PRT4165 is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. It inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. U2OS cells treated with increasing concentrations of PRT4165 show increasing numbers of cells in G2/M^[2].