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

通过非同源末端连接定义 DNA 修复中的动态蛋白质复合物

• 批准号: MR/X008754/1
• 负责人: Christine Schmidt
• 金额: $ 61.49万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FX008754%2F1
• 关键词: Defining; dynamic; protein; complexes; DNA

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.

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损害DNA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。毒性最大的损伤是DNA双链断裂，这种损伤的修复也容易出错，从而导致小头畸形、原始侏儒症和免疫缺陷等遗传缺陷，并引发癌症。如果DNA断裂能够被快速识别，从而使末端能够正确地连接在一起，那么出错的可能性就会降低。将断裂端连接在一起的细胞机制包括结构相关的XRCC4家族蛋白，其一端具有特征良好的常规折叠结构域，另一端具有内在无序区域（IDRs）。无序区域的作用尚不清楚，因为它们没有被许多实验技术检测到，但它们很可能动态地形成短暂的有序模块，调解修复机械部件之间的相互作用。确定哪些区域变得最有序，它们形成什么样的结构，以及它们与什么相互作用，是理解DNA修复功能的基础，以及它是如何被调节的。DNA修复因子的抑制可用于有针对性地治疗癌症，例如通过合成致死性，如PARP抑制剂所示，这一事实突出了这一重要性。了解XRCC4蛋白家族的动力学和结构，将允许在计算机模拟中更准确地模拟修复途径的这一部分，并可能允许开发针对这部分修复途径的新抑制剂。该项目有三个目标：1 .利用我们的新方法和核磁共振（NMR）光谱技术鉴定XRCC4家族蛋白（XRCC4， XLF和PAXX）无序区域的短寿命结构模块；2 .利用独特的基于细胞的DNA损伤修复报告定义破坏这些模块结构的后果。O3：通过应用最先进的光交联方法确定模块的相互作用。本项目中使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内在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经常参与形成这些细胞器。所提供的结构信息将标志着XRCC4家族蛋白对治疗靶向性的适应性发生了一步变化，这可能被用于癌症的治疗。