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The iron–sulfur helicase DDX11 promotes the generation of single-stranded DNA for CHK1 activation

Anna K Simon, Sandra Kummer, Sebastian Wild, Aleksandra Lezaja, View ORCID ProfileFederico Teloni, View ORCID ProfileStanislaw K Jozwiakowski, Matthias Altmeyer, View ORCID ProfileKerstin Gari  Correspondence email
Anna K Simon
1Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
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Sandra Kummer
1Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
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Sebastian Wild
1Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
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Aleksandra Lezaja
2Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
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Federico Teloni
2Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
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Stanislaw K Jozwiakowski
1Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
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Matthias Altmeyer
2Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
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Kerstin Gari
1Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
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  • For correspondence: gari@imcr.uzh.ch
Published 18 February 2020. DOI: 10.26508/lsa.201900547
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  • Figure S1.
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    Figure S1. FeS cluster binding is required for DNA binding by DDX11. Related to Fig 1.

    (A) Scheme of protein structure. In dark grey helicase domain 1 (HD1), in light grey helicase domain 2 (HD2), in orange FeS cluster domain. Roman letters depict the seven helicase motifs. The sequence of the FeS domain is expanded, and the four FeS cluster-coordinating cysteines are highlighted in orange and the conserved arginine-263 in blue. (B) Graphical representation of the multiple sequence alignment of DDX11’s iron–sulfur cluster-binding pocket. Consensus sequence was created with the help of the WebLogo3 tool (http://weblogo.threeplusone.com/create.cgi) using sequences from 20 different species. Cysteines are highlighted in orange; positively charged amino acids in blue and negatively charged amino acids in red. (C) Schematic representation of the experimental procedure of the iron incorporation assay. (D) Representative InstantBlue-stained SDS gel of pulled-down proteins in iron incorporation assay. Dotted line indicates that lanes were excised for clarity. (E) Electrophoretic mobility shift assays with 10 nM of different FAM-labelled DNA structures and 400 nM of DDX11 variants.

  • Figure 1.
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    Figure 1. FeS cluster binding is indispensable for DDX11’s biochemical activities.

    (A) Radioactive iron-55 incorporation in wild-type (wt) DDX11 and cysteine variants, as measured by liquid scintillation counting. Levels are expressed as % iron incorporation, with wild-type levels set to 100%. Error bars depict standard deviations from three independent experiments. Statistical analysis: ordinary one-way ANOVA (****P < 0.0001). (B) InstantBlue-stained SDS gel of purified DDX11 variants. (C) Electrophoretic mobility shift assays with 10 nM of FAM-labelled 5′-overhang substrate and increasing amounts of DDX11 variants. Numbers indicate the percentage of bound DNA from two independent experiments (Exp. 1 and Exp. 2). (D) ATPase activity of DDX11 variants in the presence of single-stranded DNA, as measured by the release of inorganic phosphate from radio-labelled γ-32P-ATP in TLC. Activity is depicted as % of hydrolysed ATP, with background activity in the absence of DNA subtracted. Error bars depict standard deviations from three independent experiments. Statistical analysis: ordinary one-way ANOVA (****P < 0.0001). (E) Helicase assays with 10 nM of FAM-labelled 5′-overhang substrate and increasing amounts of DDX11 variants. Numbers indicate the percentage of unwound DNA from two independent experiments (Exp. 1 and Exp. 2). See also Fig S1. MW, molecular weight.

    Source data are available for this figure.

    Source Data for Figure 1[LSA-2019-00547_SdataF1.tif]

  • Figure 2.
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    Figure 2. The positive charge at position 263 is important for FeS cluster binding and DDX11’s biochemical activities.

    (A) Radioactive iron-55 incorporation in wild-type (wt) DDX11 and arginine variants, as measured by liquid scintillation counting. Levels are expressed as % iron incorporation, with wild-type levels set to 100%. Error bars depict standard deviations from three independent experiments. Statistical analysis: ordinary one-way ANOVA (****P < 0.0001). (B) InstantBlue-stained SDS gel of purified DDX11 variants. (C) Electrophoretic mobility shift assays with 10 nM of FAM-labelled 5′-overhang substrate and increasing amounts of DDX11 variants. Numbers indicate the percentage of bound DNA from two independent experiments (Exp. 1 and Exp. 2). (D) ATPase activity of DDX11 variants in the presence of single-stranded DNA, as measured by release of inorganic phosphate from radio-labelled γ-32P-ATP in TLC. Activity is depicted as % of hydrolysed ATP, with background activity in the absence of DNA subtracted. Error bars depict standard deviations from three independent experiments. Statistical analysis: ordinary one-way ANOVA (****P < 0.0001; **P = 0.0045; ns, nonsignificant). (E) Helicase assays with 10 nM of FAM-labelled 5′-overhang substrate and increasing amounts of DDX11 variants. Numbers indicate the percentage of unwound DNA from two independent experiments (Exp. 1 and Exp. 2). See also Fig S2. MW, molecular weight.

  • Figure S2.
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    Figure S2. In the DDX11 variant R263K iron incorporation is partially restored. Related to Fig 2.

    (A) Representative InstantBlue-stained SDS gel of pulled-down proteins in iron incorporation assay.

  • Figure 3.
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    Figure 3. DDX11 interacts with Pol δ independently of its FeS cluster.

    (A) Gene Ontology term enrichment analysis of interaction partners obtained upon pull-down of YFP-DDX11 from HeLa Flp-In T-REx cells. (B) Flag pull-down of over-expressed Flag-tagged DDX11 from 293T cells and co-immunoprecipitated endogenous proteins. (C) Reciprocal co-immunoprecipitations of Flag-tagged POLD1 and untagged DDX11, and Flag-tagged DDX11 and untagged POLD1, respectively, extracted from 293T cells. (D) Co-immunoprecipitations of Flag-tagged DDX11 variants and untagged POLD1 from 293T cells. See also Fig S3, Tables S1, and S2. WB, Western blot.

  • Figure S3.
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    Figure S3. The interaction of DDX11 with WDHD1 does not depend on FeS cluster binding. Related to Fig 3.

    (A) Reciprocal co-immunoprecipitations of Flag-tagged WDHD1 and untagged DDX11, and Flag-tagged DDX11 and myc-tagged WDHD1, respectively, extracted from 293T cells. (B) Co-immunoprecipitations of Flag-tagged DDX11 variants and myc-tagged WDHD1 from 293T cells. (C) Scheme of DDX11 protein structure as in Fig S1A, and fragments used in Fig S3D. (D) Co-immunoprecipitation of YFP-tagged WDHD1 with full-length (FL) DDX11 and DDX11 fragments #1, #2, and #3 using GFP-trap beads. WB, Western blot.

  • Figure 4.
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    Figure 4. DDX11 can remove obstacles from the DNA template ahead of Pol δ.

    (A) Schematic of primer extension assay. Blue ellipse depicts 5′-FAM label on the primer that gets extended by Pol δ. Light grey circle depicts 3′-biotin on the DNA block that was added to prevent primer extension. Numbers indicate lengths of primers and gaps in nucleotides. (B) Time-resolved primer extension assay with substrate as depicted in A (15 nt-long DNA block) or without a DNA obstacle (no block). S denotes substrate only. In all other lanes, 2.5 nM of Pol δ was added in the absence or presence (25 nM) of DDX11 variants. N+7 denotes extension of primer by seven nucleotides. (C) Quantification of primer extension beyond the DNA block in the absence or presence of 25 nM DDX11 variants. Data points indicate values from two independent experiments. Lines connect the theoretical mean values.

  • Figure 5.
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    Figure 5. DDX11 promotes the formation of single-stranded DNA.

    (A, B) QIBC experiment showing the mean intensity per nucleus of native CldU indicative of single-stranded DNA (A) or chromatin-bound RPA (B) plotted against the total DAPI intensity per nucleus. The RPE-1 cells had been labelled for 24 h with the nucleotide analogue CldU followed by 1 h 30 min or 2 h of treatment with 2 mM HU and 2 μM ATRi. Control cells were left non-treated. The cells were pre-extracted before fixation. Levels of CldU and RPA are colour-coded, as indicated in the legends. (C) Software-based random selection of S-phase cells, as defined by an intermediate DNA content (>2N and <4N) and RPA positivity. Overlays of DAPI and the chromatin-bound RPA and native CIdU signals are shown. Scale bar, 20 μm. (D) Cell fractionation into cytoplasmic (Cyt), nucleoplasmic (Nuc), and chromatin (Chr) extracts in untreated cells (left) or cells treated with 2 mM HU and 2 μM ATRi for 2 h (right). Histone H3 was used as a chromatin marker. Numbers indicate the percentage of RPA on chromatin from two independent experiments (Exp. 1 and Exp. 2). A.U., arbitrary units; siC, siControl; WB, Western blot.

  • Figure 6.
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    Figure 6. DDX11 is required for CHK1 activation.

    (A, B, C) Western blots showing time course of CHK1-pS345 activation in control RPE-1 cells (siC) and cells depleted of DDX11 (siDDX11). (A, B, C) Cells were treated or not with 4 mM HU (A), 10 μM aphidicolin (Aph) (B), and 1 μM camptothecin (CPT) (C). Numbers indicate the mean values and standard deviations of the percentage of CHK1-pS345 per total CHK1 from three independent experiments. See also Fig S4. WB, Western blot.

  • Figure S4.
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    Figure S4. DDX11 is required for CHK1 activation. Related to Fig 6.

    (A) Western blots showing CHK1-pS345 activation in control RPE-1 cells (siC) and cells depleted of DDX11 (siDDX11) with two different siRNAs. Note that siRNA #1 was used throughout the study. The cells were treated or not with 4 mM HU for 2 h. Numbers indicate the percentage of CHK1-pS345 per total CHK1 from two independent experiments (Exp. 1 and Exp. 2). WB, Western blot.

Tables

  • Figures
  • Supplementary Materials
    • View popup

    Primers used for site-directed mutagenesis.

    Site-directed mutagenesis primerSequence (5′–3′)
    K50R (forward)CCAACTGGCACTGGGAGGTCCTTAAGTC
    K50R (reverse)GACTTAAGGACCTCCCAGTGCCAGTTGG
    R263Q (forward)GGTCTCCCTTGGCTCCCAGCAGAACCTTTG
    R263Q (reverse)CAAAGGTTCTGCTGGGAGCCAAGGGAGACC
    R263K (forward)GGTCTCCCTTGGCTCCAAGCAGAACCTTTGTG
    R263K (reverse)CACAAAGGTTCTGCTTGGAGCCAAGGGAGACC
    R263E (forward)GGTCTCCCTTGGCTCCGAGCAGAACCTTTGTG
    R263E (reverse)CACAAAGGTTCTGCTCGGAGCCAAGGGAGACC
    C267S (forward)CCCGGCAGAACCTTAGTGTAAATGAAGACGTG
    C267S (reverse)CACGTCTTCATTTACACTAAGGTTCTGCCGGG
    C350S (forward)GAGGCCCGGGCCAGTCCCTATTACGGG
    C350S (reverse)CCCGTAATAGGGACTGGCCCGGGCCTC
    • View popup

    DNA substrates used in study.

    SubstrateOligo No.Sequence (5′–3′)Figure
    5′-overhang5′-FAM_42GACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTGFigs 1C and E, 2C and E, and S1E
    sAS13CAAAGATGTCCTAGCAAGGC
    Competitor to 5′-overhang (helicase assay)sAS14GCCTTGCTAGGACATCTTTGFigs 1E and 2E
    ssDNA5′-FAM_XO1GACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTGCCCACCTGCAGGTTCACCCFigs 1D, 2D, and S1E
    double-stranded DNA5′-FAM_XO1GACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTGCCCACCTGCAGGTTCACCCFig S1E
    XO1cGGGTGAACCTGCAGGTGGGCAAAGATGTCCTAGCAAGGCACTGGTAGAATTCGGCAGCGTC
    Y-structure5′-FAM_42GACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTGFig S1E
    sAS36CAAAGATGTCCTAGCAAGGCTTTTTTTTTTTTTTTTTTTTTT
    3′-overhang5′-FAM_42GACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTGFig S1E
    sAS38TGGTAGAATTCGGCAGCGTC
    Primer template5′-FAM_sAS50GGGTGAACCTGCAGGTGGFig 4B and C
    XO1GACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTGCCCACCTGCAGGTTCACCC
    DNA blocksAS51_3′-biotinTGTCCTAGCAAGGCA
    Competitor (primer extension assay)XO1cGGGTGAACCTGCAGGTGGGCAAAGATGTCCTAGCAAGGCACTGGTAGAATTCGGCAGCGTC
    • View popup

    siRNAs used in study.

    siRNASequence (5′–3′)Figure
    siControlAGGUAGUGUAAUCGCCUUGttUsed throughout the study
    siDDX11 #1GGCGUUAGCUCCCGUAGGAttUsed throughout the study
    siDDX11 #2GAAUUCUGCCGGCGAAGAAttUsed in Fig S4A
    • View popup

    Primary and secondary antibodies used for Western blots.

    Protein/tagRaised inProviderCatalogue no.
    β-actin (C4)-HRPMouseSanta Cruzsc-47778
    CHK1 (pSer345)RabbitCell Signaling2348
    Total CHK1MouseSanta Cruzsc-8408
    DDX11MouseSanta Cruzsc-271711
    DNA Pol δ (p125)MouseAbcamab196561
    FLAG M2MouseSigma-AldrichF1804
    Histone H3MouseAbcamab10799
    MMS19RabbitProteintech16015-1-AP
    PCNAMouseSanta Cruzsc-56
    RPA34MouseThermo Fisher ScientificMA1-26418
    WDHD1RabbitNovus BiologicalsNBP1-89091
    Mouse IgG, HRP-linkedSheepGE HealthcareNA931
    Rabbit IgG, HRP-linkedDonkeyGE HealthcareNA934

Supplementary Materials

  • Figures
  • Tables
  • Table S1 List of putative interaction partners of YFP-tagged DDX11, as identified by mass spectrometry.

  • Table S2 Gene Ontology terms analysis using the PANTHER classification system tool.

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DDX11 promotes CHK1 activation
Anna K Simon, Sandra Kummer, Sebastian Wild, Aleksandra Lezaja, Federico Teloni, Stanislaw K Jozwiakowski, Matthias Altmeyer, Kerstin Gari
Life Science Alliance Feb 2020, 3 (3) e201900547; DOI: 10.26508/lsa.201900547

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DDX11 promotes CHK1 activation
Anna K Simon, Sandra Kummer, Sebastian Wild, Aleksandra Lezaja, Federico Teloni, Stanislaw K Jozwiakowski, Matthias Altmeyer, Kerstin Gari
Life Science Alliance Feb 2020, 3 (3) e201900547; DOI: 10.26508/lsa.201900547
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