title: The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.
abstract: The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. 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.
authors.affiliation: 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.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Miles
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.
authors.forename: Mark G
authors.initials: MG
authors.lastname: Frost
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom; Scottish Institute for Cell Signalling, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.
authors.forename: Eilis
authors.initials: E
authors.lastname: Carroll
authors.affiliation: Protein Science Group, School of Biosciences, University of Kent, Canterbury, Kent CT2 7NZ, United Kingdom.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Rowe
authors.affiliation: Protein Science Group, School of Biosciences, University of Kent, Canterbury, Kent CT2 7NZ, United Kingdom.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Howard
authors.affiliation: 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.
authors.forename: Ateesh
authors.initials: A
authors.lastname: Sidhu
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.
authors.forename: Viduth K
authors.initials: VK
authors.lastname: Chaugule
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom; Scottish Institute for Cell Signalling, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom. Electronic address: A.F.Alpi@dundee.ac.uk.
authors.forename: Arno F
authors.initials: AF
authors.lastname: Alpi
authors.affiliation: 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; Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom. Electronic address: h.walden@dundee.ac.uk.
authors.forename: Helen
authors.initials: H
authors.lastname: Walden
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004260:DNA Repair; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster; D005199:Fanconi Anemia / Q000235:genetics; D052240:Fanconi Anemia Complementation Group L Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051856:Fanconi Anemia Complementation Group Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination; D029867:Xenopus Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D029721:Drosophila Proteins; C576817:FANCD2 protein, Xenopus; D051856:Fanconi Anemia Complementation Group Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; D029867:Xenopus Proteins; C513404:UBE2T protein, human; D044763:Ubiquitin-Conjugating Enzymes; C499908:FANCL protein, human; D052240:Fanconi Anemia Complementation Group L Protein
keywords: DNA repair; E3 ubiquitin ligase; isothermal titration calorimetry (ITC); nuclear magnetic resonance (NMR); structure; ubiquitin