Our DNA is constantly being damaged by, for example, UV light, chemicals or even normal processes in our cells. The damage can take many forms, one of the most serious being breaks in both strands of the DNA (or DSBs for double-strand breaks). Our cells repair DSBs by different processes. The most accurate is called homologous recombination or HR. Most of the time, HR is successful, but when it fails it can lead to cancer. In the nucleus of our cells, DNA is part of the chromatin, which consists of many nucleosomes. In each nucleosome, the DNA is compacted as it wraps around the histone proteins. When a DSB occurs, a molecule called ubiquitin gets attached to a histone in the nucleosome and becomes a signal for the DNA damage response (DDR) proteins that localize near the DSB and take part in the repair. We are interested in understanding how three DDR proteins – the BRCA1-BARD1 complex, RAD18 and 53BP1 – regulate HR DNA repair. BRCA1-BARD1 and RAD18 activate HR DNA repair while 53BP1 inactivates HR DNA repair. A correct balance of BRCA1-BARD1, RAD18 and 53BP1 is needed for HR DNA repair to work properly. If this balance is perturbed, DNA repair does not work properly and this can cause cancer. Indeed, it is known that mutations in BRCA1-BARD1 that damage the function of this protein result in crippled HR DNA repair and ovarian cancer. With the Liz Tilberis Award, we have been able to show that to regulate HR DNA repair, BRCA1-BARD1, RAD18 and 53BP1 need to bind to the same ubiquitin signal in nucleosomes. Using a powerful electron microscope, we have studied RAD18 and BARD1 bound to ubiquitin-bearing nucleosomes. These highly magnified visualizations of RAD18-ubiquitin-nucleosome and BARD1-ubiquitin-nucleosome have allowed us to design experiments to better understand how HR DNA repair works. For the next two years, we want to understand how the BARD1 component of BRCA1-BARD1 associates with ubiquitin-nucleosome in living cells and how mutations in BARD1 affect HR DNA repair. The outcome of this work will greatly improve our understanding of HR DNA repair that could aid in designing new drugs for ovarian cancer. For example, since RAD18 activates HR, we could devise drugs to inactivate RAD18 and thus inactivate HR. Paradoxically, inactivating HR DNA repair in cancer cells could make them more sensitive to cell killing agents like PARP inhibitors, which have already shown great promise in treating ovarian cancer. With RAD18 inhibitors, we could make PARP inhibitors more lethal to cancer cells. By understanding the effects of mutations in BARD1 on HR DNA repair, as we propose, we believe that we will be able to determine whether BARD1 mutations found in patients with ovarian cancer and classified as “variants of unknown significance” are cancer-predisposing or not. This work would have important implications for ovarian cancer prevention and treatment.
This grant was made possible in part by a generous donation from Judy and Phil Messing, in memory of Carol S. Messing.
Dr. Botuyan received her B.S. in Chemistry from the University of the Philippines and Ph.D. in Chemistry from Purdue University. She undertook postdoctoral training at the Scripps Research Institute, the Beckman Research Institute of the City of Hope, the University of Toronto, and the Mayo Clinic. She is currently a Research Scientist and an Assistant Professor at the Mayo Clinic in Rochester, MN. Dr. Botuyan is interested in understanding how genome integrity is maintained and how impaired DNA double-strand break (DSB) repair can lead to cancer. As a biochemist and structural biologist, she has helped characterize the mechanisms of action of several DNA damage response (DDR) proteins. With the generous support from the OCRA, Dr. Botuyan’s current work focuses on probing how DDR proteins RAD18, BARD1 and 53BP1 function together in regulating DSB repair by homologous recombination, with implications for cancer prevention and the long-term development of new therapeutics for ovarian cancer. Dr. Botuyan’s research has also been supported by the US Department of Defense Ovarian Cancer Research Program.