Loss of BRCA1 or BRCA2 massively increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions

Loss-of-function mutations in the BRCA1 and BRCA2 genes increase the risk of tumour development. As both gene products play a role in homologous recombination repair, much research has focussed on the unstable genomic phenotype of BRCA1/2 mutant cells manifest mainly as large scale rearrangements. Here we employed whole genome sequencing of multiple isogenic chicken DT40 lymphoblast cell clones to precisely determine the consequences of BRCA1/2 loss on genomic mutagenesis. We found that spontaneous base substitution mutation rates increased eight-fold upon the disruption of either BRCA1 or BRCA2, and the arising mutation spectra were identical to a mutation signature associated with BRCA1/2 mutant tumours. We determined the mutation spectrum caused by the methylating agent methyl methanesulfonate (MMS), and showed that MMS also induces more mutations in BRCA1/2 deficient cells. Spontaneously arising and MMS-induced insertion/deletion mutations and large rearrangements were also more common in BRCA1/2 mutant cells compared to the wild type control, but BRCA2 mutants contained more short deletions, primarily in the 2-5 bp length range, which showed microhomology near the breakpoints suggesting repair by non-homologous end joining. The identical base substitution and different deletion phenotypes of BRCA1 and BRCA2 suggest that the two mutation classes are generated by different processes. An increased and prolonged gamma-H2AX signal in MMS-treated BRCA1/2 cells coupled with a reduced activation of the CHK1 kinase suggests an aberrant processing of stalled replication forks, which may lead to an increased use of mutagenic translesion synthesis for lesion bypass. The high rate of base substitution mutagenesis demonstrated by our experiments is likely to be a significant contributor to the oncogenic effect of the inactivation of BRCA1 or BRCA2.