The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.
Bottom Line: The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown.We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro.However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo.
Affiliation: From the Protein Structure and Function Laboratory, Lincoln's Inn Fields Laboratories of the London Research Institute, Cancer Research, United Kingdom, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom.Show MeSH
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Mentions: Several E2-RING E3 ligase interactions are enhanced by the presence of the ubiquitin thioester bound on the E2 (34, 35). Therefore another explanation is that FANCL interaction with ubiquitin enhances the recognition of ubiquitin-charged Ube2T (Ube2T∼Ub). To test this hypothesis, we generated a stable Ube2T∼Ub ester and assayed FANCL binding via pull-down. We observed no difference in the levels of FANCL binding in a comparison between ubiquitin-charged Ube2T and uncharged Ube2T (Fig. 7A). Furthermore, there was no difference in Ube2T∼Ub binding when the ubiquitin binding surface of ELF was mutated. These data suggest that ubiquitin binding does not enhance FANCL's interaction with E2.
Affiliation: From the Protein Structure and Function Laboratory, Lincoln's Inn Fields Laboratories of the London Research Institute, Cancer Research, United Kingdom, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom.