSB修复调节的基本见解，这将导致新的，基于机制的策略，以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.