Mechanism of Ubiquitination and Deubiquitination in the Fanconi Anemia Pathway

Sylvie van Twest; Vincent J Murphy; Charlotte Hodson; Winnie Tan; Paolo Swuec; Julienne J O'Rourke; Jörg Heierhorst; Wayne Crismani; Andrew J Deans

doi:10.1016/j.molcel.2016.11.005

Mechanism of Ubiquitination and Deubiquitination in the Fanconi Anemia Pathway

Mol Cell. 2017 Jan 19;65(2):247-259. doi: 10.1016/j.molcel.2016.11.005. Epub 2016 Dec 13.

Authors

Sylvie van Twest¹, Vincent J Murphy¹, Charlotte Hodson¹, Winnie Tan², Paolo Swuec³, Julienne J O'Rourke², Jörg Heierhorst⁴, Wayne Crismani¹, Andrew J Deans⁵

Affiliations

¹ Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia.
² Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia; Department of Medicine (St. Vincent's Health), The University of Melbourne, VIC 3010, Australia.
³ Architecture and Dynamics of Macromolecular Machines Laboratory, London Research Institute, South Mimms, Hertfordshire EN6 3LD, UK.
⁴ Molecular Genetics Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia; Department of Medicine (St. Vincent's Health), The University of Melbourne, VIC 3010, Australia.
⁵ Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia; Department of Medicine (St. Vincent's Health), The University of Melbourne, VIC 3010, Australia. Electronic address: adeans@svi.edu.au.

PMID: 27986371
DOI: 10.1016/j.molcel.2016.11.005

Abstract

Monoubiquitination and deubiquitination of FANCD2:FANCI heterodimer is central to DNA repair in a pathway that is defective in the cancer predisposition syndrome Fanconi anemia (FA). The "FA core complex" contains the RING-E3 ligase FANCL and seven other essential proteins that are mutated in various FA subtypes. Here, we purified recombinant FA core complex to reveal the function of these other proteins. The complex contains two spatially separate FANCL molecules that are dimerized by FANCB and FAAP100. FANCC and FANCE act as substrate receptors and restrict monoubiquitination to the FANCD2:FANCI heterodimer in only a DNA-bound form. FANCA and FANCG are dispensable for maximal in vitro ubiquitination. Finally, we show that the reversal of this reaction by the USP1:UAF1 deubiquitinase only occurs when DNA is disengaged. Our work reveals the mechanistic basis for temporal and spatial control of FANCD2:FANCI monoubiquitination that is critical for chemotherapy responses and prevention of Fanconi anemia.

Keywords: DNA repair; FANCB; FANCD2; Fanconi anemia; RING E3; core complex; deubiquitination; enzyme mechanism; monoubiquitination.

MeSH terms

Cell Line
DNA / genetics
DNA / metabolism
DNA-Binding Proteins / metabolism
Fanconi Anemia / genetics
Fanconi Anemia / metabolism*
Fanconi Anemia Complementation Group A Protein / metabolism
Fanconi Anemia Complementation Group C Protein / metabolism
Fanconi Anemia Complementation Group D2 Protein / genetics
Fanconi Anemia Complementation Group D2 Protein / metabolism*
Fanconi Anemia Complementation Group E Protein / metabolism
Fanconi Anemia Complementation Group G Protein / metabolism
Fanconi Anemia Complementation Group L Protein / metabolism
Fanconi Anemia Complementation Group Proteins / genetics
Fanconi Anemia Complementation Group Proteins / metabolism*
Humans
Inhibitor of Differentiation Protein 2 / metabolism
Multiprotein Complexes
Nuclear Proteins / metabolism
Protein Binding
Protein Multimerization
Recombinant Proteins / metabolism
Substrate Specificity
Time Factors
Transfection
Ubiquitin-Specific Proteases / metabolism
Ubiquitination*

Substances

DNA-Binding Proteins
FAAP100 protein, human
FANCA protein, human
FANCB protein, human
FANCC protein, human
FANCD2 protein, human
FANCE protein, human
FANCG protein, human
FANCI protein, human
Fanconi Anemia Complementation Group A Protein
Fanconi Anemia Complementation Group C Protein
Fanconi Anemia Complementation Group D2 Protein
Fanconi Anemia Complementation Group E Protein
Fanconi Anemia Complementation Group G Protein
Fanconi Anemia Complementation Group Proteins
ID2 protein, human
Inhibitor of Differentiation Protein 2
Multiprotein Complexes
Nuclear Proteins
Recombinant Proteins
USP1 associated factor 1, human
DNA
FANCL protein, human
Fanconi Anemia Complementation Group L Protein
USP1 protein, human
Ubiquitin-Specific Proteases