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

DNA双链断裂（DSB）被认为是最具细胞毒性的DNA损伤，通常会导致异常的染色体易位，促进癌变。在真核生物中，DSB修复发生在染色质（DNA和组蛋白的复合物）中，并且涉及两个主要途径，非同源末端连接（NHEJ）和同源重组（HR）。这些途径受组蛋白翻译后修饰级联调控，组蛋白翻译后修饰控制DSB修复蛋白的有序组装。该级联反应的中心是环指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建立的机制特异性地在组蛋白H2A和H2A.X（H2AK13ub和H2AK15ub）的赖氨酸残基K13和K15处泛素化染色质。RNF168对于参与调节HR和NHEJ之间平衡的几种下游修复因子的募集至关重要，但将RNF168与这些因子联系起来的潜在机制尚不清楚。我们有两个主要目标，与本建议的两个目标相对应。在目标1中，我们将探测一系列的HR和NHEJ修复蛋白的泛素化核小体核心颗粒（NCP）的识别，使用结构和细胞生物学方法。目的二：通过结构测定和生物化学方法，探讨RNF168及其相关的E2泛素结合酶的作用机制。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RING泛素连接酶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如何在生成和阅读染色质泛素化位点特异性。

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.