BRCA2, a breast and ovarian tumor suppressor, is required for replication stress suppression through its role in repair of stalled replication forks. Here we report that inefficient resolution of stalled replication forks in absence of BRCA2 results in persistent accumulation of mostly un-phosphorylated RPA32 (at S33, T21 and S4/S8) at stalled replication forks. Furthermore, this ‘persistent’ accumulation of RPA32 coated ssDNA not only results in fork collapse, as marked by increased accumulation of both 53BP1 and Mre11 at the site of stalling, but also leads to suppression of nucleotide excision repair (NER), especially in S phase population. No such persistent RPA32 coated ssDNA is observed in BRCA1 depleted cells, suggesting that this intermediate is exclusive to BRCA2 deficiency. Furthermore, generation of this intermediate was not dependent on Mre11 implying that persistent accumulation of RPA32 coated ssDNA was not a result of excessive DNA processing by Mre11 exonuclease in absence of BRCA2. This is the first report of differences between BRCA1 and BRCA2 role at stalled replication forks. In this report, we also show that BRCA2 heterozygous primary mammary epithelial cells, and not BRCA1 heterozygous cells, are haploinsufficient for nucleotide excision repair. This would imply higher predisposition to skin lesions, including melanoma in BRCA2 mutation carriers. Risk for melanoma is indeed higher in BRCA2 mutation carriers compared to BRCA1 mutation carriers. We further confirmed that there exists a difference in mutational signature in whole genome sequences derived from BRCA1 and BRCA2 tumors, in line with the kind of differences expected if there is an increased NER defect in BRCA2 mutated cells when compared to BRCA1 mutated cells.

Our findings provide first such DNA repair mechanism based evidence that could potentially explain the differences in BRCA1 and BRCA2 mutation driven risk for melanoma. This mechanistic difference between BRCA1 and BRCA2 response at stalled replication forks, and subsequently a crosstalk with nucleotide excision repair, might also contribute towards other clinical differences observed in BRCA1 and BRCA2 mutation driven breast cancer.

Citation Format: Shailja Pathania, Rachel Reed, Haohui Duan, Aedin Culhane, Judy Garber. BRCA2, and not BRCA1, dependent crosstalk between replication stress suppression and nucleotide excision repair - Clinical implications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-162.