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

范可尼贫血途径：在 DNA 链间交联修复中的作用

• 批准号: 8213405
• 负责人: Johannes Walter
• 金额: $ 41.78万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8213405
• 关键词: Address; Affect; Biochemical; Biological Assay; Biological Models; Bypass; Cell-Free System; Cells; Chemicals; Chromatin; Cisplatin; Complex; Coupled; DNA; DNA Damage; DNA Double Strand Break; DNA Interstrand Cross-Link Repair; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; DNA-dependent ATPase; Defect; Development; Disease; Egg Proteins; Environment; Event; Factor Analysis; Fanconi anemia protein; Fanconi' s Anemia; Genetic; Grant; Hereditary Disease; Human; Human Genetics; In Vitro; Incidence; Knowledge; Laboratories; Lesion; Malignant Neoplasms; Manuscripts; Measures; Mitomycins; Modeling; Molecular; Mono-S; Nature; Nucleotides; Organism; Pancytopenia; Pathway interactions; Play; Polymerase; Positioning Attribute; Predisposition; Preparation; Process; Proteins; Rana; Recruitment Activity; Resistance; Role; Set protein; Slide; Solid Neoplasm; Support System; Surgical incisions; Syndrome; System; Testing; Xenopus; base; biochemical model; crosslink; egg; helicase; insight; leukemia; mutant; public health relevance; repaired; research study; ubiquitin ligase

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.

描述（由申请人提供）：Fanconi贫血（FA）是一种人类遗传疾病，其特征是骨髓衰竭，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的区别特征是对诱导DNA链交联（ICL）的化学物质的极端敏感性。 FA是由13个粉丝蛋白缺陷引起的，其中8种形成了一种泛素连接酶，单次启发性将fanci-fancd2 Heterodimer（“ ID”复合物）呈现。间接证据强烈表明这些FA蛋白会促进ICL修复，但是它们的具体作用仍然是神秘的，这在很大程度上是由于缺乏直接测量ICL修复的分子测定法。使用爪蟾卵提取物，我们开发了第一个支持FA途径依赖性ICL修复的实验系统。我们表明维修与DNA复制密切相关。具体而言，维修涉及ICL上两个DNA复制叉的收敛。最初从ICL暂停20个核苷酸后，一条领先的链的3'末端向病变（进近）的前进，将核苷酸从受损碱基（Translesion DNA合成或TLS）上插入核苷酸，然后在DNA聚合酶6依赖的时尚（扩展）中延伸到病变中。与病变旁路同时，另一个模板链上发生了核解切口。最终，以复制依赖性方式完全修复了输入DNA的很大一部分。重要的是，从鸡蛋提取物中对FANCD2进行免疫部门会大大减少修复，并在TLS步骤之前立即减少病变旁路。该无细胞系统的开发代表了实验突破，该突破允许对包括FA蛋白在内的ICL修复中涉及的因素进行系统分析。在这笔赠款中，我们关注FA蛋白在病变旁路中的作用。在特定的目标1中，我们解决了如何完成方法步骤，包括它取决于FANCM还是FANCJ。在AIM 2中，我们解决了哪种聚合酶促进了跨质量DNA合成步骤，以便我们可以在AIM 3中阐明ID复合物如何催化这一事件。实验将阐明富康尼贫血的分子缺陷。 公共卫生相关性： Fanconi贫血（FA）是一种癌症易感障碍，是由一组蛋白质中的缺陷引起的，这些蛋白质被认为可以固定DNA间交叉链接（ICL），这是一种特别危险的DNA损伤。但是，FA蛋白通常固定ICL的机制是晦涩的。我们发现，源自青蛙卵的提取物在无细胞环境中概括了ICL修复的过程。我们将使用该系统来阐明FA蛋白在ICL修复中的功能。