Late steps of allelic break-induced replication suppress tandem duplication associated with BRCA1 deficiency

Microhomology-mediated tandem duplication (TD) is a distinct mutational signature in BRCA1-deficient tumors. While several mechanisms have been proposed for its generation, much understanding comes from repeat-based reporters, which may not represent the region of the human genome lacking neighboring repeats. To address this limitation, we developed a repeat-less TD reporter and a PCR-based endogenous site-specific TD assay to examine TDs induced by replication-coupled single-ended DNA double-strand breaks (seDSBs) in Brca1-deficient mouse embryonic stem cells. We found that TDs from seDSBs were detectable in normal cells but significantly increased in Brca1-deficient cells. This increase did not involve classical non-homologous end joining. TD formation appeared to be facilitated by a switch to microhomology-mediated non-allelic sister chromatid recombination from initial allelic DNA synthesis, suggesting that allelic DNA synthesis, a late step of allelic break-induced replication (aBIR), is more prone to premature termination in Brca1-deficient cells, promoting TDs. Disrupting RAD51 loading reduced TDs in wild-type cells but not in Brca1-deficient cells, indicating both RAD51-dependent and -independent pathways for TD formation. Additionally, RAD54 and BRCA1-BARD1 suppressed TDs in a RAD51-dependent manner, with BARD1's effect partially mediated through RAD51 interaction. These findings together implicate late steps of aBIR in TD suppression and provide novel insights into the mechanisms underlying BRCA1-linked TD formation in cancer.