Repair of DNA double-strand breaks with damaged ends

描述（由申请人提供）：电离辐射、拓扑异构酶抑制剂、拟放射性药物以及在某种程度上所有基于自由基的遗传毒素均诱导DNA双链断裂（DSB），这些断裂在末端或末端附近具有缺失或受损的碱基、断裂的糖和其他修饰。这些DSB不能简单地重新连接，而是需要复杂的加工才能重新连接，从而增强其潜在的细胞毒性、致染色体断裂和致突变作用。在G1和G 0期细胞中，修复这种断裂的主要途径是非同源末端连接，它几乎可以连接任何两个DNA末端，而不管末端序列和结构如何。该途径的体外模型已被开发，其结合了定义的位点特异性标记的底物、人细胞的核提取物和纯化的蛋白质。这种体外系统能够准确地恢复原始DNA序列的网站上的一个模型交错的自由基介导的DSB，尽管丢失的核苷酸和末端封闭的两端在两条链。所提出的研究的主要目标是阐明该过程的生物化学细节，特别是（i）酪氨酰-DNA磷酸二酯酶（hTdpl）、Artemis和Werner综合征因子（Werner’s syndrome factor，Werner’s syndrome factor，Werner’s syndrome factor，Werner’s syndrome factor）在突出和/或凹陷的3’-磷酸乙醇酸末端加工中的作用，（ii）其他末端连接因子对这种加工的调节，和（iii）在对齐的DSB末端上精确填补缺口的结构要求，包括对氧化修饰的碱基的耐受性和DNA聚合酶λ的特征使其能胜任该过程。作为这些研究的补充，将在用新制癌素和加利车霉素（特异性诱导具有突出的3 '-磷酸乙醇酸末端的DSB的试剂）处理的正常和hTdpl缺陷型淋巴母细胞中检查DSB修复和细胞存活，以评估其在体内修复中的作用。这些研究可能有助于利用各种末端连接因子作为DSB修复的药理学操作的靶点，最终目标是提高放疗/化疗的疗效，并最大限度地减少DSB的遗传毒性作用。

DESCRIPTION (provided by applicant): Ionizing radiation, topoisomerase inhibitors, radiomimetic drugs, and to some extent all free radical-based genotoxins, induce DNA double-strand breaks (DSBs) that have missing or damaged bases, fragmented sugars, and other modifications at or near the termini. These DSBs cannot be simply religated, but rather require complex processing in order to be rejoined, thus enhancing their potential cytotoxic, clastogenic and mutagenic effects. In G1 and G0-phase cells, the primary pathway for repair of such breaks is nonhomologous end joining, which can join virtually any two DNA ends, regardless of terminal sequence and structure. An in vitro model of this pathway has been developed that combines defined site-specifically labeled substrates, nuclear extracts of human cells, and purified proteins. This in vitro system is capable of accurately restoring the original DNA sequence at the site of a model staggered free radical-mediated DSB, despite missing nucleotides and terminally blocked ends in both strands. The primary goal of the proposed studies is to elucidate the biochemical details of this process, particularly (i) the roles of tyrosyI-DNA phosphodiesterase (hTdpl), Artemis, and the Werner's syndrome factor (Wrn) in processing of protruding and/or recessed 3'-phosphoglycolate termini, (ii) the regulation of such processing by other end-joining factors, and (iii) the structural requirements for accurate gap filling on aligned DSB ends, including the tolerance for oxidatively modified bases and the features of DNA polymerase lambda render it competent for this process. As a complement to these studies, DSB repair and cell survival will be examined in normal and hTdpl-deficient lymphoblastoid cells treated with neocarzinostatin and calicheamicin, agents that specifically induce DSBs with protruding 3'-phosphoglycolate termini, to assess its role in repair in vivo. Such studies may aid in the exploitation of the various end-joining factors as targets for the pharmacological manipulation of DSB repair, with the ultimate goal of improving the efficacy of radio/chemotherapy, and minimizing the genotoxic effects of DSBs.