Fig 2: Cells sensitive to ATR inhibition exhibit higher levels of endogenous RS activation and DNA-PK activation. The expression and phosphorylation of key players in ATR pathway, RS, NHEJ, and DNA damage was measured by Western blot analysis after 24 hours treatment with 1 µmol/L ATRi ceralasertib, 4 hours HU at a concentration of 2.5 mmol/L, or combination of the two agents (20 hours only ATRi followed by 4 hours ATRi+HU). Cell extracts were immunoblotted with the indicated antibodies and HSP90 was used as a loading control.

Fig 3: Inhibition of ataxia telangiectasia and rad3-related (ATR) kinase reduces pathological ventricular remodeling in Mybpc3-/- cardiomyopathy.(A) Schematic illustration of ATR kinase inhibition from postnatal day (P) 4 to P7 with the drug AZD6738. (B) M-mode echocardiography at P7 from control (Ctl) and Mybpc3-/- mice administered vehicle (Veh.) or 25 mg/kg per day AZD6738. Echocardiography assessment of interventricular septal thickness at end diastole (IVSd) (C), left ventricular posterior wall thickness at end diastole (LVPWd) (D), left ventricular internal diameter at end diastole (LVIDd) (E), and fractional shortening (FS) (F) in Ctl (n=5–7) and Mybpc3-/- (n=9–12) mice administered Veh. or AZD6738. Heart weight (HW) (G) and HW/body weight (BW) ratio (H) for Ctl (n=5–7) and Mybpc3-/- (n=9–12) mice administered Veh. or AZD6738. (I) Representative immunohistochemical staining with wheat-germ agglutinin (green) and 4',6-diamidino-2-phenylindole (blue) of left ventricular (LV) tissue from Ctl and Mybpc3-/- mice administered Veh. or AZD6738. Bar=10 µm. (J) LV cross-sectional area of Ctl (n=5) and Mybpc3-/- (n=5) mice administered Veh. or AZD6738. Minimum 50 cells/sample measured. (K) Western blot of p53 in myocardial tissue lysate from Mybpc3-/- administered Veh. or AZD6738. (L) Relative quantification of p53 protein expression from Mybpc3-/- administered Veh. (n=4) or AZD6738 (n=5) normalized to ß-actin. All results are shown as mean±SEM.

Fig 4: AKAP12 interacts with ATR. (A) Cells were either mock-treated or exposed to UVB (10 J/m2). After 30 min, whole cell lysates were sampled and reciprocal Co-IP and immunoblots of ATR and AKAP12 were performed. Input represents 10% of total cellular lysate. (B) Full-length HA-tagged AKAP12 and truncated HA-tagged AKAP12 mutants were transfected in HEK293 cells expressing FLAG-tagged ATR and exposed to UVB (10 J/m2). Co-IP with anti-HA followed by immunoblotting with either anti-ATR or anti-FLAG at 30 min post-UVB. Input represents 10% of total cellular lysate. (C) Proximity ligation confirming sub-cellular localization of the ATR–AKAP12 interaction in HEK293 cells at 30 min after UVB (10 J/m2) or mock treatment. PLA was performed with anti-AKAP12 and anti-ATR antibodies. Green detection events signify juxtaposition between AKAP12 and ATR in maximum intensity projection images. Nuclei were stained with DAPI (blue). Bar represents 50 µm. (D) Schematic diagram of wild-type AKAP12 and mutant variants that were engineered to either prevent the AKAP12–ATR interaction (AKAP12?ATR), prevent nuclear localization (AKAP12?NLS) or prevent PKA-RII-AKAP12 binding (AKAP12?PKA). (E) Co-IP of ATR and immunoblotting of HA-tagged wild-type or HA-tagged mutant AKAP12 (AKAP12?PKA, AKAP12?ATR and AKAP12?NLS). Cells were either mock-treated or exposed to UVB (10 J/m2) and cytoplasm, nucleus and chromatin fractions extracted (30 min). Input represents 10% of total cell lysate. (F) Co-IP of HA-tagged wild-type or HA-tagged mutant AKAP12 with anti-HA and immunoblotting of RII and C subunits of PKA. HEK293 cells were either mock-treated or exposed to UVB (10 J/m2) and cytoplasmic, nuclear and chromatin fractions obtained. Input represents 10% of total cellular levels of the respective fractions. Data in all panels are representative from three independent experiments.

Fig 5: ZFP161 Maintains Genomic Stability. a Generation and characterization of ZFP161 KO mice. A. Schematic representation of wild- type and gene-trapped alleles in the ZFP161 genomic locus. b PCR and Western blot analysis of genomic DNAs and lysates from ZFP161+/+, ZFP161+/-, and ZFP161-/- mice. F (exp) and F (obs) are expected and observed Mendelian frequencies, respectively, and n, the number of pups genotyped at three weeks after birth. Two-sided Fisher’s exact test, p = 0.02719. c Mice were treated with UV and ATR signaling was determined by Western blot of skin tissues. d–g Genomic stability was examined by measuring ?H2AX (e), metaphase spread (f), and micronucleated normochromic erythrocytes (Mn-NCE, CD71-PI + ) (g). n = 3 independent experiments. 3 mice per genotypes, 50 metaphases per mice. h–l TCGA database analysis. Tumor mRNA expression levels were obtained from TCGA for 32 paired patient samples (h). Correlation of ZFP161 expression levels with chromosomal instability levels. Number of telomeric allelic imbalances (i), frequency of LOH (j), large scale transition (k), and weighted genome integrity index (l). m Diagram showing that ZFP161 mediates the recruitment of ATRIP/ATR to repair stalled replication fork. The graphs represent mean ± S.D., two-tailed, paired t-test. Source data are provided as a Source Data file.