The Fanconi  Anemia pathway (FA) is critical for repair of DNA damage caused by interstrand crosslinks (ICLs). The pathway is complex, consisting currently of 15 proteins (in vertebrates). The key event of the pathway is the monoubiquitination of downstream targets FANCD2 and FANCI, which recruit the DNA repair machinery. The E3 ligase activity that carries out the monoubiquitination event resides in the Fanconi Core Complex (CC), which consists of 8 proteins.  Patient mutations in any of the CC proteins prevent the monoubiquitination of targets, FANCD2 and FANCI. Interestingly, E3 ligase activity is associated with only one of these 8 CC proteins, FANCL.

To understand the molecular details of the critical monoubiquitination event, we use both a structural and biochemical approach.

We were able to express and purify domains of the human FANCL protein and subsequently able to solve 50% of the human FANCL structure. The structure reveals that it is not a WD40 protein as predicted. Comparison of the human FANCL structure with the drosophila (previously solved by our lab) reveals a subtle secondary structural difference. Also surface chemical composition analyses reveal differences, accounting for the low sequence identity between the two (only 20%). Furthermore we have shown that the RING domain of human FANCL is sufficient for binding its E2, Ube2t, at a sub-micromolar affinity and the central URD domain is sufficient for binding substrates, FANCD2 and FANCI.

Unveiling the structure of FANCL and subsequently understanding FANCL’s protein-protein interactions will allow a greater understanding of the biochemistry of the FA pathway and the role of E3 ligases in selective monoubiquitination.