Fig. 3: PrimPol-dependent repriming requires ATR-CHK1 kinase activity.
a, b ATR-1 cells harboring doxycycline-inducible myc-PrimPolWT or myc-PrimPolCH were transfected with 5 nM of siControl or siUTR-PrimPol (siPPol #4) for 24 h and treated with 1 μg/ml of doxycycline for 24 h. The expression level of myc-tag PrimPol and α-Tubulin were analyzed by western blotting. b Dot plot and mean of fork speed in ATR-1 cells harboring doxycycline-inducible myc-PrimPolWT or myc-PrimPolCH transfected with 5 nM of siControl or siPPol #4 for 24 h and treated with 0.1 μM of 4OHT with or without 1 μg/ml of doxycycline for 3 days. Representative result of two independent reproducible experiments are shown. c Dot plot and mean of fork speed in control cells harboring doxycycline-inducible myc-PrimPolWT treated with 0.1 μM of 4OHT with or without 1 μg/ml of doxycycline for 3 days. 1 nM of ATRi (Berzosertib) was added 24 h prior to IdU/CldU labeling. Representative result of two independent reproducible experiments are shown. d Dot plot and mean of fork speed in ATR-1 cells treated with 0.1 μM of 4OHT for 3 days. Low dose of ATRi (1 nM) and Chk1i (Rabusertib, 1 nM) was added 24 h prior to IdU/CldU labeling. Representative result of two independent reproducible experiments are shown. e Dot plot and mean of fork speed in Control and RSTC #2 cells treated with 0.1 μM of 4OHT for 3 days. Low dose of ATRi (1 nM) and Chk1i (1 nM) was added 24 h prior to IdU/CldU labeling. Representative result of two independent reproducible experiments are shown. f Colony re-formation assay of RSTC #5 with long-term ATR or Chk1 inhibition. Top, the number of colonies treated at indicated concentration of ATR or Chk1 inhibitor for ~14 days. The results represent the means ± SEM of three independent experiments. Bottom, representative image of anchorage independent colonies. g Protein domain structure of PrimPol. The catalytic signature motifs (I, II, and III) of archaeal-eukaryotic primase (AEP) domain; Zinc-finger domain (Zn) and RPA binding domain (RBD) are indicated. Multiple sequence alignment of PrimPol homologs in indicated animals is shown. Phosphorylation sites (S/T) are indicated in red. h ATR-1 cells harboring doxycycline-inducible myc-PrimPolS255A or myc-PrimPolS255D were transfected with 5 nM of siControl or siPPol #4 for 24 h and treated with 1 μg/ml of doxycycline for 24 h. The expression level of myc-tag PrimPol and α-Tubulin were analyzed by western blotting. i Dot plot and mean of fork speed in ATR-1 cells harboring doxycycline-inducible myc-PrimPolS255A or myc-PrimPolS255D transfected with 5 nM of siControl or siPPol #4 for 24 h and treated with 0.1 μM of 4OHT with or without 1 μg/ml of doxycycline for 3 days. 1 nM of ATRi was added 24 h prior to IdU/CldU labeling. Representative result of four independent reproducible experiments are shown. b, c, d, e, i Black lines indicate the mean; n = 200; one-way ANOVA Tukey’s test. All source data are provided as a Source Data file.
