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（申请人提供）：范可尼贫血（FA）是一种以骨髓衰竭、发育异常、白血病和实体瘤发病率高为特征的人类遗传病。在细胞水平上，FA 的显着特征是对诱导 DNA 链间交联 (ICL) 的化学物质极其敏感。 FA 是由 13 种 Fanc 蛋白的缺陷引起的，其中 8 种形成泛素连接酶，可单泛素化 FancI-FancD2 异二聚体（“ID”复合物）。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个谜，这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，我们开发了第一个支持 FA 途径依赖性 ICL 体外修复的实验系统。我们证明修复与 DNA 复制密切相关。具体来说，修复涉及 ICL 上两个 DNA 复制叉的汇聚。最初从 ICL 暂停 20 个核苷酸后，一条前导链的 3' 末端前进到病变处（方法），在受损碱基上插入一个核苷酸（跨病变 DNA 合成或 TLS），然后以一种方式延伸到病变之外。 DNA 聚合酶 6 依赖型（延伸）。与病变旁路同时，在另一条模板链上发生溶核切口。最终，输入 DNA 的很大一部分以复制依赖性方式得到完全修复。重要的是，在 TLS 步骤之前，从鸡蛋提取物中免疫消除 FancD2 会显着减少修复并阻止病变旁路。这种无细胞系统的开发代表了一项实验性突破，可以对参与 ICL 修复的因素（包括 FA 蛋白）进行系统分析。在这笔资助中，我们重点研究 FA 蛋白在病变旁路中的作用。在具体目标 1 中，我们讨论了方法步骤是如何完成的，包括它是否依赖于 FancM 还是 FancJ。在目标 2 中，我们讨论了哪些聚合酶促进跨损伤 DNA 合成步骤，以便我们可以在目标 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背后的分子缺陷。 公共卫生相关性： 范可尼贫血 (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。我们发现青蛙卵提取物重现了无细胞环境中 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。