Mechanisms of DNA interstrand cross-link repair

DNA链间交联修复机制

• 批准号: 9247224
• 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/9247224
• 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; Dangerousness; 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; 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（由适用提供）：DNA链间交联（ICLS）共价连接DNA的Watson和Crick Strands，并且非常细胞毒性。人们认为广泛使用的化学治疗剂（例如氮芥末，顺铂化合物，丝裂霉素C）被认为是通过ICL的产生起作用的。但是，肿瘤几乎总是对这些药物的抗性，部分原因是DNA修复的上调。重要的是，ICL还由内源性代谢产物（例如反应性醛，脓肿部位）产生，并且无法修复内源性ICL似乎会导致人类疾病。例如，FANE基因中的突变使细胞对ICL敏感并引起Fanconi贫血，其特征是骨髓衰竭和对白血病和其他癌症的倾向显着升高。此外，FAN1核酸酶中的突变会引起肾脏疾病。为什么两类突变都破坏了ICL修复，导致这种不同的疾病尚不清楚。为了回答这个问题，了解ICL修复中FANC和FAN1蛋白的分子功能至关重要。我们已经发现，爪蟾青蛙蛋提取物概括了三种不同形式的ICL修复。我们先前表明，鸡蛋提取物概括了取决于粉丝蛋白的复制耦合ICL修复，我们使用这种方法表明粉丝蛋白会导致DNA切口，从而从DNA中切出或“解开” ICL。在未发表的结果中，我们发现了第二种，复制依赖性的ICL修复反应，但是与狂热蛋白无关。在其他未发表的结果中，我们概括了第三个独立于复制的ICL修复反应，该反应需要FAN1和SNM1A。我们假设这两种依赖复制的途径代表了修复增殖组织中ICL的替代方法，而第三个途径在非增殖细胞中至关重要。在此提案中，我们将利用爪蟾鸡蛋提取物的能力来阐明在依赖fanc依赖的ICL修复途径中如何解决ICL（AIM 1）。我们还将研究幻想独立的ICL修复途径的机制，并确定两种复制依赖性途径中的哪个用于修复各种外源性和内源性ICL（AIM 2）。最后，我们将阐明FAN1和SNM1A在非复制独立的ICL修复中的作用，我们将解决它们是否在哺乳动物细胞中执行相似的功能（AIM 3）。总而言之，该建议将阐明三种不同的ICL修复途径的分子机制，并在使用时探索它们。这项工作对人类健康和癌症生物学的潜在影响很大。小分子抑制促进新颖的，幻想独立的ICL修复途径的因素可能会重新敏感的幻想缺陷肿瘤，该肿瘤对ICL具有抗性。相反，对该途径的刺激可能会对缺乏依赖fanc依赖性ICL修复的Fanconi贫血患者具有治疗益处。