Fig 1: ATR activation by pRPA and pTopBP1. (A) Representative western blot of ATR-dependent phosphorylation of p53 and RPA32 and TopBP1 utilizing pRPA and/or pTopBP1. RPA and/or TopBP1 were initially phosphorylated by DNA-PK before being used in ATR kinase reactions as indicated by the scheme in the top right. Quantification of the p53 pSer15 band intensities comparing the effect of (B) pRPA (normalized to lane 5), (C) pTopBP1 (normalized to lane 8), and (D) pRPA and pTopBP1 (normalized to lane 11) on ATR activation. Quantification of the RPA32 pSer33 band intensities comparing the effect of (E) pRPA (normalized to lane 5), (F) pTopBP1 (normalized to lane 8), and (G) pRPA and pTopBP1 (normalized to lane 11) on ATR activation. Data in (B–G) are from triplicate experiments (mean ± SEM) and are analyzed by one-way ANOVA.
Fig 2: Impact of RPA-PPIi on ATR kinase activity. (A) Chemical structure of the RPA-PPIi PAME. (B) Representative EMSA of RPA ssDNA binding upon increasing amounts of PAME (12.5–200 µM). (C) Representative EMSA of RPA dsDNA unwinding upon increasing amounts of PAME (1.6–25 µM). (D) Representative western blot of ATR-dependent phosphorylation events upon increasing amounts of RPA70 OB-F inhibitors HAMNO (20–160 µM) and PAME (5–40 µM). (E) Quantification of HAMNO inhibition of ATR-dependent phosphorylation events. (F) Quantification of PAME inhibition of ATR-dependent phosphorylation events. Data in (E,F) are from duplicate experiments (mean ± range) normalized to a control reaction (D, lane 2) and are fit to a nonlinear regression.
Fig 3: ATR activation by AcRPA and RPA-DBi impact. (A) Representative western blot of ATR-dependent phosphorylation events over time utilizing Um- or Ac-RPA. (B) Quantification of band intensities from triplicate experiments as described in (A) (normalized to the highest band intensity for each blot). (C) Representative western blot of ATR-dependent phosphorylation events upon increasing amounts of RPA-DBi TDRL-551 (2.5–20 µM) utilizing Um- or Ac-RPA. (D) Quantification of band intensities from triplicate experiments as described in (C) (normalized to lanes 2 and 7 for Um- and Ac-RPA, respectively). Data in (B,D) are from triplicate experiments (mean ± SEM). (B) is fit to a linear equation and (D) is fit to a linear regression.
Fig 4: ATR inhibition at physiological ATP concentrations. (A) Representative western blot of the ATR reactions inhibited by 25 µM NERx 329 or 125 nM VE-821 at different concentrations of ATP (40 µM to 5 mM). Quantification of the ATR-dependent phosphorylation of (B) p53 Ser15, (C) RPA32 Ser33, and (D) RPA32 pThr21 normalized to control reactions at each ATP concentration. Data in (B–D) are from duplicate experiments (mean ± range).
Fig 5: ATR phosphorylation of RPA32 and RPA-DBi impact. (A) Representative western blot of control reactions of the ATR kinase reconstitution demonstrating the dependence of RPA, TopBP1, and ssDNA on ATR phosphorylation of RPA32 Ser33 and Thr21. (B) Quantification of control reactions of the ATR kinase reconstitution. (C) Representative western blot of ATR-dependent phosphorylation of RPA upon increasing amounts of TDRL-551 and NERx 329 (2.5–20 µM). (D) Quantification of TDRL-551 inhibition of ATR-dependent phosphorylation events. (E) Quantification of NERx 329 inhibition of ATR-dependent phosphorylation events. Data in (B) are from triplicate experiments (mean ± SEM) and are normalized to the highest intensity band measured from each blot. Data in (D,E) are from triplicate experiments (mean ± SEM) normalized to the control reaction (C, lane 2), and data in (E) are fit to a nonlinear regression.
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