Repair of DNA double-strand breaks with damaged ends

描述（由申请人提供）：电离辐射、拟辐射天然产物和氧化应激诱导的自由基介导的DNA双链断裂（DSBs）具有化学修饰的末端，如32-磷酸乙醇酸，必须在断裂重新连接之前去除。此外，这些断裂的精确重新连接需要通过在对齐的DSB端上填充间隙来替换碎片化的核苷酸。辐射诱发的dsb也经常伴有附近的氧化碱基损伤，从而干扰再连接。由拓扑异构酶抑制剂诱导的dsb具有蛋白质连接的末端，同样必须去除以进行修复。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进行修复，以及它们如何影响整个连接过程。用于处理受损末端的候选酶包括酪氨酸-DNA磷酸二酯酶（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敲低增强，以产生具有各种末端加工缺陷组合的细胞。这些细胞将进行细胞毒性、细胞遗传学和DSB修复试验，以及一项新开发的实时PCR试验，用于测量细胞中阻断末端的持久性。这些研究将确定这些修复因子是否为解决阻断的DNA末端提供了替代途径，以及它们之间的重叠程度。定义的DSB底物，在不同的情况下带有32-磷酸乙醇酸酯或氧化修饰的碱基，将用于确定Artemis和Metnase在修剪受损末端时的特异性。由此产生的修剪结束进展到间隙填充和细胞提取物的连接步骤的效率将被确定。DNA聚合酶对DSB端缝隙填充损伤碱基的耐受性将确定，以及聚合酶之间可能的合作与竞争；和阿尔忒弥斯解决不同类型的受损DNA末端。

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.