Fig 2: YTHDC1 is responsible for ATR activation.(A) Immunoblot analysis of activated ATR, total ATR, activated ATM, total ATM and YTHDC1 in A549 cells transfected with NC, siYTHDC1-1 or siYTHDC1-2. A549 cells were treated with VP-16 for 24 h prior to analysis. (B,C) Quantification of panel A. The relative protein p-ATR, p-ATM, ATR or ATM levels were determined by normalizing the intensities of p-ATR, p-ATM, ATR or ATM to the intensity of GAPDH. (n = 3). (D) Immunoblot analysis of activated ATR, total ATR, and YTHDC1 in A549 cells transfected with indicated siRNAs. A549 cells were treated with VP-16 or DMSO for 24 h prior to analysis. (E,F) Quantification of panel D. The relative protein p-ATR or ATR levels were determined by normalizing the intensities of p-ATR or ATR to the intensity of GAPDH. (n = 3). (G) Immunoblot analysis of activated ATR, total ATR, and METTL3 in A549 cells transfected with indicated siRNAs. A549 cells were treated with VP-16 or DMSO for 24 h prior to analysis. (H,I) Quantification of panel G. The relative protein p-ATR or ATR levels were determined by normalizing the intensities of p-ATR or ATR to the intensity of GAPDH. (n = 3). (J) Immunoblot analysis of activated ATR and total ATR in A549 cells transfected with indicated siRNAs. A549 cells were treated with VP-16 or DMSO for 24 h prior to analysis. (K,L) Quantification of panel J. The relative protein p-ATR or ATR levels were determined by normalizing the intensities of p-ATR or ATR to the intensity of GAPDH. (n = 3). (M) Immunoblot analysis of activated ATR, total ATR, and YTHDC1 in YTHDC1-deficient A549 cells overexpressed with Vector, YTHDC1-WT or YTHDC1-MUT. Cells were treated with VP-16 for 24 h prior to analysis. (N,O) Quantification of panel M. The relative protein p-ATR or ATR levels were determined by normalizing the intensities of p-ATR or ATR to the intensity of GAPDH. (n = 3). (P) Immunofluorescence (IF) detection of p-ATR foci in the lungs of C57/BL6 mice from NC or shYTHDC1 groups treated with BLM or saline (n ≥ 4 mice per group). Scale bar: 5 μm. (Q) Quantification of panel P. The average number of p-ATR foci per cell. (n ≥ 4 per group). (R) Quantification of panel P. The percentage of cells with p-ATR foci was calculated (n ≥ 4 per group). Data information: All values are mean ± SEM. The unpaired Student’s two-tailed t-test was used to determine the statistical significance between two groups. The One-way ANOVA was used to determine the statistical significance for more than two groups (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). n = number of biological replicates. Source data are available online for this figure.

Fig 3: Tolerance to platinum-induced DNA damage in ERCC1 and p53 deficient lung cancers requires ATR signaling during S phase, which serves to limit global replication fork collapse and replication catastrophe.

Fig 4: Morphological abnormalities of the RPE monolayer and increased chromosomal damage following the conditional deletion of Atr.Loss of either a single copy of Atr or both copies of Atr lead to morphological abnormalities of the RPE (A), a significant decrease in cell density (B) and a significant increase in micronuclei formation (C). i pun/un (n = 5); ii Trp1-Cretg/o (n = 7); iii Atrcond/+ (n = 9); and iv (s) Atrcond/− small (n = 23); (A, B and C). RPE whole mounts were stained for nuclear localized β-gal activity (black spots) and phalloidin (yellow) to identify nuclear material and cell boundaries, respectively. Red boxes indicate the 200 μm2 region used for cell counting, solid red circles and white arrowheads mark individual cells and micronuclei, respectively (A). Error bars indicate S.E.M. (B and C). Scale bar: 25 μm (A). For (B) α: comparison to pun/un (P<0.001); β: comparison to Trp1-Cretg/o (P<0.001) and γ: comparison to Atrcond/+ (P<0.01).

Fig 5: Model for the relationship between ATR, homologous recombination and chromosomal stability.In the presence of replication stress from endogenous lesions, ATR is activated initiating an S-phase arrest to block aberrant merger of another replication fork at the same lesion. Some stalled replication forks will be substrates for HR (depicted here is the formation of a chicken foot structure that acts as a RAD51 substrate). If HR proceeds as normal, then the replication fork will be restored. However, we propose that this progression is dependent upon sufficient time to complete the HR reaction and possibly a more direct licensing of a later step in HR by ATR kinase activity (CHK1, BRCA1 and BLM, for example – not shown). If HR does not restore the stalled fork, then it may collapse, potentially leading to chromosomal breaks and the production of micronuclei. Thick lines are parental strand DNA, thin lines are daughter strand DNA, half arrowheads represent 3′ ends, the solid black triangle represents a DNA lesion and solid black circles represent RAD51 protein.