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，这一过程对于实现有效的适应性免疫系统所需的抗体多样性至关重要。RAG1+RAG2重组酶破坏DNA的分子细节已被很好地理解，但对随后的DNA修复如何发生知之甚少，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。这种理解的缺乏是因为断裂的编码DNA末端在体外切割后复合体中迅速丢失，阻碍了结构研究。我们现在已经通过创造具有特定活性表观遗传修饰的核小体解决了这个问题，这使我们能够在体外研究裂解后复合体。该项目将使用利兹大学世界一流的冷冻电镜设备，并结合结构生物学、细胞生物学和免疫学。该研究的具体目标是：(i)确定RAG1+RAG2与修饰的核小体模板复合物的高分辨率结构；（ii）研究切割后反应中的不同构象状态，（iii）发现这些构象如何使纯化的非同源末端连接蛋白能够招募来影响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.