Fig. 3: Myosin VI cooperates with WRNIP1 to protect stalled forks from DNA2-mediated degradation.

a iPOND assays show localization of myosin VI at replication forks. U2OS cells, 30 min EdU-pulsed −/+ 4 mM HU. Chromatin-bound proteins are visualized using western blotting and Ponceau S staining. b SIRF assays confirm the presence of myosin VI at replication forks. U2OS cells, 30 min EdU-pulsed −/+ 4 mM HU, followed by click reaction with Biotin azide and standard PLA assay. c Interaction of myosin VI with WRNIP1 is enhanced upon replication stress. U2OS cells, −/+ 4 mM HU, followed by standard PLA assay. For (b, c): Left: representative images, Hoechst (blue), PLA (magenta), scale bar = 10 µm. Right: dot plots of PLA signal intensities with mean values −/+ 95% confidence intervals. d WRNIP1 interacts directly with the MIUMyUb domains of myosin VI. GST-pulldown assay with recombinant proteins, visualized by western blotting and Ponceau S staining. Input represents 5% of the 2 µM MIUMyUb sample. e K63-linked ubiquitin chains enhance the WRNIP1-myosin VI interaction. GST-pulldown as in (d), using 1 µM His-MIUMyUb and increasing concentrations of K63 poly-ubiquitin. f Depletion of ubiquitin conjugates interferes with the WRNIP1-myosin VI interaction. GST-pulldown assay with recombinant baits and cellular lysates, pre-treated for 10 min at 37 °C −/+ 5 µM USP2cc. Visualization by western blotting and Ponceau S staining. g Schematic representation of fork protection mechanisms by WRNIP1 and BRCA2 according to Porebski et al.²¹ with nascent DNA colored in green and red. h Inhibition of DNA2 restores fork stability in WRNIP1- and myosin VI-deficient cells. Fiber assays performed on siRNA- transfected U2OS cells, −/+5 h nuclease inhibitor treatment, as in Fig. 2b. IdU/CldU ratios are shown as dot plots with median values. Knockdown efficiencies are shown in Supplementary Fig. 3g. For (a–f, h): A representative experiment from three independent replicates is shown. For (b, c, h): Significance levels were calculated using the two-tailed Mann–Whitney test from the indicated number of nuclei or fibers per sample (ns: not significant, ****: p < 0.0001, ***: p < 0.001) and annotated for p > 0.0001. Source data are provided as a Source Data file.