Repair of DNA double-strand breaks with damaged ends

描述(申请人提供)：自由基介导的DNA双链断裂(DSB)由电离辐射、仿射天然产物和氧化应激诱导，具有化学修饰的末端，如32-磷酸乙醇酸盐，必须在断裂可以重新连接之前移除。此外，这些断裂的准确重新连接需要用对齐的DSB末端的缺口填充来替换碎裂的核苷酸。辐射诱导的双链断裂还经常伴随着附近的氧化碱基损伤，这可能会干扰重新连接。由拓扑异构酶抑制剂诱导的双链断裂具有蛋白质连接的末端，同样必须移除以进行修复。拟议项目的总体目标是确定这些受损的DSB末端如何通过非同源末端连接来解决修复问题，以及它们如何影响整个连接过程。处理受损末端的候选酶包括酪氨酸二酯酶(Tdp1)和DNA末端特异性内切酶Mre11、Artemis、Metnase和CtIP。在Artemis、Metnase、Tdp1、Mre11及其组合中存在遗传缺陷的小鼠和人类细胞将通过siRNA敲除来增强，以产生具有各种末端加工缺陷组合的细胞。这些细胞将接受细胞毒性、细胞遗传学和DSB修复分析，以及新开发的实时聚合酶链式反应(Real-time PCR)分析，以衡量封闭末端在细胞中的持久性。这些研究将确定这些修复因子是否提供了解决受阻DNA末端的替代途径，以及它们之间的重叠程度。确定的DSB底物，轴承末端在不同的环境中带有32-磷酸乙醇酸盐或氧化修饰的碱，将被用来确定Artemis和Metnase在修剪受损末端方面的特异性。由此产生的修剪末端进行到细胞提取液中的缺口填充和连接步骤的效率将被确定。将确定DNA聚合酶在排列的DSB末端填充缺口时对受损碱基的耐受性，以及聚合酶和Artemis在解决不同类型的受损DNA末端方面可能的合作和竞争。 与公共卫生相关：由于放射治疗和某些类型的化疗通过诱导DNA双链断裂来杀死肿瘤细胞，因此在这种断裂末端处理损伤的酶是潜在的治疗靶点，可以被利用来提高疗效和治疗指数。此外，一些修复酶在去除末端损伤的过程中会删除正常DNA的片段，因此修复的准确性可能取决于所使用的特定修复酶，这是拟议研究的重点。由于不准确的双链断裂修复可能导致基因组变化，从而促进恶性肿瘤，因此在癌症预防方面，了解细胞如何为特定类型的双链断裂选择修复酶和修复系统是至关重要的。

DESCRIPTION (provided by applicant): Free radical-mediated DNA double-strand breaks (DSBs) induced by ionizing radiation, radiomimetic natural products, and oxidative stress have chemically modified termini such as 32-phosphoglycolates that must be removed before the break can be rejoined. In addition, accurate rejoining of these breaks requires replacement of fragmented nucleotides by gap filling on aligned DSB ends. Radiation-induced DSBs are also often accompanied by nearby oxidative base damage that can interfere with rejoining. DSBs induced by topoisomerase inhibitors have protein-linked termini that likewise must be removed for repair. The overall goal of the proposed project is to determine how these damaged DSB ends are resolved for repair by nonhomologous end joining, and how they affect the overall joining process. Candidate enzymes for processing damaged ends include tyrosyl-DNA phosphodiesterase (TDP1) and the DNA end-specific endonucleases Mre11, Artemis, Metnase and CtIP. Mouse and human cells with genetic defects in Artemis, Metnase, TDP1, Mre11 and combinations thereof will be augmented with siRNA knockdown to generate cells with various combinations of end-processing deficiencies. These cells will be subjected to cytotoxicity, cytogenetic and DSB repair assays, as well as a newly developed real-time PCR assay for measuring the persistence of blocked termini in cells. These studies will determine whether these repair factors provide alternative pathways for resolving blocked DNA termini, and the degree of overlap between them. Defined DSB substrates, bearing ends with 32-phosphoglycolates or oxidatively modified bases in various contexts, will be used to determine the specificities of Artemis and Metnase in trimming damaged ends. The efficiency with which the resulting trimmed ends progress to the gap filling and ligation steps in cell extracts will be determined. Tolerance for damaged bases in gap-filling on aligned DSB ends by DNA polymerase ; will be determined, as well as the possible cooperation and competition between polymerase ; and Artemis in resolving different types of damaged DNA termini. PUBLIC HEALTH RELEVANCE: Because radiotherapy and some types of chemotherapy kill tumor cells by inducing DNA double- strand breaks, enzymes that process damage at the ends of such breaks represent potential therapeutic targets that could be exploited to improve efficacy and therapeutic index. Moreover, some repair enzymes delete segments of normal DNA in the process of removing damage from the ends, and therefore the accuracy of repair can depend on the specific repair enzymes used, which is the focus of the proposed research. Because inaccurate double-strand break repair can lead to genomic alterations that promote malignancy, it is essential in terms of cancer prevention to understand how the choice of repair enzymes and repair systems is made by the cell for specific types of double-strand breaks.