Regulation of DNA Double-Strand Break Repair

描述（由申请人提供）：暴露于环境来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，部分是通过诱导DNA双链断裂（DSB）。 DNA依赖性蛋白激酶（DNA-PK）是DSB修复的关键调节因子。 DNA-PK通过与断裂的DNA末端结合而被激活，使自身、其他修复蛋白和信号分子（如p53）磷酸化。 待检验的假设是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，形成一个被捕的复合物。 一个DNA末端与相容的相反DNA末端的突触导致激酶活化和检查点释放，可能通过DNA-PK催化亚基自磷酸化。 有人提出，DNA-PK还感测DSB何时不能在没有进一步处理的情况下被修复，并通过招募和潜在磷酸化处理连接末端所需的酶来响应。 或者，如果复合物仍然不可修复，DNA-PK可以磷酸化复合物或肿瘤抑制因子p53中的负调控位点。 三个具体目标是：（1）表征DNA-PK与分离的DNA末端结合时形成的初始复合物。 DNA-蛋白质交联点和蛋白质和DNA对化学和酶探针的可及性模式将被表征。 (2)描述检查点释放中涉及的机制。 将鉴定突触复合物中蛋白质-DNA接触和蛋白质构象相对于初始复合物的变化。 将鉴定突触复合物中DNA-PKcs磷酸化的位点并研究其功能。 (3)表征在修复前需要处理的DNA末端形成的DNA-PK复合物。 复合物将在具有发夹末端的寡核苷酸上形成，并且因此在没有核酸裂解加工的情况下不可修复。 该复合物募集和磷酸化发夹核酸内切酶Artemis的能力将被表征。 将研究在不存在Artemis的情况下使用替代底物，如p53。 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.