Pretreatment of USP1/UAF1 with C527 resulted in inhibition of its enzyme activity with an IC 50 of 0.88±0.03 μM. C527 inhibits the DUB activity of the USP12/USP46 complex and other DUB enzymes in vitro . However, the IC 50 of C527 for these DUB enzymes was higher in comparison with USP1/UAF1 complex. C527 has considerably less inhibitory effect on UCH-L1 and UCH-L3, a different subclass of DUB enzymes. C527 treatments causes an increase in the levels of Ub-FANCD2 and Ub-FANCI. Pretreatment of cells with the C527 causes an enhancement in the cytoxicity of mitomycin C and camptothecin. C527 treatments lead to an increase in ubiquitinated forms of FANCD2 and FANCI, cause a decrease in homologous recombination activity, and sensitize cells to DNA damaging agents.