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

描述(申请人提供)：Fanconi贫血(FA)是一种人类遗传性疾病，以骨髓衰竭、发育异常以及白血病和实体瘤的高发病率为特征。在细胞水平上，FA的显著特征是对引起DNA链间交联(ICL)的化学物质高度敏感。FA是由13个FANC蛋白的缺陷引起的，其中8个形成泛素连接酶，使FANCI-FANCD2异源二聚体(“ID”复合体)单一泛素化。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是未知的，很大程度上是由于缺乏直接测量ICL修复的分子检测。利用非洲爪哇的卵子提取物，我们开发了第一个支持FA途径依赖的ICL体外修复的实验系统。我们证明修复与DNA复制密切相关。具体地说，修复涉及ICL上两个DNA复制分叉的会聚。在最初暂停ICL的20个核苷酸之后，一个前导链的3‘端进入病变(接近)，在受损的碱基对面插入一个核苷酸(跨病变DNA合成或TLS)，然后以DNA聚合酶6依赖的方式延伸到病变之外(延伸)。在病变搭桥的同时，另一条模板链上也会发生核溶解切开。最终，很大一部分输入DNA以复制依赖的方式被完全修复。重要的是，鸡蛋提取物中FANCD2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项实验性的突破，使系统地分析参与ICL修复的因素，包括FA蛋白。在这项资助中，我们重点介绍FA蛋白在病变搭桥中的作用。在具体目标1中，我们讨论如何完成这一方法步骤，包括它是取决于FancM还是FancJ。在目标2中，我们解决了哪些聚合酶促进跨损伤DNA合成步骤，以便在目标3中，我们可以阐明ID复合体如何催化这一事件。这些实验将阐明范可尼贫血背后的分子缺陷。 公共卫生相关性： Fanconi贫血(FA)是一种癌症易感性疾病，由一组蛋白质缺陷引起，这些蛋白质被认为可以修复DNA链间交联(ICL)，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。我们发现，从青蛙卵中提取的提取物概括了在无细胞环境中ICL修复的过程。我们将利用这个系统来阐明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.