SUMMARY

promotes DNA repair. It causes chromatin ac-cumulation of FANCD2 and facilitates PCNA’s recruitment of translesion polymerases to stalled replication. USP1, a protease that removes monoubiquitin from FANCD2 and PCNA, was thought to reverse the DNA damage response of these substrates. We disrupted USP1 in chicken cells to dissect its role in a sta-ble genetic system. USP1 ablation increases FANCD2 and PCNA monoubiquitination but unexpectedly results in DNA crosslinker sensi-tivity. This defective DNA repair is associated with constitutively chromatin-bound, mono-ubiquitinated FANCD2. In contrast, persistent PCNA monoubiquitination has negligible im-pact on DNA repair or mutagenesis. USP1 was previously shown to autocleave after DNA dam-age. In DT40, USP1 autocleavage is not stim-ulated by DNA damage, and expressing a noncleavable mutant in the USP1 knockout strain partially rescues crosslinker sensitivity. We conclude that efficient DNA crosslink repair requires FANCD2 deubiquitination, whereas FANCD2 monoubiquitination is not dependent on USP1 autocleavage