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 也是喜树碱(CRT)的靶点，FDA批准的类似物是治疗癌症的有效新药 人类癌症的治疗。CRT通过可逆地稳定共价酶-DNA复合体而毒害TOPI。 在S期，复制叉与CPT-Top1-DNA加合物的碰撞产生的DNA损伤 发出细胞周期停止和细胞死亡的信号。尽管人们普遍认为TOPI靶向药物可以诱导DNA 在损伤的S阶段，很明显，响应损伤而激活的信号通路最终决定了 细胞的命运。以酵母为模型系统，复制机制的保守组件CDC45 和DPB11(TopBP1)，保护细胞免受TOPI损伤。雷帕霉素敏感的TOR信号也能保护 S期细胞毒性DNA损伤中的酵母细胞。我们的数据支持这样一种模型，即TOR充当 通过维持复制叉稳定性和dNTP池响应遗传毒性应激的生存途径 对于容易出错的跨损伤DNA聚合酶来说是必要的。因此，依赖TOR的细胞存活对 DNA损伤剂与突变率增加不谋而合，这可能有助于获得 抗药性。 提出了三个具体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，以响应细胞毒剂，如CRT。在目标1中，组合了 酵母遗传学和染色质免疫沉淀物查询高密度平铺阵列(ChlP芯片实验) 将研究雷帕霉素敏感的TOR信号维持复制分叉的机制 稳定性，并调节DNA损伤诱导的突变。目标2建议确定雷帕霉素- 敏感的mTOR信号调节人细胞对环毒化疗药物的敏感性和获得性 抗药性。DNA纤维标记策略将确定雷帕霉素治疗是否影响复制分叉 在DNA损伤存在时的进行性和稳定性，而诱导的DNA损伤的程度将是 由yH2 AX染色确定。基于小干扰RNA的方法将确定S阶段的检查点功能是否 对于mTOR的保护功能是必需的。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CDC45和TopBPI在调控中的相互作用 细胞对CRT和雷帕霉素的敏感性。这些研究将为TOR的功能提供关键的见解 调节细胞对DNA损伤的反应的途径，同时将影响临床的发展 雷帕霉素联合拓扑异构酶L靶向治疗。阻断药物诱导的可能性 赋予耐药性的突变代表了雷帕霉素的独特应用，具有临床意义 儿童恶性肿瘤的治疗。

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.