Understanding the high mutation frequency of cancer cells is key to finding ways to prevent or delay the onset of disease and subsequent chemoresistance. Errors in repair of DNA double-strand breaks (DSBs) can cause mutations, hence understanding how repair pathways act is of essential importance for the development of new strategies to reduce the frequency of tumors and the appearance of chemoresistance. Mammalian DSB repair is mediated by nonhomologous end-joining (NHEJ) and homologous recombination (HR). Whereas HR uses a homologous template for repair, NHEJ acts by ligation of DSBs. The pathways therefore produce different repair outcomes, and the relative rate of usage of NHEJ and HR is regulated in cells by processes which are poorly understood. Cell cycle appears to be a major determinant of usage of the HR pathway, with HR used infrequently in G1 somatic cells. A second regulator of pathway choice is the DNA damage response factor, 53BP1. 53BP1 limits resection of DSBs, a key step in HR. 53BP1 therefore promotes NHEJ at the expense of HR. Using genetic approaches in primary mouse cells, we propose to measure how 53BP1 interacts with cellular activities including BRCA1, BLM and Exo1 to mediate DSB resection, a key step regulating the use of HR versus NHEJ. We will furthermore test whether the effect of 53BP1 in limiting resection is required for suppressing genomic instability and tumorigenesis arising from defects in DNA repair in non-dividing cells. Finally, we propose to determine how BRCA1 interacts with PALB2, a repair factor that helps recruit BRCA2 to break sites. BRCA1 and PALB2 form a complex through interactions of predicted coiled-coil regions, and we will identify and characterize the exact molecular mechanism of interaction using biophysical and cell biology techniques. Together, these studies will advance our understanding of critical commitment steps to HR in primary mammalian cells, and inform strategies for the design of rational therapies for treatment of individuals with mutations in key DNA repair genes.
