Fig. 3: DDX41 opposes R-loop accumulation and can unwind hybrids in vitro.

a Proximity ligation assay (PLA) between endogenous DDX41 and GFP-tagged HBD or HBD-WKK. Representative images and quantification of nuclear PLA spots. Dots represent results from individual cells, black line indicates the median. p-value < 0.0001 derived from > 50 cells from n = 1 experiment using two-sided Student’s t-test. Scale bars—20 µm. b S9.6 and double-stranded DNA dot-blot analysis of U2OS cells expressing GFP-tagged M27-RNaseH1 upon doxycycline (dox) after 48 h of indicated knockdowns. Representative images (left). Data are represented as mean ± standard deviation (right). Black dots represent individual results from n = 2 biologically independent experiments. p-values (p = 0.437, p = 0.653, p = 0.338, p = 0.0281) derived using one-way ANOVA with Tukey correction for multiple comparisons. c HBD-GFP retention assay after indicated 48 h knockdowns in U2OS cells. Control cells were treated with 100 µM DRB for 3 h. Center of boxplots indicates the median of the population, limits the 25th–75th percentile, whiskers the 10th–90th percentile, and dots represent outliers. Representative data of n = 3 biologically independent experiments are shown. p-values (p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001) derived from n > 1000 cells using one-way ANOVA with Tukey correction for multiple comparisons. d Fluorescence polarization (FP) assay of full-length DDX41 and indicated 6-FAM-conjugated oligonucleotides in n = 2 independent experiments with individually thawed protein aliquots. The protein concentration on a log₂-scale is plotted against the FP in mP (milipolarization unit). Data are represented as mean values ± standard deviation. Colored lines represent Michaelis–Menten fits. e FRET-based RNA–DNA hybrid displacement assay. Titrated full-length (FL) DDX41 is incubated with 100 nM RNA–DNA hybrid substrate and 5 µM ATP. Displacement of the IBFQ-conjugated 38-mer DNA oligo from the 6-FAM-conjugated 13-mer RNA oligo was measured by the change in fluorescence intensity. Data of n = 3 independent experiments with individually thawed proteins are represented as mean values ± standard deviation (n = 2 for 2.5 µM, unlabeled DNA). f Displacement assay from e using titrated full-length (FL) DDX41, R525H, or 153–410 mutant. Data are represented as mean ± standard deviation. Dots indicate results of n = 3 independent experiments with individually thawed proteins. p-values (p = 0.005, p = 0.0021, p < 0.0001, p = 0.0329, p < 0.0001) derived by two-way ANOVA with Tukey correction for multiple comparisons. Source data are provided as a Source Data file.