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

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.