Abstract

Ubiquitin signaling is regulated by deubiquitinating enzymes (DUBs), which can edit or remove ubiquitin from substrates. Recently, ZUP1 was discovered as the founding and only member of a novel DUB class that cleaves long K63-linked ubiquitin chains, with a putative role in DNA repair. However, ZUP1 has poor activity on its own, suggesting additional mechanisms exist to promote its activity in cells. Here, using a range of cellular, biochemical, and structural proteomics approaches, we show that ZUP1 directly interacts with the RPA complex, a single-stranded DNA binding protein complex involved in DNA replication and multiple DNA repair processes. Functionally, the ZUP1-RPA complex interaction dramatically stimulates ZUP1 K63-linked DUB activity, which occurs through S1 and S1 site communication. Collectively, our results suggest a mechanism that couples sensing of ssDNA to the activation of K63-linked deubiquitination via the ZUP1-RPA complex.