Ubiquitin chain editing promotes p97/VCP-dependent removal of RPA from DNA.

DNA repair requires dynamic control of proteins on single-stranded DNA (ssDNA), yet how ubiquitin signaling regulates ssDNA-bound factors remains poorly understood. Here, we identify in human cells a ubiquitin chain-editing mechanism that promotes extraction of replication protein A (RPA) from ssDNA. We show that RPA stimulates the deubiquitinase ZUP1 and enhances its activity toward K63 linkages within ubiquitin chains. In response to DNA damage, RPA is modified with branched K48-K63 ubiquitin chains. ZUP1 selectively removes K63 linkages from these chains, remodeling the ubiquitin signal on RPA. We show that ZUP1-mediated editing of branched ubiquitin chains promotes p97/VCP-dependent removal of ubiquitinated RPA from ssDNA. Loss of ZUP1 causes accumulation of branched ubiquitin chains on RPA and pathological RPA trapping on ssDNA, resulting in elevated ssDNA signaling and genome instability. Together, our findings identify ubiquitin chain editing as a mechanism that controls RPA dynamics on ssDNA and enables p97/VCP-mediated protein extraction during DNA repair.