Fig 2: HUWE1 and WRNIP1 control RNAPII-dependent ATM activation upon replicative stress.a PLA with antibodies against WRNIP1 and MCM2 (n = 190, 167, 132, 140, 175, 172, 207 cells) or WRNIP1 and RNAPII (n = 171, 145, 130, 140, 137, 161, 207 cells) in HCT116 cells treated with 1 mM hydroxyurea (HU) or released from HU for the indicated time. The graph shows median±quartiles for counts of proximity pairs in nuclei. P-values were determined using Kruskal–Wallis test followed by Dunn’s multiple comparison. Displayed p-values correspond to the comparison of the respective sample to the non-treatment control. b PLA with antibodies to PCNA and RNAPII in cells with 2 h pre-treatment using 20 nM AZD4573 or 100 nM triptolide followed by the addition of 1 mM HU for 4 h (n = 187, 160, 195, 223, 198, 171 cells). c Immunoblot analysis of HCT116 cells treated with 1 mM HU for 4 h or in combination with 2 h pre-treatment using 20 nM AZD4573 or 100 nM triptolide (n = 3). d Immunoblot analysis of HCT cells expressing shCtrl or shRAD50 after treatment with 1 mM HU for 2 h (n = 2). e Immunoprecipitation (IP) of ATM in cells treated with 1 mM hydroxyurea or in combination with 20 nM triptolide for 4 h (n = 2). f Neutral comet assay in HCT116 cells treated with DMSO, 1 mM HU alone or in combination with 20 nM AZD4573 for 6 h, followed by a 24 h release phase. Scale bar: 50 µm. From left, n = 585, 517, 376, 320 cells. g Crystal violet staining of HCT116 cells treated with 1 nM AZD4573 or 2.5 nM triptolide for 3 h followed by addition of 250 µM hydroxyurea (HU) for 4 days. P-values were determined using one-way ANOVA followed by Tukey’s multiple comparison. b, f Boxplots show median±quartiles with whiskers ranging up to 1.5-fold of the inter-quartile range. P-values were determined using Kruskal–Wallis test followed by Dunn’s multiple comparison. Source data are provided as a Source Data file.

Fig 3: Proposed model of HUWE1 and WRNIP1 dependent coordination of transcription and replication.HUWE1 binds to WRNIP1 and both proteins prevent transcription-dependent TRCs. HUWE1 promotes the association of WRNIP1 with elongation-competent pS2-RNAPII. Upon induction of replication stress or in HUWE1-CS cells, WRNIP1 translocates from RNAPII to stalled replication forks, leading to stabilization of TRCs and recruitment of ATM to RNAPII via the MRN complex. Stabilization of the collision state induces ATM signaling and limits DNA breakage.

Fig 4: HUWE1 and WRNIP1 limit ATM activation at RNAPII.a Immunoblotting analysis of HUWE1-WT and HUWE1-CS cells treated with 20 nM AZD4573 or 20 nM triptolide for 6 h (n = 4). b Scheme of RNAPII-MRN-ATM interaction. See also Supplementary Fig. 5c. c Immunoblot analysis of HUWE1-WT and HUWE1-CS cells expressing shCtrl or shRAD50 (n = 3). d Immunoprecipitation (IP) of RAD50 and MRE11 in benzonase-treated lysates of HCT116 cells (n = 2). e Immunoprecipitation of ATM in benzonase-treated lysates of shWRNIP1 or shCtrl cells. A representative image (left) and densitometry-based quantification (right, n = 3, mean ± SD) are shown. f Immunoprecipitation of ATM in benzonase-treated lysates using HUWE1-WT and HUWE1-CS cells treated with DMSO (-) or 20 nM triptolide (+) for 4 h. A representative image (left) and densitometry-based quantification (right, n = 3, mean ± SD) are shown. g CRISPR-KO screen using a focused sgRNA library targeting genes within GO terms “Cell cycle”, “DNA replication” and “DNA repair” in HUWE1-WT and HUWE1-CS cells. h Crystal violet staining (left) and quantification (right, n = 3, mean ± SD) of HUWE1-WT or HUWE1-CS cells expressing shRAD50 or shCtrl after 7 days of unperturbed growth. P-values were determined with one-way ANOVA followed by Tukey’s multiple comparison. i Synergy of ATM and HUWE1 inhibitors in killing of HUWE1-WT and HUWE1-CS cells determined by WST-8 assay after 4 d of treatment. Source data are provided as a Source Data file.

Fig 5: HUWE1 interacts with WRNIP1.a LC-MS/MS analysis of HUWE1 immunoprecipitates in HUWE1-WT and HUWE1-CS cells. b Validation of WRNIP1 as an interactor f HUWE1 by immunoprecipitation (IP) and immunoblotting (n = 3). c Immunofluorescence staining of HUWE1 and WRNIP1 in HCT116 cells (n = 3). Scale bar: 5 µm. d PLA assay with HUWE1 and WRNIP1 antibodies in WT and CS cells after pulse-labeling with 25 µM EdU for 30 min. PLA foci were quantified and are displayed for EdU-positive (EdU+) and EdU-negative (EdU-) cell populations and for cells stained solely with either the HUWE1-IgG (H1) or the WRNIP1-IgG (W1). From left, n = 128, 161, 107, 92,199, 214 cells. Scale bar: 10 µm. e Mapping of the WRNIP1 domain required for HUWE1 binding. UBZ: Ubiquitin binding zinc finger; LZ: Leucine zipper; AAA: AAA+ ATPase domain. See also Supplementary Fig. 2e, f. f DNA fiber assay in HCT116 cell expressing either shCtrl or shWRNIP1. Example images (left) and quantification (right) are shown (n = 125 fibers per group). P-values were determined using the non-parametric, two-tailed Mann–Whitney test. Scale bar: 4 µm. g EdU incorporation assay in WT or CS cells expressing shCtrl or shWRNIP1 (shW1). Scale bar: 20 µm, n = 2. h Quantification of the EdU incorporation assay shown in (g). From left, n = 192,165,184,115 cells. i Quantification of RNAPII-PCNA PLA foci in control and WRNIP1-depleted cells treated with 100 µM DRB or 100 nM triptolide for 6 h (=100 cells per group). j RNAPII-PCNA PLA foci quantification of cells expressing full-length (FL) HA-WRNIP1, WRNIP1 lacking the leucine zipper domain (?LZ) or only the WRNIP1 UBZ domain (UBZ). See also Supplementary Fig. 2f. From left, n = 190,185,176,179 cells. k Quantification of RNAPII-PCNA PLA in HUWE1-WT and HUWE1-CS cells, expressing shCtrl or shWRNIP1. From left, n = 177,140,137,132 cells. d, h–k Boxplots show median±quartiles with whiskers ranging up to 1.5-fold of the inter-quartile range. P-values were determined using Kruskal–Wallis test followed by Dunn’s multiple comparison. Source data are provided as a Source Data file.