Article Figures & Data
Figures
- 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. 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. 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. 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. 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. 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. 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. 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. 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. 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
Primers used for site-directed mutagenesis.
Site-directed mutagenesis primer Sequence (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 DNA substrates used in study.
Substrate Oligo No. Sequence (5′–3′) Figure 5′-overhang 5′-FAM_42 GACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTG Figs 1C and E, 2C and E, and S1E sAS13 CAAAGATGTCCTAGCAAGGC Competitor to 5′-overhang (helicase assay) sAS14 GCCTTGCTAGGACATCTTTG Figs 1E and 2E ssDNA 5′-FAM_XO1 GACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTGCCCACCTGCAGGTTCACCC Figs 1D, 2D, and S1E double-stranded DNA 5′-FAM_XO1 GACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTGCCCACCTGCAGGTTCACCC Fig S1E XO1c GGGTGAACCTGCAGGTGGGCAAAGATGTCCTAGCAAGGCACTGGTAGAATTCGGCAGCGTC Y-structure 5′-FAM_42 GACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTG Fig S1E sAS36 CAAAGATGTCCTAGCAAGGCTTTTTTTTTTTTTTTTTTTTTT 3′-overhang 5′-FAM_42 GACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTG Fig S1E sAS38 TGGTAGAATTCGGCAGCGTC Primer template 5′-FAM_sAS50 GGGTGAACCTGCAGGTGG Fig 4B and C XO1 GACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTGCCCACCTGCAGGTTCACCC DNA block sAS51_3′-biotin TGTCCTAGCAAGGCA Competitor (primer extension assay) XO1c GGGTGAACCTGCAGGTGGGCAAAGATGTCCTAGCAAGGCACTGGTAGAATTCGGCAGCGTC siRNAs used in study.
siRNA Sequence (5′–3′) Figure siControl AGGUAGUGUAAUCGCCUUGtt Used throughout the study siDDX11 #1 GGCGUUAGCUCCCGUAGGAtt Used throughout the study siDDX11 #2 GAAUUCUGCCGGCGAAGAAtt Used in Fig S4A Primary and secondary antibodies used for Western blots.
Protein/tag Raised in Provider Catalogue no. β-actin (C4)-HRP Mouse Santa Cruz sc-47778 CHK1 (pSer345) Rabbit Cell Signaling 2348 Total CHK1 Mouse Santa Cruz sc-8408 DDX11 Mouse Santa Cruz sc-271711 DNA Pol δ (p125) Mouse Abcam ab196561 FLAG M2 Mouse Sigma-Aldrich F1804 Histone H3 Mouse Abcam ab10799 MMS19 Rabbit Proteintech 16015-1-AP PCNA Mouse Santa Cruz sc-56 RPA34 Mouse Thermo Fisher Scientific MA1-26418 WDHD1 Rabbit Novus Biologicals NBP1-89091 Mouse IgG, HRP-linked Sheep GE Healthcare NA931 Rabbit IgG, HRP-linked Donkey GE Healthcare NA934
