The Fanconi anemia pathway: role in DNA interstrand cross-link repair

描述（由申请人提供）：范可尼贫血（FA）是一种人类遗传性疾病，其特征为骨髓衰竭、发育异常以及白血病和实体瘤的高发病率。在细胞水平上，FA的显著特征是对诱导DNA链间交联（ICL）的化学物质极其敏感。FA是由十三种Fanc蛋白中的缺陷引起的，其中八种形成使FancI-FancD 2异二聚体（“ID”复合物）单泛素化的泛素连接酶。间接证据有力地表明，这些FA蛋白促进ICL修复，但它们的具体作用仍然是谜，在很大程度上是由于缺乏直接测量ICL修复的分子测定。使用非洲爪蟾卵提取物，我们已经开发出第一个实验系统，支持FA通路依赖的ICL体外修复。我们表明，修复是密切耦合到DNA复制。具体而言，修复涉及ICL上两个DNA复制叉的会聚。在最初从ICL暂停20个核苷酸后，一条前导链的3'端前进到损伤（方法），从损伤的碱基插入核苷酸（translesion DNA合成或TLS），然后以DNA聚合酶6依赖性方式延伸超过损伤（延伸）。与病变旁路同时，在另一模板链上发生溶核切口。最终，输入DNA的显著部分以复制依赖的方式被完全修复。重要的是，来自鸡蛋提取物的FancD 2的免疫耗竭显著减少了修复，并在TLS步骤之前立即阻止了病变旁路。这种无细胞系统的发展代表了一个实验性的突破，允许系统分析ICL修复中涉及的因素，包括FA蛋白。在这项资助中，我们专注于FA蛋白在病变旁路中的作用。在具体目标1中，我们讨论了如何完成方法步骤，包括它是否取决于FancM或FancJ。在目标2中，我们解决了哪些聚合酶促进translesion DNA合成步骤，这样我们就可以在目标3中阐明ID复合物如何催化这一事件。这些实验将阐明范可尼贫血的分子缺陷。 公共卫生相关性： 范科尼贫血（FA）是一种癌症易感性疾病，由一组被认为可以修复DNA链间交联（ICL）的蛋白质缺陷引起，这是一种特别危险的DNA损伤类型。然而，FA蛋白通常固定ICL的机制尚不清楚。我们已经发现，来自青蛙卵的提取物重现了无细胞环境中ICL修复的过程。我们将使用这个系统来阐明FA蛋白在ICL修复中的功能。

DESCRIPTION (provided by applicant): Fanconi anemia (FA) is a human genetic disease that is characterized by bone marrow failure, developmental abnormalities, and a high incidence of leukemias and solid tumors. At the cellular level, the distinguishing feature of FA is extreme sensitivity to chemicals that induce DNA interstrand cross-links (ICLs). FA is caused by defects in thirteen Fanc proteins, eight of which form a ubiquitin ligase that mono-ubiquitylates the FancI-FancD2 heterodimer (the "ID" complex). Indirect evidence strongly indicates that these FA proteins promote ICL repair, but their specific role remains enigmantic, in large part due to the absence of molecular assays that directly measure ICL repair. Using Xenopus egg extracts, we have developed the first experimental system that supports FA pathway-dependent ICL repair in vitro. We show that repair is intimately coupled to DNA replication. Specifically, repair involves convergence of two DNA replication forks on the ICL. After initially pausing 20 nucleotides from the ICL, the 3' end of one leading strand advances to the lesion (Approach), inserts a nucleotide across from the damaged base (translesion DNA synthesis or TLS), and is then extended beyond the lesion in a DNA polymerase 6-dependent fashion (Extension). Concurrent with lesion bypass, nucleolytic incisions occur on the other template strand. Ultimately, a significant fraction of the input DNA is fully repaired in a replication-dependent manner. Importantly, immunodepletion of FancD2 from egg extracts dramatically reduces repair and arrests lesion bypass immediately prior to the TLS step. The development of this cell-free system represent an experimental breakthrough that allows a systematic analysis of the factors involved in ICL repair, including the FA proteins. In this grant, we focus on the role of the FA proteins in lesion bypass. In Specific Aim 1, we address how the Approach step is accomplished, including whether it depends on FancM or FancJ. In Aim 2, we address which polymerases promote the translesion DNA synthesis step, so that we may, in Aim 3, elucidate how the ID complex catalyzes this event. The experiments will elucidate the molecular defects that underlie Fanconi anemia. PUBLIC HEALTH RELEVANCE: Fanconi anemia (FA) is a cancer predisposition disorder which is caused by defects in a set of proteins that are thought to fix DNA inter-strand cross-links (ICLs), a particularly dangerous type of DNA damage. However, the mechanism by which the FA proteins normally fix ICLs is obscure. We have discovered that extracts derived from frog eggs recapitulate the process of ICL repair in a cell-free environment. We will use this system to elucidate the function of FA proteins in ICL repair.