Kinase-dead ATR differs from ATR loss by limiting the dynamic exchange of ATR and RPA

Abstract

ATR kinase is activated by RPA-coated single-stranded DNA (ssDNA) to orchestrate DNA damage responses. Here we show that ATR inhibition differs from ATR loss. Mouse model expressing kinase-dead ATR (Atr^+/KD), but not loss of ATR (Atr^+/-), displays ssDNA-dependent defects at the non-homologous region of X-Y chromosomes during male meiosis leading to sterility, and at telomeres, rDNA, and fragile sites during mitosis leading to lymphocytopenia. Mechanistically, we find that ATR kinase activity is necessary for the rapid exchange of ATR at DNA-damage-sites, which in turn promotes CHK1-phosphorylation. ATR-KD, but not loss of ATR, traps a subset of ATR and RPA on chromatin, where RPA is hyper-phosphorylated by ATM/DNA-PKcs and prevents downstream repair. Consequently, Atr^+/KD cells have shorter inter-origin distances and are vulnerable to induced fork collapses, genome instability and mitotic catastrophe. These results reveal mechanistic differences between ATR inhibition and ATR loss, with implications for ATR signaling and cancer therapy.

Fig. 1

Atr^+/KD male mice display defects in spermatogenesis and meiotic sex chromosome inactivation. a Conservation and relative amino acid number of aspartate (D) residue in the PI3KK core of ATR, ATM, and DNA-PKcs in mouse and human. b The histogram reports the means ± SD of the sperm counts derived from 6 Atr^+/+, 5 Atr^+/−, and 14 Atr^+/KD male mice. Unpaired two-tailed t test was used. c Testes from several 12–37 weeks old Atr^+/+, Atr^+/−, and Atr^+/KD mice were weighed. Mann–Whitney test was used. d Representative histological sections of H&E staining of epididymis and testis (3 weeks and 15 weeks) of Atr^+/+, Atr^+/−, and Atr^+/KD mice. TUNEL assay was performed on 7 weeks old mice and representative images are reported. e The means ± SD of the ratio between diplotene and pachytene cells for Atr^+/+, Atr^+/−, and Atr^+/KD mice are reported and unpaired two-tailed t test was used for the statistical analysis. f Representative SCP3 and γH2AX immunofluorescence images of spermatocytes spreads are shown for Atr^+/+, Atr^+/−, and Atr^+/KD mice. The distribution of the mean percentages of positive γH2AX pachytenes is reported. A total of 587, 214, and 647 pachytenes were analyzed from 5 Atr^+/+, 3 Atr^+/−, and 8 Atr^+/KD mice, respectively. Unpaired two-tailed t test was used for the statistical analysis. Source data are provided as a Source Data file

Fig. 2

Mild lymphocytopenia in Atr^+/KD mice and genomic instabilities at telomeres, rDNA repeats, and fragile sites in Atr^+/KD cells. a Thymocytes were counted with Trypan Blue from several Atr^+/+, Atr^+/−, and Atr^+/KD thymuses. The means ± SD are reported and unpaired two-tailed t test was used. b The means ± SD of the ratio of IgM+B220^high re-circulated B cells vs IgM+B220^mid naive B cells in the bone marrow of Atr^+/+, Atr^+/−, and Atr^+/KD mice are shown. Unpaired two-tailed t test was used. c Purified naive CD43- B cells from spleens of Atr^+/+, Atr^+/−, and Atr^+/KD mice were stimulated to CSR in vitro. Representative flow cytometry profiles with IgG1 and B220 staining are reported at days 3.5 and 4.5. The mean percentages ± SD of IgG1+ cells are plotted. Unpaired two-tailed t test was used. P values between Atr^+/+ and Atr^+/− are 0.4592 (day 2.5), 0.0741 (day 3.5), and 0.0768 (day 4.5). P values between Atr^+/KD and controls are shown above the bar graph. d The means ± SD of fragile telomeres per metaphase were plotted for Atr^+/+, Atr^+/−, and Atr^+/KD B cells from three independent experiments and unpaired two-tailed t test was used. Representative images of fragile telomeres, indicated by the yellow arrows, are shown. e Metaphase spreads from Atr^+/+, Atr^+/−, and Atr^+/KD B cells were stained with DAPI and a rDNA probe by FISH (two independent experiments). The bars show the mean percentages ± SD of metaphases that contain a number of rDNA clusters that is different from 8. Unpaired two-tailed t test was used. Representative metaphases with 8 or ≠8 clusters are shown. f FISH on GIMAP locus was carried out on metaphase spreads from Atr^+/+ and Atr^+/KD B cells treated with 0.25 mM HU. Two independent experiments were performed and the number of metaphases analyzed by FISH and the number of chromosome aberrations at the GIMAP locus are reported in the table. An example of a normal GIMAP locus and few examples of breaks and fusions near GIMAP locus found in Atr^+/KD B cells are shown. Source data are provided as a Source Data file

Fig. 3

The rapid exchange of ATR on damaged DNA is dependent on its kinase activity. a The kinetics of GFP-ATR recruitment to sites of DNA damage. U2OS cells stably expressing GFP-ATR were monitored by confocal microscopy at the indicated time points after micro-irradiation (405 nm). Images of a representative cell are shown (scale bar is 10 µm). b FRAP analysis of GFP-ATR in U2OS cells after laser-induced foci formation, untreated or following treatment with ATRi (VE-821) or CHK1i (LY2603618) at 10 µM concentration. After photobleaching, recovery of the focus signal was monitored up to 90 s. The means ± SEM of the maximal recovery of GFP-ATR are reported for every condition analyzed and unpaired two-tailed t test was used for the statistical analysis. Representative cells are shown. Scale bar is 10 µM. c Quantification of the recovery of GFP-ATR relative intensity in an undamaged area after bleaching, in untreated or ATRi treated U2OS cells. The means ± SEM of the maximal recovery of GFP-ATR are reported and unpaired two-tailed t test was used for the statistical analysis. Representative cells are shown. Scale bar is 10 µM. Source data are provided as a Source Data file

Fig. 4

Atr^+/KD cells display HU-induced genome instability and checkpoint defects. a Metaphases from B cells treated with HU (0.25 mM) or Aphidicolin (0.5 µM) were analyzed for chromatid breaks, chromosome breaks, and breaks in fusions. The bar graphs represent the means ± SD and unpaired two-tailed t test was used. Three (HU) or two (Aphidicolin) independent experiments were performed. A total of 142 (Atr^+/+), 161 (Atr^+/−), and 156 (Atr^+/KD) metaphases in HU, and 83 (Atr^+/+), 90 (Atr^+/−), and 91 (Atr^+/KD) in Aphidicolin were analyzed. b The means ± SD of the number of complex rearrangements (fusions, quadri-radials) in HU were plotted from three independent experiments. Unpaired two-tailed t test was used. c Atr^+/+, Atr^+/−, and Atr^+/KD MEFs were pulse-labeled with BrdU for 30 min, washed and released in unchallenged media up to 6 h. Dot plots show the percentage of BrdU-positive cells and histograms show the cell cycle distribution of BrdU-positive cells. d Atr^+/+, Atr^+/−, and Atr^+/KD MEFs were pulse-labeled with BrdU for 30 min, treated with 2 mM HU for 3 h and then released from HU in unchallenged media up to 12 h. Data were plotted as in (c). e Primary Atr^+/+, Atr^+/−, and Atr^+/KD MEFs were treated with 2 mM HU for 3 h or 200 nM CPT for 2 h, with or without ATRi (10 µM, VE-821). Whole-cell extracts (WCE) were immunoblotted with the indicated antibodies. f Atr^+/+, Atr^+/−, and Atr^+/KD MEFs were treated with increasing concentrations of HU (0.01, 0.05, 0.1, 0.2 mM) for 1 h, and WCE were immunoblotted with the indicated antibodies. g Histogram shows the quantification of pCHK1/CHK1 at 0.2 mM HU derived from three independent experiments. The means ± SD are shown and unpaired two-tailed t test was used. h Atr^+/KD MEFs were treated with 0.2 mM HU for 1 h with or without pre-treatment for 1 h with the indicated checkpoint inhibitors. ATRi (VE-821) and CHK1i (LY603218) were used at 10 µM concentration, ATMi (KU-55933) at 15 µM, and DNA-PKcsi (NU7441) at 5 µM. WCE were immunoblotted with the indicated antibodies. Source data are provided as a Source Data file

Fig. 5

Hyperactivation of ATM and DNA-PKcs, increased breaks and chromatin-bound RPA in HU-treated Atr^+/KD MEFs. a Dot plot of alkaline comet tail moments of Atr^+/+, Atr^+/−, and Atr^+/KD cells left untreated, treated with 2 mM HU for 3 h or released from HU in unchallenged medium for an additional 3 h. The means ± SD are shown in the dot plot of one representative experiment and unpaired two-tailed t test was used for the statistical analysis. b The chromatin-bound RPA fractions are shown as dot plots. The dotted lines were arbitrarily set at 10³ and mark the separation between the RPA chromatin-bound positive (>10³) and negative (<10³) cells. The percentages of RPA positive cells from a representative experiment are shown. c The mean values ± SD of chromatin-bound RPA in untreated and HU (0.1 mM or 0.2 mM for 1 h) treated cells is reported from two independent experiments. Unpaired two-tailed t test was used. d Chromatin fractionation experiment was performed on Atr^+/+, Atr^+/−, and Atr^+/KD MEFs untreated or treated with 0.2 mM HU. The whole cell extracts (WCE), the soluble (SOL), and the chromatin (CHR) fractions were analyzed by SDS-PAGE and Western blot with the indicated antibodies. e, f FRAP analysis of GFP-RPA transfected U2OS cells after laser-induced foci formation, untreated or following treatment with 10 µM ATRi (VE-821) or Chk1i (LY2603618). Representative cells are shown up to 20 s after photobleaching. Scale bar is 10 µm. Recovery of the focus signal was monitored up to 90 s after photobleaching. g The means ± SEM of the maximal recovery of GFP-RPA are reported for every condition analyzed and unpaired two-tailed t test was used for the statistical analysis. Source data are provided as a Source Data file

Fig. 6

ATR-KD protein exacerbates replication stress-induced replication fork stalling and collapse. a Atr^+/+, Atr^+/−, and Atr^+/KD MEFs were treated with 2 mM HU for 3 h and then released in unchallenged media up to 6 h and the indicated time points (1, 3, and 6 h after HU release) were collected for whole-cell extracts (WCE), that were immunoblotted with the indicated antibodies. b Atr^+/+, Atr^+/−, and Atr^+/KD MEFs were treated with 2 mM HU for 6 h and then released overnight in unchallenged media. Nuclei were stained for RAD51 and DAPI. Data are representative of two independent experiments and a total of about 1000 cells were analyzed for every genotype. The mean values ± SD are shown and unpaired two-tailed t test was used. Representative immunofluorescence images are shown for Atr^+/+, Atr^+/−, and Atr^+/KD MEFs. c Atr^+/+, Atr^+/−, and Atr^+/KD MEFs were pulse-labeled with 25 µM CldU for 20 min, treated with 2 mM HU for 3 h and then released in media containing 125 µM IdU for 1 h. IdU track lengths were measured using ImageJ software. Means ± SD are shown in the dot plot of one representative experiment and Mann–Whitney test was used for the statistical analysis. d Molecular combing experiment in Atr^+/+, Atr^+/−, and Atr^+/KD MEFs in unchallenged conditions. Cells were labeled with IdU for 10 min and then with CldU for 20 min. Inter-origin distances were measured and statistical analysis was done with ANOVA test. Means ± SD are shown in the dot plot. e Model of the function of ATR kinase activity on ATR and RPA exchange describing the dominant-negative effect of ATR-KD protein in physiological conditions (meiosis, telomere, and rDNA replication) and upon replication stress challenge. Source data are provided as a Source Data file