Structural classification of NHEJ pathways; unravelling the role of Ku-binding proteins

DNA包含生命的遗传蓝图，并允许细胞在生长和分裂过程中精确复制。然而，像DNA分子这样的双螺旋弦可以以多种方式被破坏，这是生命的一个基本问题，因为这种遗传密码的改变会导致细胞死亡，最终导致癌症。然而，人类已经进化出复杂的DNA修复系统，可以识别DNA的损伤并采取行动修复它。DNA损伤的严重程度各不相同，可以简单到单个碱基对的改变或化学物质的添加，也可以是DNA的一条或两条链的断裂。DNA双链断裂被称为DNA双链断裂，被认为是最危险的DNA损伤形式，因为它最难修复。非同源末端连接（Non-homologous end joining， NHEJ）是人类修复dsb的两个关键机制之一。该机制依赖于几个核心蛋白，即DNA- pkcs、Ku70/80、DNA连接酶IV、XRCC4和XLF。生物分子，如DNA和它们结合的蛋白质可以通过几种方法可视化，然而，NHEJ中涉及的蛋白质通常被证明很难使用这些结构生物学技术来研究。然而，最近，一种被称为冷冻电子显微镜的方法的进步使我们能够看到一些核心蛋白质和复合物，并确定它们如何相互作用和DNA。然而，也有许多接合蛋白参与NHEJ，在某些条件下对有效的DNA修复至关重要。这些接头蛋白如何与核心NHEJ机制相互作用尚不清楚。我们最近的低温电镜结构表明，核心NHEJ蛋白XLF对于有效DNA修复所需的大型多蛋白复合物的形成至关重要。我们相信其他NHEJ接头蛋白也可能在大型NHEJ组装的形成中发挥重要作用，为了支持这一观点，我们有初步数据表明新鉴定的接头蛋白PAXX可以稳定NHEJ蛋白的另一种排列。因此，本研究将研究四种接头蛋白及其在NHEJ过程中形成大蛋白复合物中的作用。具体来说，我们将研究蛋白质PAXX、WRN、CYREN和APLF -，并使用冷冻电子显微镜观察它们与DNA-PKcs和Ku70/80等蛋白质结合时的情况。了解这些蛋白质如何决定NHEJ复合物的组装将使我们能够解开这个复杂机制中的多个步骤。用药物靶向NHEJ是一种行之有效的策略，可与化疗和放疗联合用于治疗许多癌症。然而，对于目前可用的非特异性NHEJ抑制剂，长期以来一直存在担忧。我们希望揭开一个复杂的多步骤、多途径过程，将允许设计靶向NHEJ抑制剂，提供更具体和个性化的治疗。

DNA contains the genetic blueprint for life and allows for cells to be accurately replicated during growth and division. The double-helical string like DNA molecules can however be damaged in multiple ways and this is a fundamental problem for life as alterations to this genetic code can cause cell death and eventually cancer. However, humans have evolved intricate DNA repair systems that can recognise the damage to DNA and act to repair it. DNA damage varies in severity and can be as simple as single base-pair changes or chemical additions, through to breaks across one or both strands of the DNA. A break across both strands of DNA called a DNA-double strand-break (DSBs), is considered to be the most dangerous form of DNA damage as it is the most difficult to repair. Non-homologous end joining (NHEJ), is one of two key mechanisms in humans which repairs DSBs. This mechanism is dependent upon a few core proteins, namely DNA-PKcs, Ku70/80, DNA Ligase IV, XRCC4 and XLF. Biological molecules such as DNA and the proteins they bind to can be visualised using a few methods, however the proteins involved in NHEJ have typically proven to be difficult to study using these structural biology techniques. Recently however, advances in one such method termed cryo-electron microscopy have allowed us to visualise some of these core proteins and complexes and determine how they interact with each other and DNA. Nevertheless, there are also numerous adaptor proteins involved in NHEJ, that under certain conditions become essential for efficient DNA repair. How these adaptor proteins interact with the core NHEJ machinery is not known. Our recent cryo-EM structures have shown that XLF, a core NHEJ protein is essential for formation of a large multiprotein complex that is required for efficient DNA repair. We believe it is possible that other NHEJ adaptor proteins may also play important roles in the formation of large NHEJ assemblies, and to support this idea we have preliminary data that shows the newly identified adaptor protein, PAXX can stabilise an alternative arrangement of NHEJ proteins.Therefore, this proposal will examine four adaptor proteins and their role in the formation of large protein complexes during NHEJ. Specifically, we will investigate the proteins PAXX, WRN, CYREN and APLF - and visualise these when they bind to proteins such as DNA-PKcs and Ku70/80 using cryo-electron microscopy. Understanding how these proteins dictate NHEJ complex assembly will allow us to unravel the multiple steps in this intricate mechanism. Targeting NHEJ with drugs is a well-established strategy used in combination with chemo- and radiotherapy for the treatment of many cancers. However, there is long standing concern over the non-specific NHEJ inhibitors that are currently available. We hope that unravelling what may be an intricate multiple step, multiple pathway process, will allow the design of targeted NHEJ inhibitors that could provide more specific and personalised treatments.