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，并允许在生长和分裂过程中准确复制细胞。然而，像DNA分子这样的双螺旋串可能会以多种方式损坏，这是生命的基本问题，因为对这种遗传密码的改变会导致细胞死亡，最终导致癌症。但是，人类已经发展出复杂的DNA修复系统，可以识别DNA的损害并作用进行修复。 DNA损伤在严重程度上有所不同，并且可以像单碱基对变化或化学添加一样简单，以至于在DNA的一条或两条链之间断裂。在两组DNA中的突破称为DNA双重链破裂（DSB），被认为是最难修复的DNA损伤的最危险形式。非同源末端连接（NHEJ）是人类维修DSB的两个关键机制之一。该机制取决于一些核心蛋白，即DNA-PKC，KU70/80，DNA连接酶IV，XRCC4和XLF。可以使用几种方法可视化生物分子，例如DNA和与之结合的蛋白质，但是使用这些结构生物学技术很难研究涉及NHEJ的蛋白质。然而，最近，一种称为冷冻电子显微镜的方法中的进步使我们能够可视化其中一些核心蛋白和复合物，并确定它们如何相互相互作用和DNA。然而，在NHEJ中也有许多辅助蛋白，在某些条件下对于有效的DNA修复至关重要。这些衔接蛋白如何与核心NHEJ机械相互作用。我们最近的冷冻EM结构表明，XLF，核心NHEJ蛋白对于形成有效的DNA修复所需的大型多蛋白络合物至关重要。我们认为，其他NHEJ适配器蛋白可能在形成大型NHEJ组装中也起着重要作用，为了支持这个想法，我们拥有的初步数据显示了新鉴定的衔接子蛋白，PAXX可以稳定NHEJ蛋白的替代排列NHEJ蛋白质。此建议将其在大量的蛋白质中进行四个拟合蛋白，并在四个适应性的范围内进行了研究，并且在其中的角色构建了nhej蛋白。具体而言，我们将研究蛋白质PAXX，WRN，CYREN和APLF的蛋白质 - 并使用冷冻电子显微镜与蛋白质（例如DNA-PKCS和KU70/80）结合时可视化它们。了解这些蛋白质如何决定NHEJ复合物组装将使我们能够在这种复杂机制中揭示多个步骤。用药物将NHEJ靶向NHEJ是一种结合化学和放射疗法来治疗许多癌症的良好策略。但是，人们对目前可用的非特异性NHEJ抑制剂有很长的关注。我们希望阐明可能是复杂的多步骤，多个途径过程，将允许设计有针对性的NHEJ抑制剂，这些抑制剂可以提供更具体和个性化的治疗方法。

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.