Repair of DNA double-strand breaks with damaged ends

说明(申请人提供)：电离辐射、拓扑异构酶抑制剂、仿射药物，在某种程度上，以及所有基于自由基的遗传毒素，都会导致DNA双链断裂(DSB)，碱基缺失或损坏，糖链碎裂，以及末端或附近的其他修饰。这些DSB不能简单地宗教化，而是需要复杂的处理才能重新加入，从而增强它们潜在的细胞毒性、致分裂和致突变作用。在G1和G0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，而无论末端序列和结构如何。这一途径的体外模型已经被开发出来，它结合了确定的位点特异性标记底物、人类细胞的核提取物和纯化的蛋白质。这个体外系统能够准确地在交错的自由基介导的DSB模型的位置恢复原始的DNA序列，尽管两条链上都缺少核苷酸和末端被阻断。拟议研究的主要目的是阐明这一过程的生化细节，特别是(I)酪氨酸I-DNA磷酸二酯酶(HTdpl)、Artemis和Werner综合征因子(WRN)在加工突出和/或凹陷的3‘-磷酸乙醇酸末端中的作用，(Ii)其他末端连接因子对这种加工的调节，以及(Iii)准确填充排列的DSB末端缺口的结构要求，包括对氧化修饰碱基的耐受性和DNA聚合酶lambda的特性使其能够胜任这一过程。作为这些研究的补充，DSB修复和细胞存活将在正常和hTdpl缺陷的淋巴母细胞中进行检测，以评估其在体内修复中的作用。这类研究可能有助于开发各种末端连接因子作为DSB修复的药理操作靶点，最终目的是提高放/化疗的疗效，并将DSB的遗传毒性效应降至最低。

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.