The study investigates the processing of single-stranded DNA (ssDNA) gaps generated by PRIMPOL during replication stress, using hydroxyurea and cisplatin treatments. The authors found that these gaps are expanded bidirectionally by the MRE11 and EXO1 nucleases, leading to their conversion into double-stranded DNA (DSBs). They identified USP1 as a critical regulator of PRIMPOL-generated ssDNA gaps, promoting gap accumulation and expansion by MRE11 and EXO1. USP1's role is linked to its de-ubiquitinating activity on Proliferating Cell Nuclear Antigen (PCNA), suggesting that PCNA ubiquitination suppresses gap accumulation. Depletion of USP1 suppressed DSB formation in PRIMPOL-overexpressing cells, highlighting its unexpected role in promoting genomic instability under these conditions. The findings provide insights into the mechanisms of ssDNA gap processing and their impact on genomic stability.The study investigates the processing of single-stranded DNA (ssDNA) gaps generated by PRIMPOL during replication stress, using hydroxyurea and cisplatin treatments. The authors found that these gaps are expanded bidirectionally by the MRE11 and EXO1 nucleases, leading to their conversion into double-stranded DNA (DSBs). They identified USP1 as a critical regulator of PRIMPOL-generated ssDNA gaps, promoting gap accumulation and expansion by MRE11 and EXO1. USP1's role is linked to its de-ubiquitinating activity on Proliferating Cell Nuclear Antigen (PCNA), suggesting that PCNA ubiquitination suppresses gap accumulation. Depletion of USP1 suppressed DSB formation in PRIMPOL-overexpressing cells, highlighting its unexpected role in promoting genomic instability under these conditions. The findings provide insights into the mechanisms of ssDNA gap processing and their impact on genomic stability.