CELLULAR RESPONSE TO TOPOISOMERASE I

DNA拓扑异构酶I（Topi）在DNA复制、转录和重组中起重要作用 并且也是喜树碱（CRT）的靶点，FDA批准的喜树碱类似物是治疗糖尿病的有效新药。 治疗人类癌症。CRT通过可逆地稳定共价酶-DNA复合物来毒害Topi。 在S期，复制叉与CPT-Top1-DNA加合物的碰撞产生DMA损伤， 信号细胞周期停滞和细胞死亡。虽然普遍认为Topi靶向药物诱导DNA 在S期的损伤中，很明显，响应损伤而激活的信号通路最终决定了 细胞命运使用酵母作为模型系统，复制机制的保守组分CDC 45 和DPB 11（TopBP 1），保护细胞免受Topi损伤。雷帕霉素敏感的TOR信号传导也保护 酵母细胞的细胞毒性DNA损伤在S期。我们的数据支持一个模型，即TOR作为一个 通过维持复制叉稳定性和dNTP库响应遗传毒性应激的存活途径 这是易错跨损伤DNA聚合酶所必需的。因此，TOR依赖性细胞存活响应于 DNA损伤剂与增加的突变率相一致，这可能有助于获得 耐药性 提出了三个具体的目标来研究复制机制和TOR的保守方面 维持细胞存活的信号，以响应细胞毒性剂，例如CRT。在目标1中， 酵母遗传学和染色质免疫沉淀查询高密度平铺阵列（ChIP芯片实验） 将研究雷帕霉素敏感的TOR信号维持复制叉的机制， 稳定性和调节DNA损伤诱导的诱变。目的2提出确定雷帕霉素是否- 敏感的mTOR信号传导调节人细胞对细胞毒性化疗药物的敏感性， 抗药性的证据。DNA纤维标记策略将确定雷帕霉素治疗是否影响复制叉 在DNA损伤的存在下的进展和稳定性，而诱导的DNA损伤的程度将是 通过yH 2AX染色来定义。基于siRNA的方法将确定S期检查点功能是否 这是mTOR保护功能所必需的。在目标3中，对合成致命相互作用的分析将定义 保守的人类DNA复制蛋白，CDC 45和TopBPI，在调节 细胞对CRT和雷帕霉素的敏感性。这些研究将为职权范围的职能提供重要的见解 在调节细胞对DNA损伤的反应的途径，而将影响临床发展， 雷帕霉素与拓扑异构酶L靶向治疗剂的组合。阻断药物诱导的 赋予耐药性的突变代表了雷帕霉素的独特应用，其对于以下疾病具有临床重要性： 儿科恶性肿瘤的治疗

DMA topoisomerase I (Topi) plays important roles in DMAreplication, transcription and recombination and is also the target of camptothecin (CRT), FDA approved analogs of which are effective new agents in the treatment of human cancers. CRT poisons Topi by reversibly stabilizing a covalent enzyme-DNA complex. During S-phase, the collision of replication forks with CPT-Top1-DNA adducts produces DMAlesions that signal cell cycle arrest and cell death. Although it is generally accepted that Topi targeted drugs induce DNA damage in S-phase, it is clear that signaling pathways activated in response to damage ultimately dictate cellular fate. Using yeast as a model system, conserved components of the replication machinery, CDC45 and DPB11(TopBP1), protect cells from Topi damage. Rapamycin-sensitive TOR signaling also protects yeast cells from cytotoxic DNA lesions during S-phase. Our data support a model whereby TOR acts as a survival pathway in response to genotoxic stress by maintaining replication fork stability and the dNTP pools necessary for error-prone translesion DNA polymerases. Thus, TOR-dependent cell survival in response to DNA damaging agents coincides with increased mutation rates, which may contribute to the acquisition of drug resistance. Three specific aims are proposed to investigate conserved aspects of the replication machinery and TOR signaling that maintain cell survival in response to cytoxic agents, suca at CRT. In Aim 1, a combination of yeast genetics and chromatin immunoprecipitates to query high-density tiling arrays (ChlP-chip experiments) will investigate the mechanism by which rapamycin-sensitive TOR signaling maintains replication fork stability and regulates DNA damage-induced mutagenesis. Aim 2 proposes to determine if rapamycin- sensitive mTOR signaling regulates human cell sensitivity to cyotoxic chemotherapeutics and the acquisition of drug resistance. A DNA fiber labeling strategy will determine if rapamycin treatment affects replication fork progression and stability in the presence of DNA damage, while the extent of DNA damage induced will be defined by yH2AX staining. siRNA-based approaches will determine if S-phase checkpoint function is required for the protective function of mTOR. In Aim 3, an analysis of synthetic lethal interactions will define pathway interactions of the conserved human DNA replication proteins, CDC45 and TopBPI, in regulating cell sensitivity to CRT and rapamycin. These studies will provide critical insights into the function of the TOR pathway in modulating cellular responses to DNA damage, while will impact the clinical development of rapamycin in combination with topoisomerase l-targeted therapeutics. The potential to block drug-induced mutations that confer resistance represents a unique application of rapamycins with clinical importance for the treatment of pediatric malignancies.