Fig. 2: APE1 is critical for ATM activation and SSB repair in the HSS system.

A The SSB plasmid was added to HSS supplemented with the ATM inhibitor KU55933 at a final concentration of 1 mM. After different incubation times, the DNA repair products were isolated and analyzed on an agarose gel. B Quantification results from Fig. 2A. Data are presented as mean values ± SD. **p(5 min)=0.0007; **p(15 min)=0.0011; **p(30 min)=0.0022; **p(60 min)=0.0087; two-tailed, paired t-test, n = 3. C The SSB plasmid was added to Mock- or ATM-depleted HSS. After different incubation times, the DNA repair products were isolated and analyzed on an agarose gel. D Quantification results from Fig. 2C. Data are presented as mean values ± SD. **p(5 min)=0.0002; *p(15 min)=0.0443, *p(30 min)=0.0337; **p(60 min)=0.0035; two-tailed, unpaired t-test, n = 3. E CTL or SSB plasmid was added to Mock- or APE1-depleted HSS. After different incubation times (5 and 15 min), the egg extracts were examined via immunoblotting analysis as indicated. F CTL or SSB plasmid was added to Mock- or APE1-depleted HSS at 40 ng/μL. After a 10-min incubation, the total egg extracts (“Input”) and DNA-bound fractions were examined via immunoblotting. G Schematic diagram of mutants and truncations of GST-APE1 proteins and His-APE1-NT34. H GST, WT or ΔNT34 GST-APE1 was added to HSS. After 30-min incubation, GST beads were added into the mixture and incubated at 4 °C. The total egg extracts (“Input”) and GST-Pulldown fractions were examined via immunoblotting. I CTL or SSB plasmid was added to Mock-depleted HSS. SSB plasmid was added to APE1-depleted HSS supplemented with negative control (−), WT, ΔNT34, and D306A Myc-APE1 protein. After incubation, the total egg extracts (“Extracts”) and DNA-bound fractions were examined via immunoblotting.] indicates mobility shift of Mre11. The data presented in Panel A, C, E, F, H, I are representative of three biological replicates. } indicates non-specific bands. Source data are provided as a Source Data file.