1. Chromosomes and Gene Expression
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Monoubiquitination by the human Fanconi anemia core complex clamps FANCI:FANCD2 on DNA in filamentous arrays

  1. Winnie Tan
  2. Sylvie van Twest
  3. Andrew Leis
  4. Rohan Bythell-Douglas
  5. Vincent J Murphy
  6. Michael Sharp
  7. Michael W Parker
  8. Wayne Crismani
  9. Andrew J Deans  Is a corresponding author
  1. Genome Stability Unit, St. Vincent’s Institute of Medical Research, Australia
  2. Department of Medicine (St. Vincent’s Health), The University of Melbourne, Australia
  3. Bio21 Institute, University of Melbourne, Australia
  4. Structural Biology Unit, St. Vincent’s Institute of Medical Research, Australia
Research Article
Cite this article as: eLife 2020;9:e54128 doi: 10.7554/eLife.54128
7 figures, 2 tables and 2 additional files

Figures

Mono-ubiquitination does not alter interaction of FANCI:FANCD2 with DNA repair proteins.

(a) 35S-labelled FAN1, SLX4, FAAP20, RAP80, SMARCAD, FANCJ, PSMD4, SF3B1, TRIM25, MCM5, BRE, BRCC, SNM1A or luciferase (control) inputs were expressed using reticulocyte extracts. (b–c) The inputs prepared from (a) were incubated with the indicated FLAG-ID2 (b) or FLAG-IubD2ub (c) followed by FLAG pull-down and elution. The complexes were subjected to SDS-PAGE, and radiolabelled proteins were detected by autoradiography (representative experiment of n = 2). (d) Quantification showing percentage of ID2, IubD2ub or FLAG resin binding to inputs.

Monoubiquitination locks FANCI:FANCD2 on DNA.

(a) Schematic of the electrophoretic mobility shift assay (EMSA) using IRDye-700 labeled dsDNA. (b) Coomassie stained SDS-PAGE gel showing monoubiquitination of FANCI:FANCD2 using recombinant FA core complex and IR-dye700 labeled dsDNA. 25, 50 and 100 nM of ID2 or IKRD2KR were incubated with 25 nM of the IR-dye700 dsDNA for 90 min. The respective percentage of FANCI or FANCD2 monoubiquitination were calculated and shown under SDS-PAGE gel. (c) Monoubiquitination reactions from (b) were resolved on 6% native PAGE gel for EMSA analysis. The percentage of ID2 binding to DNA was calculated and shown under native PAGE gel.

Monoubiquitinated FANCI:FANCD2 binds to any type of dsDNA.

EMSA gels showing binding of monoubiquitinated or unmodified ID2 complex to different oligo-based DNA substrates. Above each panel, a schematic representing the tested DNA substrate is shown. 25, 50 and 100 nM of ID2 or IKRD2KR were incubated with 25 nM of the indicated DNA substrate and the protein:DNA complexes were resolved on 6% PAGE gels (top). The percentage of DNA binding was calculated and shown under each EMSA gel. Coomassie stained SDS-PAGE gel (bottom) showing the ubiquitination reactions used in the EMSA. The percentage of FANCD2 monoubiquitination was calculated and shown under each SDS-PAGE gel.

FANCD2 monoubiquitination is sufficient for FANCI:FANCD2 locking to DNA, but stimulated by FANCI monoubiquitination.

(a) Western blots of the EMSA gels containing 50, 100 and 200 nM of IubD2ub, ID2 or IKRD2KR in the presence of 25 nM IRDye-700 labeled dsDNA (red). Left panels correspond to anti-FANCI antibody (green) and right panels correspond to anti-FANCD2 antibody (green) (b) StrepII affinity purification of mono-ubiquitinated (+ATP) and non-ubiquitinated ID2 (-ATP). (c) EMSA gels (top) and western blots (bottom) showing the monoubiquitination of 25, 50 and 100 nM IWTD2WT, IKRD2WT, IWTD2KR or IKRD2KR in the presence of 25 nM IRDye-700 labeled dsDNA. (d) Western blots of the EMSA gels showing monoubiquitination of 50, 100 and 200 nM IWTD2WT, IKRD2WT, IWTD2KR or IKRD2KR in the presence of 25 nM IRDye-700 labeled dsDNA. FANCI (left, green) and FANCD2 (right, green) remained bound to IRDye-700 labeled DNA (red) after mono-ubiquitination.

Mutations in different ubiquitin patches do not affect ID2 mono-ubiquitination or DNA binding.

(a) Crystal structure of ubiquitin with ubiquitin mutant sites depicted (PDB: 1UBQ). Hydrophobic binding pockets are indicated in blue and pink. (b) Western blots showing the time course ubiquitination assays of ID2 using wild-type ubiquitin or ubiquitin F4A, V70A, I44A, D58A, K11R and L73P mutants. (c) EMSA gels showing 25, 50 and 100 nM monoubiquitinated ID2 binding to 25 nM IRDye-700 dsDNA using various ubiquitin mutants (top). Western blots of ID2 ubiquitination products were shown at the bottom and the percentage of FANCI and FANCD2 ubiquitination were shown at the bottom of each western blot panel.

Mono-ubiquitinated FANCI:FANCD2 complex assemble into filament-like arrays.

(a) Schematic of purification of monoubiquitinated FANCI:FANCD2 using Avi-ubiquitin. (b) Coomassie stained SDS-PAGE gel (top) and western blots (bottom) showing the purification of monoubiquitinated FANCI:FANCD2 complex eluted using PreScission protease (lanes 1–7) compared to without PreScission protease (lanes 8–14). (c–e) Representative negative-stained EM image of purified FANCIub:FANCD2ub complex bound to 2.7 kb plasmid, unmodified FANCI:FANCD2 incubated with 2.7 kb plasmid and unmodified FANCI:FANCD2 complex. Pseudo-colored regions are shown to highlight particular filament-like arrays in FANCIub:FANCD2ub but not other samples.

Monoubiquitinated FANCI:FANCD2 assembles into filamentous arrays along the length of dsDNA.

(a–d) Representative EM image of monoubiquitinated FANCI:FANCD2 bound to (a) 60 bp dsDNA, (b) 150 bp dsDNA, (c) 2.7 kb dsDNA, and (d) 2.7 kb dsDNA and Benzonase-treated. Scale bar, 100 nm. Arrows indicate formation of 1–2 ID2 array; boxes indicate multiple ID2 arrays. (e) Representative 2D class average of ID2. Views of the side and top of ID2 are shown, framed in blue for comparison. (f) Representative 2D class average of IubD2ub bound to 60 bp DNA. Views of the side and top of IubD2ub are shown, framed in red for comparison. (g–h) Example comparison of the length (y) and width (x) of class average images ID2 and IubD2ub (likely an overestimate, because uranyl formate staining increases apparent particle size).

Tables

Table 1
List of proteins containing ubiquitin binding domain that are described or predicted to bind to ubiquitinated FANCD2.
ProteinFunctionDomainReference
FAN1NucleaseUBZ4(Kratz et al., 2010)
SLX4NucleaseUBZ1(Lachaud et al., 2014)
FAAP20FANCA partnerUBZ(Hein et al., 2015)
RAP80BRCA1 partnerUIM(Castillo et al., 2014)
SMARCAD1Chromatin remodelerCUE(Densham et al., 2016)
FANCJHelicase-(Raghunandan et al., 2015)
PSMD4ProteaseUIM(Jacquemont and Taniguchi, 2007)
SF3B1RNA binding proteinUBZ(Moriel-Carretero et al., 2017)
TRIM25E3 ligaseRING finger(Lossaint et al., 2013)
MCM5CMG component-(Lossaint et al., 2013)
BREBRCA1 partner-(Wang, 2007)
BRCCBRCA1 partner-(Wang, 2007)
SNM1ANucleaseUBZ(Yang et al., 2010)
CtIP*Nuclease activatorC2H2 zinc finger(Murina et al., 2014)
Rev1*Translesion polymeraseUBZ3(Moldovan et al., 2010)
  1. *Indicates not tested in our experiments, because protein not produced in TnT system.

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Recombinant DNA reagent (X. laevis)pFastbac1-FLAG-xFANCI(Klein Douwel et al., 2014)Gift from Puck Knipscheer
Recombinant DNA reagent (X. laevis)pFastbac1-StrepII-xFANCD2(Klein Douwel et al., 2014)Gift from Puck Knipscheer
Recombinant DNA reagent (H. sapiens)pFL-EGFP-His-hFANCI(Tan et al., 2020a)
Recombinant DNA reagent (H. sapiens)pFastbac1-FLAG-hFANCD2opt(Tan et al., 2020a)RRID:Addgene_ 134904Gift from Angelos Constantinou
Recombinant DNA reagent (H. sapiens)pFL/pSPL-EGFP-FLAG-B-L-100(van Twest et al., 2017)Codon optimized FANCB
Recombinant DNA reagent (H. sapiens)pFL-MBP-C-E-F(van Twest et al., 2017)
Recombinant DNA reagent (H. sapiens)pGEX-KG-GST-UBE2T(van Twest et al., 2017)Codon optimized
Recombinant DNA reagent (E. coli)pet16b-Avi-ubiquitin_rbs_BirA(Tan et al., 2020a)RRID:Addgene_134897
Recombinant DNA reagent (E. coli)pSRK2706-GST-HRV-3Cprotease(Raran-Kurussi and Waugh, 2016)RRID:Addgene_78571A gift from David Waugh
Recombinant DNA reagent (E. coli)pUC19 plasmidNew England BioLabsN3041S
Strain, strain background (E. coli)BL21 (DE3)Agilent Technologies200131
Cell line (Spodoptera frugiperda)Sf9Thermo Fisher ScientificRRID:CVCL_0549Maintained in Sf-900 II SFM
Cell line (Trichoplusia ni)High FiveThermo Fisher ScientificRRID:CVCL_C190Maintained in Sf-900 II SFM
AntibodyRabbit polyclonal antibodies against StrepIIAbcamRRID:AB_76949one in 3000 dilution
AntibodyRabbit polyclonal antibodies against FANCIAbcamRRID:AB_74332one in 3000 dilution
AntibodyRabbit polyclonal antibodies against FANCD2AbcamRRID:AB_10862535one in 3000 dilution
AntibodyMouse monoclonal antibodies against FLAGAviva BiosciencesRRID:AB_10884242one in 3000 dilution
Peptide, recombinant proteinFLAG peptideSigma-AldrichF3290
Peptide, recombinant proteinRecombinant Human His6-Ubiquitin E1 Enzyme carrier freeBoston BiochemE-304–050
Peptide, recombinant proteinUbiquitin and associated mutant variantsBoston BiochemU-110H, UM-I44A, UM-D58A, UM-F4A, UM-L73P, UM-K11R
Commercial assay or kitTNT T7 Quick Coupled Transcription/Translation SystemPromega CorporationL1170
Commercial assay or kitAnti-FLAG-M2 affinity gelSigma AldrichRRID:AB_10063035
Chemical compound, drugEasyTagL-[35S]-MethioninePerkinElmer Life SciencesNEG709A500UC
Software, algorithmXMIPP(de la Rosa-Trevín et al., 2013)

Additional files

Supplementary file 1

(A) DNA oligonucleotides used in this study. The following oligonucleotides were ordered from IDTDNA. (B) Combination of oligonucleotides annealed to generate DNA substrates used in this study.

https://cdn.elifesciences.org/articles/54128/elife-54128-supp1-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/54128/elife-54128-transrepform-v2.docx

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