USP3 has been characterized as a functional DUB in vitro, and it is the human DUB most homologous to S. cerevisiae Ubp8, which regulates H2B deubiquitination2-4.

USP3 dynamically associates with chromatin and deubiquitinates H2A/H2B in vivo. The ZnF-UBP domain of USP3 mediates uH2AUSP3 interaction. Functional ablation of USP3 by RNAi leads to delay of S phase progression and to the accumulation of DNA breaks, with ensuing activation of DNA damage checkpoint pathways. In response to ionizing radiation, (1) uH2A redistributes and colocalizes in g-H2AX DNA repair foci, and (2) USP3 is required for full deubiquitination of ubiquitin-conjugates/uH2A and g-H2AX dephosphorylation. USP3 is a novel regulator of H2A and H2B ubiquitination, highlight its role in preventing replication stress and suggest its involvement in the response to DNA double-strand breaks1.