Repair of DNA double-strand breaks with damaged ends

描述（由申请方提供）：电离辐射、拟放射性天然产物和氧化应激诱导的自由基介导的DNA双链断裂（DSB）具有化学修饰的末端，如32-磷酸乙醇酸酯，在断裂重新连接之前必须去除。此外，这些断裂的精确重新连接需要通过对齐的DSB末端上的缺口填充来替换片段化的核苷酸。辐射诱导的DSB也经常伴随着附近的氧化性碱基损伤，这可能会干扰重新连接。由拓扑异构酶抑制剂诱导的DSB具有蛋白质连接的末端，其同样必须被移除以进行修复。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酰-DNA磷酸二酯酶（TDP 1）和DNA末端特异性内切核酸酶Mre 11、Artemis、Metnase和CtIP。在Artemis、Metnase、TDP 1、Mre 11及其组合中具有遗传缺陷的小鼠和人细胞将用siRNA敲低增强，以产生具有末端加工缺陷的各种组合的细胞。这些细胞将接受细胞毒性、细胞遗传学和DSB修复试验，以及新开发的实时PCR试验，用于测量细胞中封闭末端的持久性。这些研究将确定这些修复因子是否为解决阻断的DNA末端提供替代途径，以及它们之间的重叠程度。定义的DSB底物，在各种情况下具有32-磷酸乙醇酸酯或氧化修饰的碱基的末端，将用于确定Artemis和Metnase在修剪受损末端中的特异性。将测定所得修剪末端在细胞提取物中进行差距填充和连接步骤的效率。将确定DNA聚合酶在对齐的DSB末端的缺口填充中对受损碱基的耐受性，以及聚合酶和Artemis在解析不同类型的受损DNA末端时可能的合作和竞争。

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.