Defining dynamic protein complexes in DNA repair by non-homologous end-joining

紫外线、电离辐射 (IR)、食品中的化学物质、药物和烟草烟雾等环境暴露威胁主要通过 DNA 损伤而导致疾病。因此，生物系统已经进化到最小化或逆转这种损害。毒性最强的损伤是DNA双链断裂，这种损伤的修复也容易出错，可能导致小头畸形、原始侏儒症和免疫缺陷等遗传性缺陷，甚至引发癌症。如果快速识别 DNA 断裂，从而可以将末端正确地组合在一起并重新连接，则出错的可能性就会降低。将断裂末端连接在一起的细胞机制包括结构相关的 XRCC4 家族蛋白，该蛋白一端具有特征明确的传统折叠结构域，另一端具有本质无序区域 (IDR)。人们对无序区域的作用知之甚少，因为许多实验技术没有检测到它们，但它们很可能动态形成短暂的有序模块，介导修复机械部件之间的相互作用。确定哪些区域变得最有序、它们形成什么结构以及它们与什么相互作用，对于理解 DNA 修复功能及其调控方式至关重要。 DNA 修复因子的抑制可用于有针对性地治疗癌症，例如癌症治疗，这一事实凸显了这一重要性。通过合成致死作用，如 PARP 抑制剂所示。了解 XRCC4 蛋白家族的动力学和结构，将使修复途径的这一组成部分在计算机模拟中更准确地建模，并可能允许开发针对修复途径这一部分的新抑制剂。该项目有三个目标：O1：使用我们的新方法和核磁共振 (NMR) 光谱鉴定 XRCC4 家族蛋白无序区域（XRCC4、XLF 和 PAXX）中的短寿命结构模块O2：定义使用独特的基于细胞的 DNA 损伤修复报告器破坏这些模块中的结构。O3：通过应用最先进的光交联方法识别模块的相互作用者该项目中要利用和开发的技术将适用于广泛的系统，特别是那些涉及具有本质上无序区域的蛋白质的系统。越来越清楚的是，许多细胞过程位于无膜细胞器中，而 IDR 经常参与这些细胞器的形成。所提供的结构信息将标志着 XRCC4 家族蛋白在治疗靶向方面的适应性发生了一步变化，可用于癌症治疗。

Environmental exposure threats like UV light, ionizing radiation (IR), chemicals in food, drugs and tobacco smoke cause disease principally through damage to DNA. Consequently, biological systems have evolved to minimise or reverse this damage. The most toxic damage is double-strand breakage of DNA, and the repair of such damage is also prone to error, which can lead to hereditary defects such as microcephaly, primordial dwarfism and immune deficiencies, as well as causing cancers. The likelihood of error is lower if DNA breaks are identified quickly so that the ends can be brought together and rejoined correctly. The cellular machinery that brings the broken ends together include the structurally related XRCC4 family proteins, which have well-characterised conventional folded domains at one end, and intrinsically disordered regions (IDRs) at the other. The role for the disordered regions is poorly understood, as they are not detected by many experimental techniques, but they are likely to dynamically form short-lived ordered modules that mediate interactions between the repair machinery components. Identifying which regions become most ordered, what structures they make, and what they interact with is fundamental to understanding how DNA repair functions, and how it is regulated. This importance is highlighted by the fact that inhibition of DNA repair factors can be used to treat cancers in targeted ways e.g. via synthetic lethality, as illustrated for example by PARP inhibitors. Understanding the dynamics and structures of the XRCC4 family of proteins, will allow this component of the repair pathway to be modelled more accurately in computer simulations, and may allow new inhibitors to be developed that target this part of the repair pathway.The project has three objectives:O1: Identification of modules of short-lived structure in the disordered regions of XRCC4 family proteins (XRCC4, XLF and PAXX), using our novel approach and nuclear magnetic resonance (NMR) spectroscopyO2: Defining the consequence of disrupting the structure in these modules using a unique cell-based reporter of DNA damage repair.O3: Identify interactors for the modules by applying a state-of-the-art photo-crosslinking approach The techniques to be utilised and developed in this project will be applicable to a wide range of systems, particularly those which involve proteins with intrinsically disordered regions. It is becoming increasingly clear that many cellular processes are located in membrane-less organelles, and IDRs are frequently involved in forming these. The structural information provided will mark a step change towards the amenability of the XRCC4 family proteins for therapeutic targeting, which could be exploited in the treatment of cancers.