Mechanisms of DNA interstrand cross-link repair

摘要 DNA链间交联(ICL)将DNA双螺旋的两条链共价连接在一起，并且非常 细胞毒性。广泛使用的化疗药物(如氮芥、顺铂化合物)被认为起作用 通过ICL的产生。然而，肿瘤几乎总是对这些药物产生抗药性，部分原因是 由于DNA修复的上调。ICL也是由内源性代谢物(如反应性代谢物)产生的 醛，碱性部位)，以及不能修复内源性ICL似乎导致人类疾病。为 例如，22个不同FANC基因中任何一个的突变使细胞对ICL敏感，并导致Fanconi 贫血(FA)，其特征是骨髓衰竭和显著增加的易感性 白血病和其他癌症。FANC蛋白对ICL的修复发生在S期，但它们的功能是 未知。为了阐明ICL修复的机制，我们在青蛙体内复制了含有位点特异性ICL的质粒 鸡蛋提取物。我们先前证明，这些提取物支持FANC蛋白对顺铂ICL的修复。 修复需要两个复制分叉汇聚在ICL上，从而触发泛素化和染色质 P97ATPase卸载复制的CMG解旋酶。移除CMG后，FANCI-FANCD2 复合体刺激病变两侧的磷酸二酯主干上的双重切口处，从而解决 (“脱钩”)ICL并产生一个双链DNA断裂(DSB)，随后由 同源重组。在上一个资助期，我们发现E3泛素连接酶TRAIP是 在分叉收敛时CMG泛素化所需的。有趣的是，尽管TRAIP总是 与复制分叉相关，它仅在分叉收敛时泛化CMG。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，这种机制作用于补骨脂素和AP-ICL。在这 机制，CMG泛素化导致NEIL3糖基酶的募集，这是由TRAIP引起的 断裂形成ICL的一个糖基键。解钩在一条链中创建基本(AP)站点，但 避免形成DSB。在目前的方案中，我们使用鸡蛋提取物和哺乳动物细胞来解决关键问题 从我们之前的工作中产生的问题。在目标1中，我们询问TRAIP的CMG泛素化是如何与 复制分叉收敛，这是TRAIP功能的一个关键特征，可避免复制体过早分解。 在目标2中，我们讨论了NEIL3通路如何避免DSB的形成，DSB是区别于 它来自FA途径。在目标3中，我们研究了对ICL的另一种反应机制，称为“Traverse，” 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创造坚实的基础 化疗，以及改善范可尼贫血症状的药物。

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.