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: The ELF domain of FANCL shares significant structural homology with E2-conjugating enzymes (19). E2s form a catalytic intermediate with ubiquitin via a thioester between the catalytic cysteine and the C terminus of ubiquitin (29). The ELF domain does not possess a catalytic cysteine. However, an additional feature of E2s is that they can also interact non-covalently with ubiquitin (30, 31). Therefore we hypothesized that the ELF domain of FANCL might interact with ubiquitin in a similar manner. To test this hypothesis, we performed a pull-down binding assay using 6× His-tagged ubiquitin as bait (Fig. 1A). His-ubiquitin pulls down both full-length Drosophila FANCL and the isolated ELF domain. In contrast, FANCL lacking the ELF domain (ΔELF) is not pulled down. To further characterize the interaction between the ELF domain and ubiquitin, we measured the affinity of binding using isothermal titration calorimetry (ITC), and determined a dissociation constant of 42 ± 12.0 μm (Fig. 1B).
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.