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: 7766535
• 负责人: Johannes Walter
• 金额: $ 42.38万
• 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/7766535
• 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）：范可尼贫血（Fanconi anemia， FA）是一种人类遗传性疾病，以骨髓衰竭、发育异常、白血病和实体瘤的高发为特征。在细胞水平上，FA的显著特征是对诱导DNA链间交联（ICLs）的化学物质极度敏感。FA是由13种fanc2蛋白缺陷引起的，其中8种形成泛素连接酶，使fanc2 - fanc2异源二聚体（“ID”复合物）单泛素化。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。利用爪蟾卵提取物，我们开发了第一个支持FA通路依赖性ICL体外修复的实验系统。我们发现修复与DNA复制密切相关。具体来说，修复涉及到ICL上两个DNA复制叉的聚合。在最初从ICL中暂停20个核苷酸后，一条导链的3'端向前移动到病变处（Approach），在受损碱基上插入一个核苷酸（translesion DNA synthesis或TLS），然后以DNA聚合酶6依赖的方式延伸到病变处（Extension）。在病变旁路的同时，另一条模板链上出现了溶核切口。最终，很大一部分输入DNA以依赖复制的方式被完全修复。重要的是，鸡蛋提取物中的FancD2免疫缺失显著减少修复，并在TLS步骤之前立即阻止病变旁路。这种无细胞系统的开发代表了一项实验突破，可以系统地分析ICL修复中涉及的因素，包括FA蛋白。在本研究中，我们主要关注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Approach步骤，包括它是依赖于FancM还是FancJ。在Aim 2中，我们讨论了哪些聚合酶促进翻译DNA合成步骤，因此我们可以在Aim 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背后的分子缺陷。