Biophysical and Structural Analysis of Recombination Repair Proteins

Our genomic material, DNA, is continually attacked by agents in the environment (radiation, pollution), chemicals that we ingest in our diet, and it also shows an inherent instability as it is replicated, or transcribed, or divided during cell division. Indeed, it is estimated that a single human cell will suffer more than 70,000 single strand breaks or base damages, and around 20 double strand breaks, each day. Without efficient repair, these lesions in DNA will accumulate and lead to diseases such as cancer or progressive neurodegenerative disorders. To cope with such high levels of damage, our cells are therefore equipped with a number of DNA repair mechanisms, each of which is specialised to target and remove different types of lesions.

However, many individuals carry inheritable mutations that affect the efficiency of these DNA repair pathways. For example, individuals with mutations in the BRCA1, BRCA2, PALB2, or RAD51 paralog genes, are predisposed to breast and ovarian cancers which occur with a high frequency. Mutations in the same genes can also cause Fanconi anemia, a genetic disorder characterised by congenital abnormalities, progressive bone marrow failure and predisposition to head, neck and blood cancers. These genes encode proteins that promote the repair of DNA double-strand breaks, which represent possibly the most dangerous form of damage, as their inefficient repair can lead to DNA translocations or loss of part of a chromosome.

This proposal aims to provide new insights into the mechanisms of DNA double-strand break repair, through structural, biophysical and biochemical analysis of some of the key factors in the process. In particular, we will determine the near atomic structure of RAD52, both alone and bound to DNA, and also that of a RAD51 paralog complex composed of four proteins, RAD51B, RAD51C, RAD51D and XRCC2 (abbreviated to BCDX2). These factors are key players in homologous recombinational repair, the process that promotes the repair of DNA double strand breaks, and are important for cancer avoidance. The structural analyses will be supported by mechanistic analyses (biophysical and biochemical) that will shed new light into their mechanism of action. Together, our studies will provide detailed insights into why patient-derived mutations in these important repair factors lead to human disease.

The structures of these proteins will be determined using a state-of-the-art technique called cryo-electron microscopy (cryo-EM). To do this, the proteins we are interested in will be purified away from all other cellular components, and then frozen in ice so that they can be bombarded with electrons to produce microscope images of individual molecules. These are used to reconstruct the 3D shape, or structure, of the molecule. Once we know their structure, we can start to understand why mutations compromise their activity and cause human disease. At the Francis Crick Institute, which opened in 2015 and represents the largest biomedical research facility in Europe, we are fortunate to have one of the most powerful microscopes, and superb technical backup, that will enable us to bring these studies to fruition.

All cells employ elaborate and effective DNA repair processes that are essential for the maintenance of genome integrity. Some individuals, however, are genetically predisposed to cancers that are the direct result of mutations in genes involved in DNA repair. Breast cancer is the most common cancer in women worldwide with over two million new cases diagnosed each year. Approximately 5-10% of all breast cancers show familial inheritance, and many are caused by germline mutations in the BRCA (BRCA1 or BRCA2), PALB2 (Partner and Localiser of BRCA2) or RAD51 paralog (RAD51B, RAD51C, RAD51D, XRCC3 and XRCC3) genes which are required for the recombinational repair of DNA double-strand breaks (DSBs).

This proposal will provide new understanding of the process of recombinational repair, by elucidating the near atomic structures of two key players: RAD52 and the RAD51B, RAD51C, RAD51D and XRCC2 complex (BCDX2). Currently, our understanding of RAD52 is limited to an X-ray crystal structure of the N-terminal domain of RAD52, which forms an 11-subunit ring. However, new studies indicate that this form of the protein is inactive, and the active form is an open ring that interacts with high affinity to single stranded DNA (ssDNA) bound to RPA. We will therefore solve the near atomic structure of the RAD52 open ring, and the RAD52-ssDNA-RPA complex using cryo-EM. Nothing is currently known about the structure of BCDX2, so our work will provide the first structure-function analysis of the RAD51 paralogs. The work described in the proposal builds on solid preliminary studies of the cryo-EM structures of RAD52 and BCDX2, which are both very encouraging, leading us to believe that full structural determinations will be possible. The structural analyses will be supported by the biophysical analysis of BCDX2, and the biochemical reconstitution of recombination repair using BRCA2, PALB2, RAD51, RPA and the RAD51 paralog complexes, all of which are now available in the lab.