Single-stranded DNA gaps are postulated to be fundamental to the mechanism of anti-cancer drugs. Gaining insights into their induction could therefore be pivotal for advancing therapeutic strategies. For poly (ADP-ribose) polymerase inhibitors (PARPi) to be effective, the presence of FANCJ helicase is required. However, the relationship between FANCJ dependent gaps and PARP1 catalytic inhibition or trapping-both linked to PARPi toxicity in BRCA deficient cells-is yet to be elucidated. Here, we find that the efficacy of PARPi is contingent on S-phase PARP1 activity, which is compromised in FANCJ deficient cells because PARP1, along with MSH2, is "sequestered" by G-quadruplexes. PARP1's replication activity is also diminished in cells missing a FANCJ-MLH1 interaction, but in such cells, depleting MSH2 can release sequestered PARP1, restoring PARPi-induced gaps and sensitivity. Our observations indicate that sequestered and trapped PARP1 are different chromatin-bound forms, with FANCJ loss increasing PARPi resistance in cells susceptible to canonical PARP1 trapping. However, in BRCA1 null cells, the loss of FANCJ mirrors the effects of PARP1 loss or inhibition, with the common detrimental factor being the loss of PARP1 activity during DNA replication, not trapping. These insights underline the crucial role of PARP1 activity during DNA replication in BRCA deficient cells and emphasize the importance of understanding drug mechanisms for enhancing precision medicine.