Ras18-mediated Fanconi Anemia pathway activation in response to camptothecin

描述(申请人提供)：这项提案的长期目标是了解DNA拓扑异构酶I(Top1)-靶向抗癌药物，如喜树碱(CPT)及其类似物-修复DNA损伤的分子机制。这些研究将确定新的机制，细胞通过这些机制将DNA修复与细胞周期进展结合起来，以维持基因组的稳定性。Top1通过在DNA复制、转录和染色质重塑等重要细胞过程中分解拓扑菌株，在维持基因组完整性方面发挥着重要作用。CPT共价捕获Top1-DNA共价复合体可导致复制和转录介导的致死性DNA损伤(如DSB)。然而，这些损伤修复的潜在机制还不是很清楚。初步研究表明，DNA修复蛋白Rad18与Fanconi贫血(FA)途径之间存在一种新的联系，以响应CPT诱导的DNA损伤。RAD18缺陷细胞或表达突变型RAD18的细胞E3连接酶活性缺陷，不能有效地将FANCD2(FA途径激活的关键成分)募集到染色质上，使细胞对CPT敏感。许多研究还表明，Rad18在细胞周期不同时相CPT诱导的DNA损伤修复中起重要作用。然而，将这些通路与细胞周期的特定阶段协调起来的分子网络却知之甚少。本研究旨在了解Rad18在Top1抑制剂诱导的DNA损伤中的作用及其对不同DNA修复途径的调控。在特定的Aim1项下提出的研究将集中于利用各种生化和遗传工具来确定Rad18激活FA途径以响应CPT的机制。具体目标2将验证Rad18和FA途径组件影响细胞从CPT诱导的DNA损伤中恢复的假设。RAD18和FA通路在调节S期检查点、分叉稳定性以及DNA复制的起始和延伸事件中所起的作用也将被确定。一些尖端技术，如蛋白质-DNA标记和分子梳理，将被用来检验这一假说。在特定目标3下的实验将确定Rad18在细胞周期不同阶段的作用，以响应Top1的CPT中毒。本研究的结果将有助于理解细胞维持基因组稳定的机制。对RAD18‘S与FA通路的相互作用及其在细胞周期中的调控的研究将可能揭示新的分子靶点，从而为加强Top1抑制剂对癌细胞的靶向性破坏提供新的治疗组合。

DESCRIPTION (provided by applicant): The long term goal of this proposal is to understand the molecular mechanisms that repair DNA damage induced by DNA topoisomerase I (Top1) -targeting anticancer drugs such as camptothecin (CPT) and its analogues. These studies will identify novel mechanisms by which cells integrate DNA repair with cell cycle progression to maintain genome stability. Top1 plays an important role in maintenance of genome integrity by resolving topological strain during vital cellular processes such as DNA replication, transcription and chromatin remodeling. Covalently trapping of Top1-DNA covalent complexes by CPT induces replication and transcription mediated lethal DNA lesions (such as DSBs). However, mechanisms underlying repair of these lesions are not well understood. Preliminary studies reported here, describe a novel connection between DNA repair protein Rad18 and Fanconi Anemia (FA) pathway in response to CPT induced DNA damage. The Rad18 deficient cells or cells expressing mutant Rad18 defective in E3 ligase activity, fail to efficiently recruit FANCD2 (a key component of FA pathway activation) to chromatin making cells hypersensitive to CPT. Many studies also suggest that Rad18 plays an important role in repair of CPT induced DNA damage in different phases of the cell cycle. However, the molecular networks that orchestrate these pathways with specific stages of the cell cycle are poorly understood. The specific aims in this proposal are designed to understand the roles of Rad18 in Top1 inhibitors induced DNA damage and its regulation of different DNA repair pathways. Studies proposed under Specific Aim1 will focus on determining mechanisms by which Rad18 activates FA pathway in response to CPT, using a variety of biochemical and genetic tools. The Specific Aim 2 will test the hypothesis that Rad18 and FA pathway components affect cells' recovery from CPT induced DNA damage. The roles that Rad18 and FA pathway play in regulation of S-phase checkpoint, fork stability, and the initiation and elongation events of DNA replication will also be determined. Some cutting edge techniques such as protein-DNA labeling and molecular combing will be used to test this hypothesis. Experiments under Specific Aim 3 will determine the roles of Rad18 in different phases of the cell cycle in response to CPT poisoning of Top1.Outcomes from this study will significantly contribute to understanding the mechanisms by which cells maintain genome stability. Proposed investigations into Rad18's interaction with FA pathway and its regulation during cell cycle will potentially reveal new molecular targets and thereby, novel therapeutic combinations for enhancing targeted destruction of cancer cells by Top1 inhibitors.