EXO1-mediated ssDNA gap expansion is essential for ATR activation and to maintain viability in BRCA1-deficient cells.

Abstract

DNA replication faces challenges from DNA lesions originated from endogenous or exogenous sources of stress, leading to the accumulation of single-stranded DNA (ssDNA) that triggers the activation of the ATR checkpoint response. To complete genome replication in the presence of damaged DNA, cells employ DNA damage tolerance mechanisms that operate not only at stalled replication forks but also at ssDNA gaps originated by repriming of DNA synthesis downstream of lesions. Here, we demonstrate that human cells accumulate post-replicative ssDNA gaps following replicative stress induction. These gaps, initiated by PrimPol repriming and expanded by the long-range resection factors EXO1 and DNA2, constitute the principal origin of the ssDNA signal responsible for ATR activation upon replication stress, in contrast to stalled forks. Furthermore, we show that EXO1-deficient cells exhibit marked sensitivity to translesion synthesis inhibition, a distinctive characteristic of mutations in proteins essential for repairing ssDNA gaps via template switching, such as BRCA1/2. Strikingly, EXO1 loss results in synthetic lethality when combined with BRCA1 deficiency, but not BRCA2. Indeed, BRCA1-deficient cells become addicted to the overexpression of EXO1 DNA2 or BLM. This dependence on long-range resection unveils a new vulnerability of BRCA1-mutant tumors, shedding light on potential therapeutic targets for these cancers.