The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL

Jennifer A Miles; Mark G Frost; Eilis Carroll; Michelle L Rowe; Mark J Howard; Ateesh Sidhu; Viduth K Chaugule; Arno F Alpi; Helen Walden

doi:10.1074/jbc.M115.675835

The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL

J Biol Chem. 2015 Aug 21;290(34):20995-21006. doi: 10.1074/jbc.M115.675835. Epub 2015 Jul 6.

Authors

Jennifer A Miles¹, Mark G Frost², Eilis Carroll³, Michelle L Rowe⁴, Mark J Howard⁴, Ateesh Sidhu¹, Viduth K Chaugule², Arno F Alpi⁵, Helen Walden⁶

Affiliations

¹ 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.
² Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.
³ 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.
⁴ Protein Science Group, School of Biosciences, University of Kent, Canterbury, Kent CT2 7NZ, United Kingdom.
⁵ 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.
⁶ 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.

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.

Keywords: DNA repair; E3 ubiquitin ligase; isothermal titration calorimetry (ITC); nuclear magnetic resonance (NMR); structure; ubiquitin.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Animals
Binding Sites
DNA Repair*
Drosophila Proteins / chemistry*
Drosophila Proteins / genetics
Drosophila Proteins / metabolism
Drosophila melanogaster
Fanconi Anemia / genetics
Fanconi Anemia Complementation Group L Protein / chemistry*
Fanconi Anemia Complementation Group L Protein / genetics
Fanconi Anemia Complementation Group L Protein / metabolism
Fanconi Anemia Complementation Group Proteins / chemistry*
Fanconi Anemia Complementation Group Proteins / genetics
Fanconi Anemia Complementation Group Proteins / metabolism
Gene Expression Regulation
Humans
Models, Molecular
Molecular Sequence Data
Protein Binding
Protein Folding
Protein Interaction Domains and Motifs
Protein Structure, Secondary
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Sequence Alignment
Signal Transduction
Ubiquitin / chemistry*
Ubiquitin / genetics
Ubiquitin / metabolism
Ubiquitin-Conjugating Enzymes / genetics
Ubiquitin-Conjugating Enzymes / metabolism
Ubiquitination
Xenopus Proteins / chemistry*
Xenopus Proteins / genetics
Xenopus Proteins / metabolism
Xenopus laevis

Substances

Drosophila Proteins
FANCD2 protein, Xenopus
Fanconi Anemia Complementation Group Proteins
Recombinant Proteins
Ubiquitin
Xenopus Proteins
UBE2T protein, human
Ubiquitin-Conjugating Enzymes
FANCL protein, human
Fanconi Anemia Complementation Group L Protein

Associated data

PDB/1FBV
PDB/3K1L

Abstract

Publication types

MeSH terms

Substances

Associated data

Grants and funding