FANCD2–FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair

Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2–FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryoEM to determine the structure of recombinant chicken FANCD2 and FANCI complexes. FANCD2–FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA. Ubiquitin is positioned at the interface of FANCD2 and FANCI, and acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2–FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors.