DNA Double Strand Break Repair Complexes in Space and Time with Applications to Druggability

双链断裂（DSBs）是DNA损伤中最危险的形式。dsb的修复主要通过两种途径，即同源重组（homologous recombination， HR）和非同源末端连接（non-homologous end joining， NHEJ），这两种途径都需要多蛋白复合物在时间和空间上的组装。众所周知，癌细胞表现出更高水平的DNA损伤，因此靶向DSB修复在药理学上是一种有用的治疗形式。该项目旨在建立在先前对DNA修复中涉及的多蛋白复合物的结构理解的基础上，并作为Blundell和Pellegrini实验室之间的合作，在结构指导药物设计中利用这些信息。作为佩莱格里尼实验室的一部分，该项目旨在在同源重组过程中发展对RAD51细丝的结构理解。先前在实验室的工作已经产生了突触前RAD51丝与ssDNA复合物的低温电镜结构，但是中间复合物的结构仍然未知。这个项目的一个目的是用低温电镜来解决中间复合物的结构。此外，该项目将重点关注RAD51平行体的功能和结构特征，尽管它们在人力资源中发挥着重要作用，但鲜为人知。各种结构和生化方法将用于表征这些蛋白质在同源重组过程中与RAD51功能相关的功能。作为Blundell实验室的一部分，该项目将以BBSRC DTP轮换项目的一部分进行的研究为基础，进一步探索DNA连接酶IV和Artemis在NHEJ中的作用。Artemis片段与DNA连接酶IV复合物的结构先前已经通过实验室使用x射线晶体学解决了2.4Å分辨率，为理解蛋白质-蛋白质相互作用（PPI）位点提供了一个框架。先前的计算分析表明，该PPI界面具有高度可药物性，并且高通量虚拟筛选（HTVS）分子库已经确定了候选结合物。该项目将继续进行额外的筛选和实验验证，目的是开发一种成功的这种相互作用的抑制剂。此外，该项目将专注于解决连接酶IV与NHEJ系统中其他相互作用伙伴结合的结构，特别是与DNA-PK复合物。在实验室之前的工作已经产生了DNA- pkcs和DNA- pk （DNA- pkcs与Ku70/80和DNA的复合物）的单体和二聚体形式的低温电镜结构，建立了一个工作流程，可以适应整合连接酶IV，以产生在DNA直端连接的最后阶段所涉及的复合物的结构。拟议的项目由Pellegrini和Blundell实验室合作，将核心结构生物学与结构导向药物设计的实际应用相结合。同源重组和非同源末端连接的双重关注为研究通路串扰和决策提供了广泛的范围，并将重点放在阐明和比较每个涉及的多蛋白复合物的空间和时间组织上。

PhD project strategic theme: Biosciences for an integrated understanding of healthDouble strand breaks (DSBs) are the most dangerous forms of DNA damage. DSBs are repaired through the action of two main pathways, both of which require the assembly of multi-protein complexes in time and space: homologous recombination (HR) and non-homologous end joining (NHEJ). Cancerous cells are known to exhibit higher levels of DNA damage, thus targeting DSB repair pharmacologically is a useful form of therapy. This project will aim to build on previous structural understanding of the multiprotein complexes involved in DNA repair and exploit this information in the form structure-guided drug design as a collaboration between the Blundell and Pellegrini labs.As part of the Pellegrini Lab this project seeks to develop structural understanding of RAD51 filaments during homologous recombination. Previous work in the lab has produced the cryo-EM structure of a presynaptic RAD51 filament in complex with ssDNA, however the structure of the intermediate complex remains unknown. One aim of this project will be to solve the structure of the intermediate complex using cryo-EM. In addition, this project will focus on the functional and structural characterisation of RAD51 paralogs, little of which is known, despite their playing essential roles in HR. A variety of structural and biochemical approaches will be used to characterise the functions of these proteins in relation to RAD51 function during homologous recombination.As part of the Blundell Lab this project will build on research conducted as part of the BBSRC DTP rotation project to further explore the role of DNA Ligase IV and Artemis in NHEJ. The structure of an Artemis fragment in complex with DNA Ligase IV has been previously solved to 2.4Å resolution by the lab using X-ray crystallography, providing a framework for understanding the protein-protein interaction (PPI) site. Previous computational analysis suggests this PPI interface is highly druggable and high-throughput virtual screening (HTVS) of molecule libraries has identified candidate binders. This project will be continued through additional screening and experimental validation of leads with the aim of developing a successful inhibitor of this interaction. In addition, the project will focus on solving the structures of Ligase IV bound to its other interacting partners within the NHEJ system, notably with the DNA-PK complex. Previous work in the lab has produced the cryo-EM structure of DNA-PKcs and DNA-PK (the complex of DNA-PKcs with Ku70/80 and DNA) in monomeric and dimeric forms, establishing a workflow which can be adapted to integrate Ligase IV to produce a structure of the complex involved during the final stage of DNA blunt-end ligation.The proposed projects in a collaboration between the Pellegrini and Blundell labs integrate core structural biology with a practical application in structure-guided drug design. The dual focus on homologous recombination and non-homologous end joining provides a broad scope for investigating pathway crosstalk and decision-making and will have an emphasis on elucidating and comparing both the spatial and temporal organisation of the multiprotein complexes involved in each.