Structural basis of FANCD2 deubiquitination by USP1-UAF1
Martin L Rennie1, Connor Arkinson2,3, Viduth K Chaugule2,3
1Institute of Molecular Cell and Systems Biology, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK. Martin.Rennie@glasgow.ac.uk.
Abstract:
Ubiquitin-specific protease 1 (USP1) acts together with the cofactor UAF1 during DNA repair processes to specifically remove monoubiquitin signals. One substrate of the USP1-UAF1 complex is the monoubiquitinated FANCI-FANCD2 heterodimer, which is involved in the repair of DNA interstrand crosslinks via the Fanconi anemia pathway. Here we determine structures of human USP1-UAF1 with and without ubiquitin and bound to monoubiquitinated FANCI-FANCD2. The crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1, two related proteases. A cryo-EM reconstruction of USP1-UAF1 in complex with monoubiquitinated FANCI-FANCD2 highlights a highly orchestrated deubiquitination process, with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for the requirement of both proteins, despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1-UAF1 regulation and substrate recognition.
Insights
Ubiquitin-specific protease 1 (USP1) and its cofactor UAF1 remove ubiquitin signals during DNA repair. Structural studies reveal how USP1-UAF1 interacts with and modifies the FANCI-FANCD2 complex for DNA interstrand crosslink repair.
Area of Science:
- Molecular Biology
- Structural Biology
- DNA Repair Mechanisms
Background:
- Ubiquitin-specific protease 1 (USP1) is crucial for DNA repair, acting with cofactor UAF1 to remove monoubiquitin.
- USP1-UAF1 targets the FANCI-FANCD2 heterodimer, essential for repairing DNA interstrand crosslinks via the Fanconi anemia pathway.
Purpose of the Study:
- To determine the structures of the human USP1-UAF1 complex.
- To elucidate the mechanism of USP1-UAF1 in complex with its substrate, monoubiquitinated FANCI-FANCD2.
- To understand the molecular basis of USP1-UAF1 regulation and substrate recognition.
Main Methods:
- X-ray crystallography to determine USP1-UAF1 structures with and without ubiquitin.
- Cryo-electron microscopy (cryo-EM) for the USP1-UAF1/monoubiquitinated FANCI-FANCD2 complex.
- Mutagenesis and biochemical assays to confirm protein interfaces.
Main Results:
- Crystal structures revealed USP1 plasticity and differences compared to related proteases (USP12-UAF1, USP46-UAF1).
- Cryo-EM showed USP1-UAF1 induces conformational changes in FANCI-FANCD2 during deubiquitination.
- An extensive UAF1-FANCI interface was identified, explaining the necessity of both proteins.
Conclusions:
- The study provides detailed molecular insights into the regulation and substrate recognition of the USP1-UAF1 deubiquitinating enzyme.
- Structural and biochemical data illuminate the mechanism of FANCI-FANCD2 modification in DNA repair.
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