DNA replication stress leads to fork arrest, a major threat to genomic integrity. One mechanism for replication restart involves PRIMPOL repriming downstream of the arrested fork, leaving behind a single-stranded DNA (ssDNA) gap. Accumulation of these gaps is linked to hypersensitivity to genotoxic agents in certain genetic backgrounds, such as BRCA deficiency. This study investigates how PRIMPOL-generated ssDNA gaps are processed under replication stress induced by hydroxyurea and cisplatin. It shows that gaps are expanded in the 3'-5' direction by MRE11 exonuclease and in the 5'-3' direction by EXO1 exonuclease, ultimately converting them into double-stranded DNA breaks (DSBs). The de-ubiquitinating enzyme USP1 is identified as a critical regulator of these gaps. USP1 promotes gap accumulation during S-phase and their expansion by MRE11 and EXO1 nucleases. This activity is linked to USP1's role in de-ubiquitinating PCNA, suggesting that PCNA ubiquitination suppresses gap accumulation during replication. USP1 depletion suppresses DSB formation in PRIMPOL-overexpressing cells, highlighting an unexpected role for USP1 in promoting genomic instability under these conditions. The study also shows that USP1 de-ubiquitination activity promotes TLS-mediated gap filling. The findings suggest that USP1, through PCNA de-ubiquitination, promotes gap expansion by suppressing TLS-mediated gap filling, leading to DSB formation. These results highlight the importance of USP1 in maintaining genomic stability during replication stress.DNA replication stress leads to fork arrest, a major threat to genomic integrity. One mechanism for replication restart involves PRIMPOL repriming downstream of the arrested fork, leaving behind a single-stranded DNA (ssDNA) gap. Accumulation of these gaps is linked to hypersensitivity to genotoxic agents in certain genetic backgrounds, such as BRCA deficiency. This study investigates how PRIMPOL-generated ssDNA gaps are processed under replication stress induced by hydroxyurea and cisplatin. It shows that gaps are expanded in the 3'-5' direction by MRE11 exonuclease and in the 5'-3' direction by EXO1 exonuclease, ultimately converting them into double-stranded DNA breaks (DSBs). The de-ubiquitinating enzyme USP1 is identified as a critical regulator of these gaps. USP1 promotes gap accumulation during S-phase and their expansion by MRE11 and EXO1 nucleases. This activity is linked to USP1's role in de-ubiquitinating PCNA, suggesting that PCNA ubiquitination suppresses gap accumulation during replication. USP1 depletion suppresses DSB formation in PRIMPOL-overexpressing cells, highlighting an unexpected role for USP1 in promoting genomic instability under these conditions. The study also shows that USP1 de-ubiquitination activity promotes TLS-mediated gap filling. The findings suggest that USP1, through PCNA de-ubiquitination, promotes gap expansion by suppressing TLS-mediated gap filling, leading to DSB formation. These results highlight the importance of USP1 in maintaining genomic stability during replication stress.