DNA Mismatch and Double-Strand Break Repair

描述（由申请人提供）：同源重组是一种允许细胞耐受各种DNA损伤剂产生的DNA损伤的必要机制。长期目标是确定顺铂、一氧化氮和甲基化剂等药物诱导DNA双链断裂形成及其修复的机制。我们首次使用单细胞微凝胶电泳证明了顺铂诱导这种断裂的形成，并且在第一个目标中提出了检查其他药物的能力，并检查DNA复制在该过程中的作用。在第二个目标中，将寻找一类依赖同源重组存活的新型突变细胞，特别是它们对DNA损伤剂的反应。肿瘤细胞中DNA错配修复的缺失导致耐药性，相反，错配修复的熟练程度导致药物致敏。第三个目标是基于发现关键错配修复蛋白MutS的c端是药物致敏所必需的，并且提出了实验来确定MutS的多聚态是否起作用。我们最近表明，顺铂诱导的重组和双链断裂修复需要DNA聚合酶I，在第四个目标中，我们将通过灭活其外切酶或聚合酶活性来确定其在这些过程中的作用。我们只能使用大肠杆菌进行这些研究，因为我们对DNA复制、修复和重组的了解比其他任何生物体都多，而且它的基因组有能力产生多种突变。这项基础研究影响了临床相关的几个领域，包括抗肿瘤药物杀死细胞的机制以及耐药肿瘤如何在癌症化疗中出现。它还影响致病菌对抗生素产生耐药性的机制以及这种耐药性如何传播。

DESCRIPTION (provided by applicant): Homologous recombination is a mechanism that is essential to allow cells to tolerate DNA damage produced by various DNA damaging agents. The long term goals are to define the mechanisms by which agents such as cisplatin, nitric oxide and methylators induce the formation of DNA double-strand breaks and their repair. We have demonstrated, for the first time, using single cell microgel electrophoresis that cisplatin induces the formation of such breaks and it is proposed in the first aim to examine the other agents for their ability to do so and to examine the role of DNA replication in the process. In the second aim, a new class of mutant cells which are dependent on homologous recombination for survival will be sought and characterized especially for their response to DNA damaging agents. Loss of DNA mismatch repair in tumor cells results in drug resistance while, conversely, mismatch repair proficiency leads to drug sensitization. The third aim is based on the finding that the C-terminal end of a key mismatch repair protein, MutS, is needed for drug sensitization and experiments are proposed to determine if the multimeric state of MutS is responsible. We recently showed that cisplatin-induced recombination and double-strand break repair require DNA polymerase I and in the fourth aim, we will determine its role in these processes by inactivating either its exonuclease or polymerase activities. We can use only E. coli for these studies because more is known about DNA replication, repair and recombination than in any other organism and because of the ability to construct multiple mutations in its genome. This basic research impacts several areas of clinical relevance, including mechanisms by which antitumor agents kill cells and how drug-resistant tumors emerge in cancer chemotherapy. It also impacts the mechanism by which pathogenic bacteria become resistant to antibiotics and how this resistance is disseminated.