Mechanisms of DNA interstrand cross-link repair

 描述(由申请人提供)：DNA链间交叉连接(ICL)共价连接DNA的Watson和Crick链，具有极强的细胞毒性。广泛使用的化疗药物(如氮芥、顺铂化合物、丝裂霉素C)被认为是通过产生ICL发挥作用的。然而，肿瘤几乎总是对这些药物产生抗药性，部分原因是DNA修复上调。重要的是，ICL也是由内源性代谢物(如活性醛、碱性部位)产生的，如果不能修复内源性ICL，似乎会导致人类疾病。例如，FANC基因的突变使细胞对ICL敏感，并导致Fanconi贫血，其特征是骨髓衰竭，极大地增加了患白血病和其他癌症的易感性。此外，FAN1核酸酶的突变还会导致肾脏疾病。为什么两类突变都会破坏ICL修复，导致如此不同的疾病，目前尚不清楚。要回答这个问题，了解FANC和FAN1蛋白在ICL修复中的分子功能将是至关重要的。我们发现，非洲爪哇蛙卵提取物概括了三种不同的ICL修复形式。我们先前证明了鸡蛋提取物重述依赖于FANC蛋白的复制偶联ICL修复，我们使用这一方法来证明FANC蛋白导致DNA切口，从而将ICL从DNA中切断或“解钩”。在未发表的结果中，我们发现了第二个依赖复制的ICL修复反应，但它独立于FANC蛋白。在其他未发表的结果中，我们概述了第三个不依赖复制的ICL修复反应，该反应需要核酸酶FAN1和SNM1A。我们假设，这两条复制依赖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是关键的。在这项提案中，我们将使用非洲爪哇卵提取液的力量来阐明ICL是如何在FANC依赖的ICL修复途径(目标1)中分解的。我们还将研究FANC非依赖性ICL修复途径的机制，并确定两条复制依赖途径中的哪一条被用于修复各种外源性和内源性ICL(目标2)。最后，我们将阐明FAN1和SNM1A在复制非依赖性ICL修复中的作用，并讨论它们是否在哺乳动物细胞中发挥类似的功能(目标3)。综上所述，这项建议将阐明三种不同的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。对促进新的FANC非依赖ICL修复途径的因子的小分子抑制可能会使对ICL产生抗药性的FANC缺陷肿瘤重新敏感。相反，刺激这一通路可能对FANC依赖的ICL修复缺陷的Fanconi贫血患者有治疗益处。

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 In unpublished results, we discovered a second, replication-dependent ICL repair reaction that is however independent of the FANC proteins.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