Mutation of aspartic acid 199 in USP1 disrupts its deubiquitinating activity and impairs DNA repair

[摘要] The deubiquitinating enzyme USP1 contains highly conserved motifs forming its catalytic center. Recently, the COSMIC mutation database identified a mutation in USP1 at Asp-199 in endometrial cancer. Here, we investigated the role of Asp-199 for USP1 function. The mutation of aspartic acid to alanine (D199A) resulted in failure of USP1 to undergo autocleavage and form a complex with ubiquitin, indicating D199A Usp1 is catalytically inactive. The D199A mutation did not affect the interaction with Uaf1. Moreover, D199A Usp1 had defects in deubiquitination of FANCD2 and PCNA and displayed reduced FANCD2 foci formation and DNA repair efficiency. Furthermore, mutation of Asp-199 to glutamic acid resulted in phenotypes similar to the D199A mutation. Collectively, our findings demonstrate the importance of Asp-199 for USP1 activity and suggest the implications of USP1 downregulation in cancer.

[关键词] aspartic acid 199;deubiquitinating enzyme;FANCD2;homologous recombination repair;PCNA;USP1