Project Title: Structural studies of DNA repair required for human immune diversity using cryoEM

细胞竭尽全力保护其基因组的完整性，但矛盾的是，淋巴细胞在V（D）J重组过程中故意破坏其DNA，这是实现有效适应性免疫系统所需的抗体多样性所必需的过程。RAG 1 + RAG 2重组酶破坏DNA的分子细节已经很好地理解，但对随后的DNA修复如何发生知之甚少，而这一过程对维持基因组稳定性至关重要，因此对维持正常的细胞健康至关重要。这种缺乏理解是因为断裂的编码DNA末端在体外迅速从切割后复合物中丢失，阻碍了结构研究。我们现在已经解决了这个问题，通过创造核小体轴承特定的活性表观遗传修饰，使我们能够研究后裂解复合物在体外。该项目将使用利兹大学的世界一流的冷冻电镜设备，并将结构生物学、细胞生物学和免疫学联合收割机结合起来。该研究的具体目标是（i）确定RAG 1 + RAG 2与修饰的核小体模板复合的高分辨率结构;（ii）研究切割后反应期间的不同构象状态;（iii）发现这种构象如何使纯化的非同源末端连接蛋白的招募能够影响DNA修复。

Cells go to enormous lengths to protect the integrity of their genome but paradoxically, lymphocytes deliberately break their DNA during V(D)J recombination, a process that is essential to achieve the antibody diversity required for an effective adaptive immune system. The molecular details by which the RAG1+RAG2 recombinase breaks DNA are well understood but little is known about how the subsequent repair of DNA occurs, and this process is vital to maintain genome stability and therefore to the maintenance of normal cellular health. This lack of understanding is because broken coding DNA ends are rapidly lost from the post-cleavage complex in vitro, hampering structural studies. We have now solved this problem by creating nucleosomes bearing specific active epigenetic modifications, that allow us to study the post-cleavage complex in vitro. This project will use the world-class facilities for cryoEM at the University of Leeds and combine structural biology, cell biology and immunology. The specific objectives of this studentship are to (i) determine the high-resolution structure of RAG1+RAG2 complexed with the modified nucleosome templates; (ii) investigate the distinct conformational states during the post-cleavage reaction and (iii) discover how such conformations enable recruitment of purified non-homologous end joining proteins to effect DNA repair.