Ras18-mediated Fanconi Anemia pathway activation in response to camptothecin

描述（由申请人提供）：本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）及其类似物诱导的DNA损伤修复的分子机制。这些研究将确定细胞将DNA修复与细胞周期进程结合以维持基因组稳定性的新机制。在DNA复制、转录和染色质重塑等重要细胞过程中，Top1通过解决拓扑应变在维持基因组完整性方面发挥重要作用。CPT捕获Top1-DNA共价复合物可诱导复制和转录介导的致死性DNA病变（如DSBs）。然而，这些损伤的修复机制尚不清楚。本文报道的初步研究描述了DNA修复蛋白Rad18与范可尼贫血（FA）通路之间的新联系，以响应CPT诱导的DNA损伤。Rad18缺陷细胞或表达突变Rad18的细胞E3连接酶活性缺陷，不能有效募集FANCD2 （FA通路激活的关键成分）到染色质，使细胞对CPT过敏。许多研究也表明，在细胞周期的不同阶段，Rad18在CPT诱导的DNA损伤的修复中发挥重要作用。然而，在细胞周期的特定阶段协调这些通路的分子网络却知之甚少。本提案的具体目的是了解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Specific Aim1下提出的研究将集中于确定Rad18激活FA通路响应CPT的机制，使用各种生化和遗传工具。Specific Aim 2将验证Rad18和FA通路组分影响细胞从CPT诱导的DNA损伤中恢复的假设。Rad18和FA通路在调控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。一些前沿技术，如蛋白质- dna标记和分子梳理将被用来检验这一假设。在Specific Aim 3下的实验将确定Rad18在细胞周期不同阶段对CPT中毒Top1的反应中的作用。这项研究的结果将有助于理解细胞维持基因组稳定性的机制。对Rad18与FA通路的相互作用及其在细胞周期中的调控的研究将有可能揭示新的分子靶点，从而发现新的治疗组合，以增强Top1抑制剂对癌细胞的靶向破坏。

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.