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

NHEJ通路的结构分类；

• 批准号: MR/X00029X/1
• 负责人: Amanda Chaplin
• 金额: $ 90.87万
• 依托单位: University of Leicester
• 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%2FX00029X%2F1
• 关键词: Structural; classification; NHEJ; pathways; unravelling

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.

DNA包含生命的遗传蓝图，并允许细胞在生长和分裂期间准确复制。然而，像DNA分子一样的双螺旋链会以多种方式受到破坏，这是生命的一个基本问题，因为这种遗传密码的改变可能会导致细胞死亡，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损伤并采取行动进行修复。DNA损伤的严重程度各不相同，可以像单碱基对变化或化学添加一样简单，一直到DNA的一条或两条链断裂。跨越DNA双链的断裂被称为DNA双链断裂(DSB)，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(NHEJ)是人类修复双链断裂的两种关键机制之一。这一机制依赖于几个核心蛋白，即DNA-PKcs、Ku70/80、DNA Ligase IV、XRCC4和XLF。生物分子，如DNA及其结合的蛋白质，可以用几种方法可视化，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进行研究。然而，最近，一种名为冷冻电子显微镜的方法的进展使我们能够可视化一些核心蛋白质和复合体，并确定它们如何相互作用和与DNA相互作用。然而，在NHEJ中也有许多接头蛋白，在某些条件下对有效的DNA修复是必不可少的。这些接头蛋白如何与核心NHEJ机制相互作用尚不清楚。我们最近的冷冻EM结构表明，XLF是一种核心NHEJ蛋白，对于形成有效的DNA修复所需的大型多蛋白复合体是必不可少的。我们认为，其他NHEJ接头蛋白也可能在形成大的NHEJ组装中发挥重要作用，为了支持这一观点，我们有初步数据表明，新发现的接头蛋白PAXX可以稳定NHEJ蛋白的另一种排列。因此，本提案将研究四种接头蛋白及其在NHEJ过程中形成大蛋白复合体中的作用。具体地说，我们将研究蛋白质PAXX、WRN、CYREN和APLF，并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。了解这些蛋白质是如何决定NHEJ复杂组装的，将使我们能够揭开这一复杂机制中的多个步骤。以NHEJ为靶点的药物是一种成熟的策略，与化疗和放射治疗相结合用于许多癌症的治疗。然而，人们对目前可用的非特异性NHEJ抑制剂的担忧由来已久。我们希望，揭开可能是一个复杂的多步骤、多途径的过程，将允许设计靶向NHEJ抑制剂，从而提供更特异和个性化的治疗。

项目成果

PAXX binding to the NHEJ machinery explains functional redundancy with XLF.

• DOI: 10.1126/sciadv.adg2834
• 发表时间: 2023-06-02
• 期刊: SCIENCE ADVANCES
• 影响因子: 13.6
• 作者: Seif-El-Dahan, Murielle;Kefala-Stavridi, Antonia;Frit, Philippe;Hardwick, Steven W.;Chirgadze, Dima Y.;De Oliviera, Taiana Maia;Britton, Sebastien;Barboule, Nadia;Bossaert, Madeleine;Pandurangan, Arun Prasad;Meek, Katheryn;Blundell, Tom L.;Ropars, Virginie;Calsou, Patrick;Charbonnier, Jean -Baptiste;Chaplin, Amanda K.
• 通讯作者: Chaplin, Amanda K.