Ras18-mediated Fanconi Anemia pathway activation in response to camptothecin

Ras18 介导的范可尼贫血途径响应喜树碱的激活

• 批准号: 8518869
• 负责人: Komaraiah Palle
• 金额: $ 19.55万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8518869
• 关键词: Affect; Antineoplastic Agents; Biochemical Genetics; Camptothecin; Cell Cycle; Cell Cycle Progression; Cell Cycle Stage; Cell physiology; Cells; Chromatin; Comb animal structure; Complex; Coupled; DNA; DNA Damage; DNA Maintenance; DNA Repair; DNA Repair Pathway; DNA Topoisomerases; DNA biosynthesis; DNA lesion; DNA repair protein; Data; Enzymes; Event; Fanconi' s Anemia; Genetic Transcription; Genome; Genome Stability; Goals; Human; Investigation; Label; Lesion; Maintenance; Malignant Neoplasms; Mediating; Molecular; Molecular Target; Monoubiquitination; Outcome Study; Pathway interactions; Pharmaceutical Preparations; Phase; Play; Poisoning; Process; Proteins; Recovery; Recruitment Activity; Regulation; Reporting; Resistance; Role; S Phase; Techniques; Testing; Therapeutic; Topotecan; Type I DNA Topoisomerases; Ubiquitination; analog; anti-cancer therapeutic; base; cancer cell; chromatin remodeling; design; environmental agent; inhibitor/antagonist; irinotecan; killings; mutant; next generation; novel; novel therapeutics; repaired; research study; response; tool; tumor; ubiquitin-protein ligase

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.

描述（由申请人提供）：该提案的长期目标是了解修复 DNA 拓扑异构酶 I (Top1) 诱导的 DNA 损伤的分子机制，靶向抗癌药物如喜树碱 (CPT) 及其类似物。这些研究将确定细胞将 DNA 修复与细胞周期进程整合起来以维持基因组稳定性的新机制。 Top1 通过解决 DNA 复制、转录和染色质重塑等重要细胞过程中的拓扑应变，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 共价复合物会诱导复制和转录介导的致死性 DNA 损伤（例如 DSB）。然而，这些损伤修复的机制尚不清楚。本文报道的初步研究描述了 DNA 修复蛋白 Rad18 和范可尼贫血 (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 E3 连接酶活性缺陷的突变体 Rad18 的细胞，无法有效地将 FANCD2（FA 途径激活的关键成分）募集到染色质，从而使细胞对 CPT 过敏。许多研究还表明，Rad18 在细胞周期不同阶段修复 CPT 诱导的 DNA 损伤中发挥着重要作用。然而，人们对在细胞周期特定阶段协调这些途径的分子网络知之甚少。本提案的具体目标是了解 Rad18 在 Top1 抑制剂诱导的 DNA 损伤中的作用及其对不同 DNA 修复途径的调节。 Specific Aim1 下提出的研究将侧重于使用各种生化和遗传工具确定 Rad18 响应 CPT 激活 FA 途径的机制。具体目标 2 将检验 Rad18 和 FA 途径成分影响细胞从 CPT 诱导的 DNA 损伤中恢复的假设。 Rad18 和 FA 通路在 S 期检查点、叉稳定性以及 DNA 复制的起始和延伸事件的调节中所发挥的作用也将被确定。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设。具体目标 3 下的实验将确定 Rad18 在细胞周期不同阶段中响应 Top1 CPT 中毒的作用。这项研究的结果将极大地有助于理解细胞维持基因组稳定性的机制。对 Rad18 与 FA 途径的相互作用及其在细胞周期中的调节的拟议研究将有可能揭示新的分子靶点，从而揭示新的治疗组合，以增强 Top1 抑制剂对癌细胞的靶向破坏。