53BP1-independent Shieldin-BRCA1 antagonism at replication-coupled double-strand breaks

BRCA1-deficient tumors are hypersensitive to PARP inhibitors (PARPi) due to impaired homologous recombination (HR). The 53BP1-Shieldin complex inhibits end resection at replication-independent DNA double-strand breaks (DSBs), promoting PARPi sensitivity in BRCA1-deficient cells. However, its role in replication-coupled DSBs is less clear. Here, we show that loss of Shld2 or Shld3, but not 53bp1, confers PARPi resistance in mouse embryonic stem cells lacking a functional BRCA1 BRCT domain. Unlike 53bp1 loss, deletion of Shld2 or Shld3 partially restores HR at replication-coupled DSBs, reduces BRCA1-linked insertion/deletion signatures, and promotes RAD51 loading. This 53BP1-independent function requires the CST complex and counteracts residual BRCA1 coiled-coil domain activity in RAD51 loading. Shld2 loss also confers PARPi resistance in Bard1-null cells retaining residual BRCA1, but not in cells expressing RING-less BRCA1 with degraded BARD1. These findings identify a 53BP1-independent function for Shieldin in sustaining HR deficiency in BRCA1-deficient cells, providing new mechanistic insights into PARPi resistance.