LAUSR

Abstract 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. While TDs induced by seDSBs were detectable in wild-type cells, they were significantly elevated in Brca1-deficient cells, independent of classical non-homologous end joining. Some of these TDs arose from allelic DNA synthesis followed by a shift to microhomology-mediated non-allelic sister chromatid recombination, suggesting increased premature termination of allelic DNA synthesis, a late step of allelic break-induced replication (aBIR), in Brca1-deficient cells. Disrupting RAD51 loading reduced TDs in wild-type cells but not in Brca1-deficient cells, indicating both RAD51-dependent and -independent TD formation. Additionally, RAD54 and BRCA1–BARD1 suppressed TDs in a RAD51-dependent manner, with the effect of BARD1 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.