Mechanisms of DNA interstrand cross-link repair

总结 DNA链间交联（ICL）共价连接DNA双螺旋的两条链，并且是极其重要的。 细胞毒性。广泛使用的化学治疗剂（例如氮芥、顺铂化合物）被认为起作用于 通过ICL的产生。然而，肿瘤几乎总是对这些药物产生耐药性，部分原因是 因为DNA修复的上调ICL也由内源性代谢物（例如反应性代谢物）产生。 醛，脱碱基位点），并且不能修复内源性ICL似乎引起人类疾病。为 例如，22种不同FANC基因中的任何一种突变都会使细胞对ICL敏感，并导致Fanconi 贫血（FA），其特征是骨髓衰竭和显著升高的易感性， 白血病和其他癌症。FANC蛋白对ICL的修复发生在S期，但它们如何发挥作用还不清楚。 未知为了阐明ICL修复的机制，我们在青蛙中复制了含有位点特异性ICL的质粒 鸡蛋提取物我们以前表明，这些提取物支持顺铂ICL修复的FANC蛋白。 修复需要两个复制叉会聚在ICL上，这触发了泛素化和染色质 通过p97 ATP酶卸载复制型CMG解旋酶。CMG移除后，FANCI-FANCD 2 复合物刺激病变两侧磷酸二酯骨架的双切口， （"解开"）ICL并产生双链DNA断裂（DSB），随后通过 同源重组在上一个资助期，我们发现E3泛素连接酶TRAIP是 所需的泛素化的CMG分叉收敛。有趣的是，尽管TRAIP总是 与复制分叉相关联，它只在分叉聚合时使CMG无处不在。在上一个周期中，我们还 发现了一种独特的复制偶联修复机制，该机制作用于pepsilen和AP-ICL。在这 通过TRAIP的CMG泛素化机制，导致NEIL 3 DNA糖基化酶的募集， 切割形成ICL的糖基键之一。脱钩在一条链中产生脱碱基（"AP"）位点，但 避免形成DSB。在目前的建议中，我们使用卵子提取物和哺乳动物细胞来解决关键问题。 我们之前工作中出现的问题在目标1中，我们问TRAIP如何将CMG泛素化与 复制叉收敛，TRAIP功能的一个关键特征，避免过早的复制体解体。 在目标2中，我们讨论了NEIL 3通路如何避免DSB的形成，DSB是区分NEIL 3和DSB的核心特征。 从FA途径。在目标3中，我们研究了对ICL的替代反应机制，称为"遍历"， 其中单个复制分叉绕过完整的ICL。这些研究对人类健康的潜在影响 是相当可观的，因为它们将有助于为更有效的癌症的发展奠定坚实的基础。 化疗以及改善范科尼贫血症状的药物。

Summary DNA interstrand cross-links (ICLs) covalently link the two strands of the DNA double helix and are extremely cytotoxic. Widely used chemotherapeutics (e.g. nitrogen mustards, cisplatin compounds) 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. 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 any one of 22 different FANC genes renders cells sensitive to ICLs and causes Fanconi anemia (FA), which is characterized by bone marrow failure and dramatically elevated predisposition to leukemia and other cancers. ICL repair by the FANC proteins occurs in S phase, but how they function was unknown. To elucidate mechanisms of ICL repair, we replicate plasmids containing site-specific ICLs in frog egg extracts. We showed previously that these extracts support cisplatin ICL repair by the FANC proteins. Repair requires that two replication forks converge on the ICL, which triggers the ubiquitylation and chromatin unloading of the replicative CMG helicase by the p97 ATPase. After CMG removal, the FANCI-FANCD2 complex stimulates dual incisions in the phosphodiester backbone on either side of the lesion, which resolves ("unhooks") the ICL and creates a double-stranded DNA break (DSB) that is subsequently repaired by homologous recombination. In the last funding period, we discovered that the E3 ubiquitin ligase TRAIP is required for the ubiquitylation of CMG upon fork convergence. Interestingly, although TRAIP is always associated with replication forks, it only ubiquitylates CMGs upon fork convergence. In the last cycle, we also discovered a distinct mechanism of replication-coupled repair that operates on psoralen- and AP-ICLs. In this mechanism, CMG ubiquitylation by TRAIP leads to the recruitment of the NEIL3 DNA glycosylase, which cleaves one of the glycosyl bonds that form the ICL. Unhooking creates an abasic ("AP") site in one strand but avoids formation of a DSB. In the current proposal, we use egg extracts and mammalian cells to address key questions that arise from our prior work. In Aim 1, we ask how CMG ubiquitylation by TRAIP is coupled to replication fork convergence, a critical feature of TRAIP function that avoids premature replisome disassembly. In Aim 2, we address how the NEIL3 pathway avoids formation of DSBs, the central feature that distinguishes it from the FA pathway. In Aim 3, we study the mechanism of an alternative response to ICLs called "traverse," in which a single replication fork bypasses an intact ICL. The potential impact of these studies on human health is considerable because they will help create a solid foundation for the development of more effective cancer chemotherapies, as well as drugs that ameliorate the symptoms of Fanconi anemia.