A STUDY OF THE BRCA1 COILED-COIL REGION IN MOUSE DEVELOPMENT, TUMOR SUPPRESSION, AND DRUG RESISTANCE.

Abstract

Homologous Recombination (HR) is a major double-stranded break (DSB) DNA repair pathway. It utilizes the sister chromatid as a template, thus promoting error-free repair. The initial HR step is DNA end resection with the creation of RPA coated single-stranded DNA (ssDNA) overhangs. This is followed by RAD51 loading onto the resected DNA, triggering strand invasion, and homology search. The BRCA1 protein is fundamental for HR mediated DNA repair, and patients with germline BRCA1 mutations have a high risk of developing cancer. These BRCA1 mutant tumors are HR deficient, and as a result, sensitive to PARP inhibitors (PARPi). BRCA1 likely promotes DNA end resection by displacing 53BP1 from the DSBs and also promotes RAD51 loading by enabling the formation of the larger BRCA1-PALB2-BRCA2 complex trough its coiled-coil region. Although BRCA1 functions in both HR steps, its essential role in each step is unclear, with previous reports showing that RAD51 loading after DNA end resection occurred could be BRCA1 independent. Here, we demonstrated that full HR restoration after activation of end resection mediated by 53BP1 knockout (KO), requires the expression of a BRCA1 protein that retained the coiled-coil region and its protein interactions. We observed that although 53bp1 KO can restore the Mendelian frequencies of a Brca1 null mouse model, mice were still tumor prone, PARPi sensitive, and had low levels of RAD51 γ-irradiation-induced foci (IRIF), suggesting that HR levels were minimal. Moreover, in order to significantly increase PARPi resistance in human cancer cell lines, 53BP1 loss of function (LoF) needs to be associated with the expression of a hypomorphic BRCA1 protein that retained the PALB2 interaction. We also demonstrated that Brca1 compound heterozygosity could rescue the developmental defects observed in Brca1 mutant homozygous mice, as long as the alleles retained complementary HR-related functions. Additionally, these compound heterozygotes mice were not tumor prone and had a normal lifespan, suggesting that HR was restored. In contrast, when the allele combination did not exhibit complementary functions, the developmental defects persisted. Overall, we demonstrated that to fully function in mouse development, tumor suppression, and drug resistance, BRCA1 role is critical in both the DNA end resection and the RAD51 loading HR steps.