Structural basis of RNF168-mediated ubiquitin signaling at chromosomal DNA breaks

染色体 DNA 断裂时 RNF168 介导的泛素信号传导的结构基础

• 批准号: 9147614
• 负责人: Georges Mer
• 金额: $ 31.4万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-22 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9147614
• 关键词: Address; Area; BRCA1 gene; Binding; Biochemical; Biochemistry; Biological Models; Cancer Etiology; Cells; Cellular biology; Chromatin; Chromosomal translocation; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Gene; DNA lesion; Double Effect; Double Strand Break Repair; Elements; Ensure; Enzymes; Equilibrium; Eukaryota; Fingers; Genome Stability; Goals; Health; Hereditary Breast Carcinoma; Histone H2A; Histone H2B; Histones; Human; Immunodeficiency and Cancer; In Vitro; Ionizing radiation; Knowledge; Link; Lysine; Maintenance; Malignant Neoplasms; Malignant neoplasm of ovary; Mediating; Methylation; Molecular; Mono-S; Mutation; NMR Spectroscopy; Nonhomologous DNA End Joining; Nucleosome Core Particle; Nucleosomes; Organism; Outcome Study; Pathway interactions; Phosphorylation; Polyubiquitin; Post-Translational Protein Processing; Proteins; Public Health; Reader; Reading; Research; Resolution; Roentgen Rays; Series; Signal Transduction; Site; Specificity; Structure; Surface; Syndrome; Testing; Time; Ubiquitin; Ubiquitin-Conjugating Enzymes; Work; X-Ray Crystallography; base; cancer therapy; carcinogenesis; cell killing; chemotherapy; cytotoxic; dimer; fascinate; homologous recombination; interest; novel therapeutic intervention; p53-binding protein 1; recombinational repair; repaired; research study; response; structural biology; treatment response; ubiquitin ligase; ubiquitin-protein ligase

DNA double-strand breaks (DSBs) are considered the most cytotoxic DNA lesions, often resulting in aberrant chromosomal translocations that promote carcinogenesis. In eukaryotes, DSB repair takes place in chromatin, a complex of DNA and histone proteins, and involves the two main pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). These pathways are regulated by a cascade of histone post-translational modifications that control the ordered assembly of DSB repair proteins. Central to this cascade is the RING-finger E3 ubiquitin ligase RNF168. By a mechanism that remains to be established, RNF168 and its cognate E2 ubiquitin-conjugating enzyme ubiquitylate chromatin specifically at lysine residues K13 and K15 of histones H2A and H2A.X (H2AK13ub and H2AK15ub). RNF168 is crucial for the recruitment of several downstream repair factors involved in regulating the balance between HR and NHEJ, but the underlying mechanisms linking RNF168 to these factors are unclear. We have two main objectives corresponding to two aims in this proposal. In Aim 1, we will probe the recognition of the ubiquitylated nucleosome core particle (NCP) by a series of HR and NHEJ repair proteins using structural and cell biology approaches. In Aim 2, we will explore the enzymatic mechanism of RNF168 and associated E2 ubiquitin-conjugating enzyme through structure determination and biochemistry. High-resolution structures of RNF168 complexes, as we propose, have the potential to reveal unprecedented details on the mechanism of action of a monomeric RING ubiquitin ligase. Collectively, in addition to providing a basic mechanistic understanding of an important DNA damage response enzyme, this research will help us understand how site specificity is conferred in both generating and reading chromatin ubiquitylation.

DNA双链断裂（DSBs）被认为是最具细胞毒性的DNA病变，通常导致异常染色体易位，促进癌变。在真核生物中，DSB修复发生在染色质中，染色质是DNA和组蛋白的复合体，涉及两种主要途径，非同源末端连接（NHEJ）和同源重组（HR）。这些途径受一系列组蛋白翻译后修饰的调控，这些修饰控制着DSB修复蛋白的有序组装。这个级联反应的核心是无名指E3泛素连接酶RNF168。RNF168及其同源的E2泛素结合酶通过一种尚待确定的机制，特异性地在组蛋白H2A和H2A的赖氨酸残基K13和K15上泛素化染色质。X （H2AK13ub和H2AK15ub）。RNF168对于参与调节HR和NHEJ之间平衡的几个下游修复因子的募集至关重要，但RNF168与这些因子之间的潜在机制尚不清楚。我们有两个主要目标，对应于这项建议的两个目的。在Aim 1中，我们将使用结构和细胞生物学方法探讨一系列HR和NHEJ修复蛋白对泛素化核小体核心颗粒（NCP）的识别。在Aim 2中，我们将通过结构测定和生物化学方法探索RNF168及其相关E2泛素偶联酶的酶促机制。正如我们所提出的，RNF168复合物的高分辨率结构有可能揭示单体环泛素连接酶的作用机制的前所未有的细节。总的来说，除了提供一个重要的DNA损伤反应酶的基本机制的理解，这项研究将帮助我们了解位点特异性是如何被赋予在生成和读取染色质泛素化。