title: Mechanism of Ubiquitination and Deubiquitination in the Fanconi Anemia Pathway.
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.
authors.affiliation: Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia.
authors.forename: Sylvie
authors.initials: S
authors.lastname: van Twest
authors.affiliation: Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia.
authors.forename: Vincent J
authors.initials: VJ
authors.lastname: Murphy
authors.affiliation: Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Hodson
authors.affiliation: 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.
authors.forename: Winnie
authors.initials: W
authors.lastname: Tan
authors.affiliation: Architecture and Dynamics of Macromolecular Machines Laboratory, London Research Institute, South Mimms, Hertfordshire EN6 3LD, UK.
authors.forename: Paolo
authors.initials: P
authors.lastname: Swuec
authors.affiliation: 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.
authors.forename: Julienne J
authors.initials: JJ
authors.lastname: O'Rourke
authors.affiliation: 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.
authors.forename: Jörg
authors.initials: J
authors.lastname: Heierhorst
authors.affiliation: Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia.
authors.forename: Wayne
authors.initials: W
authors.lastname: Crismani
authors.affiliation: 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.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Deans
mesh_terms: D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005199:Fanconi Anemia / Q000235:genetics / Q000378:metabolism*; D052217:Fanconi Anemia Complementation Group A Protein / Q000378:metabolism; D052218:Fanconi Anemia Complementation Group C Protein / Q000378:metabolism; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000235:genetics / Q000378:metabolism*; D052239:Fanconi Anemia Complementation Group E Protein / Q000378:metabolism; D052241:Fanconi Anemia Complementation Group G Protein / Q000378:metabolism; D052240:Fanconi Anemia Complementation Group L Protein / Q000378:metabolism; D051856:Fanconi Anemia Complementation Group Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051797:Inhibitor of Differentiation Protein 2 / Q000378:metabolism; D046912:Multiprotein Complexes; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection; D064570:Ubiquitin-Specific Proteases / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D004247:DNA; D004268:DNA-Binding Proteins; D052217:Fanconi Anemia Complementation Group A Protein; D052218:Fanconi Anemia Complementation Group C Protein; D052236:Fanconi Anemia Complementation Group D2 Protein; D052239:Fanconi Anemia Complementation Group E Protein; D052241:Fanconi Anemia Complementation Group G Protein; D052240:Fanconi Anemia Complementation Group L Protein; D051856:Fanconi Anemia Complementation Group Proteins; D051797:Inhibitor of Differentiation Protein 2; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D064570:Ubiquitin-Specific Proteases; C523207:FAAP100 protein, human; C498471:FANCA protein, human; C515775:FANCB protein, human; C497651:FANCC protein, human; C499905:FANCD2 protein, human; C499907:FANCE protein, human; C499909:FANCG protein, human; C519921:FANCI protein, human; C497450:ID2 protein, human; C526165:WDR48 protein, human; C499908:FANCL protein, human; C579228:USP1 protein, human
keywords: DNA repair; FANCB; FANCD2; Fanconi anemia; RING E3; core complex; deubiquitination; enzyme mechanism; monoubiquitination