Regulation of DNA Double-Strand Break Repair

DNA双链断裂修复的调控

• 批准号: 6825277
• 负责人: William S. Dynan
• 金额: $ 26.38万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-26 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6825277
• 关键词: DNA damage; DNA repair; SDS polyacrylamide gel electrophoresis; crosslink; enzyme activity; enzyme complex; gel mobility shift assay; intermolecular interaction; ionizing radiation; nucleic acid probes; nucleic acid structure; p53 gene /protein; phosphorylation; protein kinase; surface plasmon resonance; synapses

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.

描述（由申请人提供）：从环境来源接触电离辐射是普遍的。 此外，大多数癌症患者接受放射治疗。 电离辐射部分通过诱导DNA双链断裂（DSB）杀死细胞。 DNA依赖性蛋白激酶（DNA-PK）对于调节DSB修复至关重要。 DNA-PK通过与断裂的DNA末端结合而激活，磷酸化，其他修复蛋白和信号分子（如p53）。 要测试的假设是DNA-PK具有决策或“检查点”功能。 建议DNA-PK最初与DNA末端结合以形成被捕的复合物。 一个DNA的突触与兼容，相反的DNA末端导致激酶激活和检查点释放，可能通过DNA-PK催化亚基自磷酸化。 有人提出，当无法进行进一步加工的情况下无法修复DSB时，DNA-PK还会感觉到，并通过募集和潜在地磷酸化的酶做出响应，这些酶是处理结扎端的末端所需的酶。 或者，如果该复合物保持不可修复，DNA-PK可能会在复合物或肿瘤抑制p53中磷酸化负调控位点。 三个特定的目的是：（1）表征当DNA-PK与孤立的DNA端结合时形成的初始复合物。 DNA-蛋白交联的点以及蛋白质和DNA对化学和酶探针的可及性模式的表征。 （2）表征检查点释放中涉及的机制。 相对于初始复合物，蛋白质-DNA接触和蛋白质构象的变化将被鉴定出来。 将确定突触复合物中DNA-PKCS磷酸化的位点并研究了其功能。 （3）表征在DNA端形成的DNA-PK复合物，该端需要在修复前进行处理。 复合物将在具有发夹末端的寡核苷酸上形成，因此在没有核解性处理的情况下无法修复。 这种复合物能够招募和磷酸化发夹核酸内切酶Artemis的能力。 在没有p53等阿耳emi虫的情况下，使用替代底物的使用将进行研究。 一个总体目标是获得对DSB修复调节的基本见解，这将导致基于机制的新策略，以提高放射治疗的疗效。