Fig. 6: Model for DDX41 function in R-loop homeostasis.

a Immunofluorescence analysis of HSPCs after 24 h of indicated knockdowns. Cells were nucleofected with plasmids encoding GFP and respective shRNAs. GFP-positive cells were sorted via FACS and seeded on coverslips. Representative images of 53BP1 (red) staining in HSPCs (left). DNA was counterstained with DAPI (blue). Quantification of nuclear 53BP1 intensity (right). Each dot represents a single measured value. The black line indicates the median. At least 80 cells across n = 2 biologically independent experiments were measured per condition. p-values (p < 0.0001, p < 0.0001, p = 0.2227) were calculated by one-way ANOVA with Tukey correction for multiple comparisons. Scale bars—20 µm. Source data are provided as a Source Data file. b Immunofluorescence analysis of HSPCs after expression of DDX41 WT, L237F + P238T or R525H mutants tagged with GFP. GFP-positive cells were sorted by FACS and used for the analysis. Representative images of 53BP1 (red) staining in HSPCs (left). DNA was counterstained with DAPI (blue). Quantification of nuclear 53BP1 intensity (right). Dots represent results from individual cells. The median is indicated by the black line. p-values (p = 0.0237, p = 0.624, p = 6.018) were derived from at least 30 cells across n = 1 experiment using one-way ANOVA with Tukey correction for multiple comparisons. Scale bars—20 µm. c Wild-type DDX41 associates with R-loops in promoters of active genes and balances R-loop levels by unwinding RNA–DNA hybrids. Pathogenic DDX41 variants found in acute myeloid leukemia (AML) display impaired RNA–DNA hybrid unwinding activity, leading to the accumulation of R-loops at promoters. Accumulation of R-loops at promoters results in increased replication stress, DSBs, and inflammatory signaling, rendering DDX41 mutated AML cells vulnerable to ATR inhibitors.