Mechanisms of DNA interstrand cross-link repair

DNA链间交联修复机制

• 批准号: 9099349
• 负责人: Johannes Walter
• 金额: $ 42.38万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9099349
• 关键词: Address; Aldehydes; Cancer Biology; Cell-Free System; Cells; Chronic Kidney Failure; Cisplatin; Cleaved cell; Complex; Coupled; DNA; DNA Double Strand Break; DNA Interstrand Cross-Link Repair; DNA Repair; DNA glycosylase; DNA replication fork; Defect; Disease; Enzymes; Event; Failure; Fanconi' s Anemia; Generations; Genes; Genomic Instability; Health; Human; Kidney Diseases; Lead; Lesion; Link; Malignant Neoplasms; Mammalian Cell; Maps; Mechlorethamine; Mitomycins; Molecular; Mutation; Pancytopenia; Pathway interactions; Patients; Predisposition; Proliferating; Proteins; Rana; Reaction; Recruitment Activity; Reporting; Resistance; Ribose; Role; S Phase; Side; Site; Source; Surgical incisions; Syndrome; Testing; Therapeutic; Tissues; Up-Regulation; Work; Xenopus; base; crosslink; cytotoxic; egg; endonuclease; human DNA damage; human disease; leukemia; new therapeutic target; novel; nuclease; programs; protein function; public health relevance; reduce symptoms; repaired; small molecule; tumor

 DESCRIPTION (provided by applicant): DNA interstrand cross-links (ICLs) covalently link the Watson and Crick strands of DNA and are extremely cytotoxic. Widely used chemotherapeutics (e.g. nitrogen mustards, cisplatin compounds, mitomycin C) are thought to act through the generation of ICLs. However, tumors almost invariably become resistant to these agents, in part due to upregulation of DNA repair. Importantly, ICLs are also generated by endogenous metabolites (e.g. reactive aldehydes, abasic sites), and failure to repair endogenous ICLs appears to cause human disease. For example, mutation in FANC genes renders cells sensitive to ICLs and causes Fanconi anemia, which is characterized by bone marrow failure and dramatically elevated predisposition to leukemia and other cancers. In addition, mutations in the FAN1 nuclease cause kidney disease. Why two classes of mutations, both of which disrupt ICL repair, cause such different diseases is unknown. To answer this question, it will be critical to understand the molecular functions of the FANC and FAN1 proteins in ICL repair. We have discovered that Xenopus frog egg extracts recapitulate three distinct forms of ICL repair. We previously showed that egg extracts recapitulate replication-coupled ICL repair that depends on the FANC proteins, and we used this approach to show that the FANC proteins lead to DNA incisions that cut-out or "unhook" the ICL from DNA. In unpublished results, we discovered a second, replication-dependent ICL repair reaction that is however independent of the FANC proteins. In other unpublished results, we recapitulated a third, replication-independent ICL repair reaction that requires the nucleases FAN1 and SNM1A. We hypothesize that the two replication-dependent pathways represent alternative means of repairing ICLs in proliferating tissues, whereas the third pathway is critical in non-proliferating cells. In this proposal, we wil use the power of Xenopus egg extracts to elucidate how ICLs are resolved in the FANC-dependent ICL repair pathway (Aim 1). We will also investigate the mechanism of the FANC-independent ICL repair pathway and determine which of the two replication-dependent pathways are utilized to repair a variety of exogenous and endogenous ICLs (Aim 2). Finally, we will elucidate the roles of FAN1 and SNM1A in replication-independent ICL repair, and we will address whether they perform similar functions in mammalian cells (Aim 3). In summary, this proposal will elucidate the molecular mechanisms of three distinct ICL repair pathways and explore when they are utilized. The potential impact of the work for human health and cancer biology is significant. Small molecule inhibition of factors that promote the novel, FANC-independent ICL repair pathway might re-sensitize FANC-deficient tumors that became resistant to ICLs. Conversely, stimulation of this pathway may have therapeutic benefits for Fanconi anemia patients who are deficient in FANC-dependent ICL repair.

 描述（由申请人提供）：DNA 链间交联 (ICL) 共价连接 DNA 的 Watson 和 Crick 链，并且具有极强的细胞毒性。广泛使用的化疗药物（例如氮芥、顺铂化合物、丝裂霉素 C）被认为在整个世代中发挥作用。然而，肿瘤几乎总是对这些药物产生耐药性，部分原因是 DNA 修复的上调。也由内源性代谢物（例如活性醛、脱碱基位点）产生，并且内源性 ICL 修复失败似乎会导致人类疾病。例如，FANC 基因突变使细胞对 ICL 敏感并导致范可尼贫血，其特征是骨髓衰竭。此外，FAN1 核酸酶突变会导致肾脏疾病，这两种突变都会破坏 ICL 修复。要回答这个问题，了解 FANC 和 FAN1 蛋白在 ICL 修复中的分子功能至关重要，我们发现非洲爪蟾卵提取物概括了我们之前展示的三种不同形式的 ICL 修复。鸡蛋提取物重现了依赖于 FANC 蛋白的复制耦合 ICL 修复，我们使用这种方法来证明 FANC 蛋白会导致 DNA 切口，从而将 ICL 从 DNA 中切掉或“脱钩”。在未发表的结果中，我们发现了第二种依赖于复制的 ICL 修复反应，但它独立于 FANC 蛋白。在其他未发表的结果中，我们概括了第三种依赖于复制的 ICL 修复反应，该反应需要核酸酶 FAN1 和 SNM1A。两条依赖于复制的途径代表了在增殖组织中修复 ICL 的替代方法，而第三条途径在非增殖细胞中至关重要。在本提案中，我们将利用非洲爪蟾卵的力量。我们还将研究 FANC 依赖性 ICL 修复途径的机制，并确定利用两种复制依赖性途径中的哪一种来修复多种情况。最后，我们将阐明 FAN1 和 SNM1A 在复制无关的 ICL 修复中的作用，并讨论它们是否在修复中发挥相似的功能。总之，该提案将阐明三种不同的 ICL 修复途径的分子机制，并探讨该工作对人类健康和癌症生物学的潜在影响。促进新型、独立于 FANC 的 ICL 修复途径可能会使 FANC 缺陷的肿瘤重新变得对 ICL 产生耐药性，刺激该途径可能对缺乏 FANC 的贫血患者具有治疗益处。 FANC 依赖性 ICL 修复。