Mechanisms of DNA interstrand cross-link repair

 描述（由申请方提供）：DNA链间交联（ICL）共价连接DNA的沃森和克里克链，具有极强的细胞毒性。广泛使用的化学治疗剂（例如氮芥、顺铂化合物、丝裂霉素C）被认为通过ICL的产生起作用。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。重要的是，ICL也由内源性代谢物（例如反应性醛、脱碱基位点）产生，并且不能修复内源性ICL似乎引起人类疾病。例如，FANC基因的突变使细胞对ICL敏感，并导致范可尼贫血，其特征是骨髓衰竭和白血病和其他癌症的易感性显著增加。此外，FAN1核酸酶的突变会导致肾脏疾病。为什么两类突变都破坏ICL修复，导致如此不同的疾病尚不清楚。为了回答这个问题，了解FANC和FAN1蛋白在ICL修复中的分子功能至关重要。我们发现爪蟾卵提取物概括了三种不同形式的ICL修复。我们以前表明，卵提取物重现了依赖于FANC蛋白的复制偶联ICL修复，我们使用这种方法表明FANC蛋白导致DNA切口，将ICL从DNA中切除或“脱钩”。在未发表的结果中，我们发现了第二种复制依赖性ICL修复反应，但它不依赖于FANC蛋白。在其他未发表的结果中，我们概括了第三个，不依赖复制的ICL修复反应，需要核酸酶FAN 1和SNM1A。我们假设这两种复制依赖性途径代表了增殖组织中ICL修复的替代手段，而第三种途径在非增殖细胞中至关重要。在这个提议中，我们将使用爪蟾卵提取物的力量来阐明ICL是如何在FANC依赖的ICL修复途径中被解析的（目的1）。我们还将研究FANC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一种用于修复各种外源性和内源性ICL（目的2）。最后，我们将阐明FAN1和SNM1A在复制无关的ICL修复中的作用，我们将讨论它们是否在哺乳动物细胞中执行类似的功能（目的3）。总之，本研究将阐明三种不同的ICL修复途径的分子机制，并探索它们何时被利用。这项工作对人类健康和癌症生物学的潜在影响是巨大的。小分子抑制促进新的非FANC依赖性ICL修复途径的因子可能会使对ICL产生耐药性的FANC缺陷肿瘤重新敏感。相反，刺激该通路可能对缺乏FANC依赖性ICL修复的Fanconi贫血患者具有治疗益处。

 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 In unpublished results, we discovered a second, replication-dependent ICL repair reaction that is however independent of the FANC proteins.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.