Abstract
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.
Highlights
Vertebrates repair DNA interstrand crosslinks through a complex and essential process[1]
We find that FANCD2 itself forms a homodimer that is unable to interact with DNA, but this can interchange with FANCI, to create a D2–I heterodimer that interacts with DNA
The precise role of DNA in monoubiquitination of FANCD2 was unknown. It had remained unclear how the monoubiquitinated lysine on FANCD2 was accessed by UBE2T and the Fanconi anemia (FA) core complex, since it was occluded at the interface of FANCD2 and FANCI in a previous crystal structure[31]
Summary
Vertebrates repair DNA interstrand crosslinks through a complex and essential process[1]. Autosomal recessive mutations in any one of 20 genes (FANCA to FANCQ) result in this genetic illness, and collectively the FANC gene products function in a FA DNA crosslink repair pathway[3]. An understanding of this pathway is emerging from cell biological and biochemical reconstitution studies[4,5,6,7,8,9]. These indicate that a number of the FANC gene products form a large nuclear E3 monoubiquitin ligase complex (the FA core complex). Genetic studies indicate that reversal of the monoubiquitination step by the deubiquitinating enzyme USP1, in complex with UAF1, is required to complete DNA crosslink repair[21,22,23]
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