The Fanconi anemia (FA) pathway maintains genome stability by promoting the accumulation of monoubiquitinated heterodimers of FANCI and FANCD2 (ID2) at blocked DNA replication forks. Ubiquitination clamps the ID2 complex onto DNA to help promote DNA repair, and tolerate treatment with DNA damaging chemotherapies. USP1:UAF1 is the canonical ID2 deubiquitinating enzyme complex; and for this reason USP1 is a promising therapeutic target for cancer treatment. But we and others have shown that USP1:UAF1 is not fully sufficient for the deubiquitination of the FANCI subunit, and only acts on FANCD2.
To explore ubiquitination and deubiquitination, we developed a complete biochemical reconstitution of the FA pathway – requiring at least 16 recombinant proteins, specific DNA molecules, and unique phosphorylation states. We show that when bound to DNA, phosphorylated ID2 complex is rapidly monoubiquitinated in our in vitro reactions. Two DNA-clamped products are formed: complex 1 of FANCD2-Ub:FANCI and complex 2 of FANCD2-Ub:FANCI-Ub. Importantly, we see that recombinant USP1:UAF1 can rapidly deubiquitinate complex 1, but not complex 2.
By screening a panel of deubiquitinating enzymes in cell-based and biochemical experiments, we identified USP36 as a candidate second ubiquitin-removing enzyme in the FA pathway. We show, using optimised conditions, that USP36 can rapidly convert complex 2 to complex 1. A catalytically inactive variant of USP36 cannot perform the reaction. USP36 cooperates with USP1:UAF1 to convert the ID2 complex to a fully unubiquitinated state. Importantly, we show that overexpression of USP36, or highly active truncated variants found in some cancers, leads to the suppression of the FA pathway in cancer cells.
Our results show how ubiquitination and deubiquitination of ID2 complex controls DNA repair outcomes in a temporally and spatially controlled fashion to maintain genome stability. USP36, like USP1:UAF1, may be an important therapeutic target in the promotion of chemotherapy sensitivity.