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，通常会导致染色体异常易位，从而促进癌症的发生。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，它涉及两条主要途径，即非同源末端连接(NHEJ)和同源重组(HR)。这些通路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。这个级联反应的中心是无名指E3泛素连接酶RNF168。通过一个尚待确定的机制，RNF168及其同源泛素结合酶泛素化染色质，特异性地位于组蛋白H_2A和H_2A.X(H_2AK13ub和H_2AK15ub)的赖氨酸残基K13和K15处。RNF168对几个下游修复因子的招募至关重要，这些下游修复因子参与调节HR和NHEJ之间的平衡，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，与这项提案中的两个目标相对应。在目标1中，我们将利用结构和细胞生物学的方法，探索一系列HR和NHEJ修复蛋白对泛素化核小体核心颗粒(NCP)的识别。在目标2中，我们将通过结构测定和生物化学来探索RNF168及其相关的E2泛素结合酶的酶机制。我们提出的RNF168复合体的高分辨结构，有可能揭示单体环泛素连接酶作用机制的前所未有的细节。总的来说，除了提供对一种重要的DNA损伤反应酶的基本机制的理解外，这项研究还将帮助我们理解如何在产生和阅读染色质泛素化过程中赋予位点专一性。

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.