Fig 1: PrimPol is rapidly recruited to ICLs in an RPA‐dependent manner Immunoblots show the levels of the indicated proteins in whole cell extracts (WCE), soluble and chromatin‐enriched fractions. Tubulin and CTCF are shown as controls for soluble and chromatin‐bound proteins, respectively. The position of the ubiquitylated form of FANCD2 (Ub‐FANCD2) is indicated. Ponceau S staining is shown as a loading control.Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP‐UVA‐laser irradiated (10 µM TMP, 2 h followed by UVA laser irradiation) WT and PRIMPOL KO cells. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol‐positive cells and SD of three assays (n ≥ 100 cells per TMP‐treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one‐way ANOVA followed by Bonferroni post‐test. P‐values of individual comparisons are indicated.Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP‐UVA‐treated cells following control or RPA2 downregulation. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average PrimPol‐positive cells and SD of three assays (n ≥ 100 cells per TMP‐treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one‐way ANOVA followed by Bonferroni post‐test. P‐values of individual comparisons are indicated.Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP‐UVA‐treated PRIMPOL KO cells stably expressing V5‐tagged WT or RPA binding domain mutant (RBDm) PrimPol versions. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol‐positive cells and SD of two assays (n ≥ 100 cells per condition in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one‐way ANOVA followed by Bonferroni post‐test. P‐values of individual comparisons are indicated.Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP‐UVA‐treated WT and BLM KO cells upon downregulation of TOP3A, RMI1, and RMI2 (siTR) when indicated. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol‐positive cells and SD of three assays (n ≥ 250 cells per condition). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one‐way ANOVA followed by Bonferroni post‐test.Immunoblots show the levels of BLM, TOP3A, RMI1, RMI2, and PrimPol after TOP3A, RMI1, and RMI2 downregulation in WT and BLM KO cells. Ponceau S is shown as loading control. Source data are available online for this figure.
Fig 2: PrimPol interacts with factors involved in ICL traverse Experimental design for the proteomic identification of PrimPol‐interacting factors in S phase‐synchronized cells. Immunoblot showing PrimPol levels in WT and KO cells. MEK2 is shown as loading control.Volcano plot showing statistical significance (−log P‐value) vs. enrichment ratio (log2) of identified proteins after PrimPol immunoprecipitation in WT and KO cells. The positions of PrimPol, RPA1‐3, BLM, RMI1, RMI2, MHF1, SSBP1, RUNX1, HERC2, and BCCIP proteins are indicated.Volcano plot showing statistical significance (−log P‐value) vs. enrichment ratio (log2) of identified proteins after PrimPol immunoprecipitation in WT cells with or without MMC (2 µg/ml; 5 h). BLM, RMI1, RMI2, MHF1, SSBP1, RUNX1, HERC2, and BCCIP proteins are indicated.Immunoblot detection of the indicated proteins in whole cell extracts (WCE), PrimPol immunoprecipitates (IP), and non‐precipitated flow‐through fractions (FT). IgG shows the presence of anti‐PrimPol antibody in the IP fractions (lanes 4–6). Protein names in black and blue indicate the two different gels used. Source data are available online for this figure.
Fig 3: (related to Fig 2). Independent binding of PRIMPOL, BTR, FANCM, and FANCD2 to ICL‐containing DNA AConfocal microscopy images of PrimPol and γH2AX immunofluorescence (IF) in control (UVA) or TMP‐UVA‐treated cells following downregulation of the indicated factors. Nuclear DNA is counterstained with DAPI. The UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average and SD of the percentage of cells that are PrimPol‐positive in the laser path in three assays (n ≥ 100 cells per TMP‐treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one‐way ANOVA followed by Bonferroni post‐test. The p‐values of the indicated comparisons are shown.BImmunoblots showing the levels of the indicated proteins after siRNA‐mediated downregulation. siBTR is a combination of siBLM, siTOP3A, siRMI1, and siRMI2. MEK2 is shown as loading control. Protein names in black and blue indicate the two different gels used.C–FConfocal microscopy images of BLM (C), RMI1 (D), FANCM (E), or FANCD2 (F) together with γH2AX IF staining in control (UVA) or TMP‐UVA‐laser irradiated WT or PRIMPOL KO cells, as in (A). Scale bar, 10 µm. Histograms (right panels) show the average percentage of cells positive for the recruitment of the indicated factor to the laser path. Average and SD of at least two assays are shown (n ≥ 100 cells per TMP‐treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one‐way ANOVA followed by Bonferroni post‐test. P‐values of the indicated comparisons are included. Source data are available online for this figure.
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