Figure 5.

NEIL3 Zf-GRF repeat compromises APE1 endonuclease activity on ssDNA but not dsDNA. A, GST-APE1 endonuclease activity detected with or without the presence of different doses of GST-NEIL3-ZF1&2 using FAM-dsDNA39-AP as substrate. B, effect of GST-NEIL3-ZF1&2 on GST-APE1 endo activity targeting FAM-dsDNA39-AP using different concentrations of GST-APE1. C, endonuclease assay using 5′-FAM–ssDNA39-AP in the presence of GST-APE1 with or without GST-NEIL3-ZF1&2 at different doses. D, a similar assay repeated using different doses of GST-NEIL3-ZF1 instead for detecting any effects on APE1 endonuclease activity. E, 5′-FAM–ssDNA39-AP with or without GST-NEIL3-ZF1&2 incubated at 37 °C for 3 min before the addition of GST-APE1 at various doses as indicated, then incubated at 37 °C for 60 min for the shielding effect of NEIL3-ZF1&2 on ssDNA-AP. F, different doses of GST-APE1 incubated with or without GST-NEIL3-ZF1&2 at 37 °C for 3 min before the addition of FAM-ssDNA39-AP for another 60-min incubation for the inhibition effect through direct interaction between two proteins. Endo.(%) was calculated by the percentage of intensity of catalyzed products divided by the intensity of catalyzed products and uncatalyzed substrates. Intensity of bands was measured with ImageJ.