Specific aim 1: Mechanistic and structural analyses of pre-recombination complexes
DNA strand breaks are repaired by a process called homologous recombination repair. This requires a number of cellular factors including the BRCA2 and PALB2 tumour suppressor proteins, RAD51, RAD52, RAD54, the five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) and RPA. All proteins have been purified and we have carried out biochemical and structural analyses of the proteins in order to determine the precise roles that they play in DNA repair. Our work revealed that RAD52 forms two distinct structures. One is an eleven subunit ring, the structure of which has been solved at high resolution, and the second is an open ring structure. We find that the open ring interacts with RPA and DNA, and that this is the functional form of the protein in DNA repair. In addition, we have analysed the coordinated actions of BRCA2-PALB2, RAD52, RAD51 and RPA in assembling pre-recombination complexes in vitro, and have developed even more complete biochemical reactions to determine their mode of action. Importantly, we have obtained a high resolution structure of the RAD51B-RAD51C-RAD51D-XRCC2 complex by cryo-EM.
In the clinic, breast and ovarian cancer patients with BRCA germline mutations show a good response rate to treatment with a PARP inhibitor (Olaparib) and PARPi are now used as a first-line therapy against certain ovarian cancers. Unfortunately, after 3-4 years, cancer cells develop resistance to PARPi treatment. We therefore sought to find new mechanism by which the potency of PARPi therapy could be increased. We found that inactivation of a nucleotide salvage pathway protein DNPH1 significantly sensitised BRCA cells to the PARPi, and are moving forward in studies to discover drugs that can inhibit this pathway. We believe it may provide a promising strategy for more efficient killing of BRCA-deficient cancers.
Specific Aim 2: Structure-Selective Endonucleases
For several years, we have been interested in understanding the roles of structure-selective endonucleases such as SLX1-SLX4, MUS81-EME1 and GEN1 in the resolution of recombination intermediates. We discovered that SLX1-SLX4-MUS81-EME1-XPF-ERCC1 (SMX) complex forms at prometaphase, in response to CDK/PLK1 phosphorylation events and that this complex plays a key role in the resolution of DNA recombination intermediates. Mutations in key components of this complex lead to an inheritable disease known as Fanconi anaemia, and individuals with this disease are predisposed to cancer. We also discovered that the MutSβ mismatch repair protein interacts with recombination intermediates and with SLX4. Together MutSβ and SMX are significantly more active than SMX alone, and these interactions are required for the resolution of recombination intermediates. These are remarkable observations as cell cycle-dependent formation and activation of this tri-nuclease complex appears to provide a unique mechanism by which cells ensure chromosome segregation and preserve genome integrity. Additionally, we found a new role for GEN1 nuclease, in the cleavage of DNA loci that have been identified as common fragile sites.
Specific Aim 3: Senataxin and the Maintenance of Genome Stability in Neuronal Cells
The neurodegenerative disorders ataxia with oculomotor apraxia-2 is caused by mutations in the SETX gene. Individuals with this autosomal recessive cerebellar ataxia exhibit motor neuron degeneration, together with progressive muscle weakness and atrophy. Typically, motor coordination is affected at an early age (2 to 6 years) and progressive disability continues leading to confinement to a wheelchair in adolescent life. We found that mutations in the gene, SETX, that causes AOA2 are sensitive to agents that cause oxidative stress (H2O2), DNA replication stress (HU, aphidicolin), or induce the formation of R-loops (Diospyrin D1). In Senataxin- depleted cells, and also in AOA2 patient-derived cells, we observed increased DNA break formation at mitosis. Over-expression of RNase H1, or treatment with the transcription chain terminator Cordycepin, reduced the frequency of these breaks indicating that R-loops were the underlying cause of genome instability after Senataxin deficiency. We have now extended these studies by combining genomic and transcriptomic approaches and found that loss of Senataxin from cells derived from AOA2 patients leads to a genome-wide increase in RNA polymerase II (RNAPII) pausing events, R-loop accumulation, altered gene expression and transcription stress-induced genomic instability across genes and at fragile sites.
All results generated during this project have been published in international journals or are currently being written up for publication. The work has been presented at numerous conferences around the world. The use of DNPH1 inhibitors, in combination with PARPi, as a potential therapy for the specific killing of BRCA cancers has been patented.
