Polymerase iota Promotes Mitotic DNA Synthesis in BRCA2-Deficient Mammary Epithelial Cells

BRCA2 deficiency induces chronic replication stress, yet how epithelial cells adapt to this stress prior to malignant transformation remains poorly defined. Mammary epithelial cells from BRCA2 mutation carriers exhibit early replication-associated genome instability, suggesting that stress-tolerance mechanisms operate before overt cancer development. Here, we identify polymerase iota (Pol ι) as a context-dependent regulator of replication stress processing in BRCA2-deficient mammary epithelium.Analysis of publicly available single-cell transcriptomic data from BRCA2 mutation carriers revealed a selective upregulation of POLI, which we mechanistically validated using primary and non-transformed mammary epithelial cells with experimentally reduced BRCA2 expression. We show that BRCA2 loss induces Pol ι expression and creates a selective dependence on Pol ι for mitotic DNA synthesis (MiDAS), a pathway that completes replication of under-replicated DNA.Unexpectedly, loss of Pol ι in BRCA2 deficient cells does not exacerbate genome instability but instead reduces canonical markers of replication-associated damage, including 53BP1 nuclear bodies, micronuclei, and chromosomal breaks. Mechanistically, Pol ι restrains replication fork progression under stress in BRCA2-deficient cells, increasing the persistence of under-replicated DNA carried into mitosis and promoting MiDAS engagement. Loss of Pol ι releases this constraint, accelerating fork progression and reducing reliance on mitotic replication.Together, these findings define a previously unrecognized, BRCA2-dependent replication stress program in which Pol ι governs how under-replicated DNA is processed across the S-phase-to-mitosis transition. This work reveals a context-specific vulnerability in BRCA2-deficient mammary epithelium that precedes malignant transformation and reshapes our understanding of early genome instability in BRCA2 mutation carriers.