APE1 Activity is Controlled by Non-G-Quadruplex Conformations in Single- and Double-Stranded G-Quadruplex Constructs.

Apurinic/apyrimidinic endonuclease-1 (APE1) is a repair enzyme that efficiently cleaves abasic (AP) site damage in duplex DNA. Reports of in vitro activity assays between APE1 and single-stranded G-quadruplex (ssG4) reveal significant decreases in the endonuclease activity. Here, we identify that the low yields observed represent cleavage of the noncanonical folds that did not adopt a complete G4 fold. This conclusion is supported through circular dichroism analysis and activity assays analyzing the cleavage rate, folding impact on cleavage, and product inhibition. Studies were performed on AP-containing ssG4 and duplex-embedded G4 scaffolds. The CD spectra of a non-G4 containing potential quadruplex sequence reveal a noncanonical structure. APE1 can cleave an AP in these non-G4 conformation(s) in high yields comparable to the preferred duplex substrate. There is direct evidence of decreasing APE1 activity with increasing G4 folding in ssG4 and duplex-G-quadruplex-duplex (DGD) systems. Also observed is a positional dependency on yield in the non-G4 DGD scaffolds, but not in the non-G4 ssG4 scaffolds. In conclusion, our studies provide evidence that APE1 efficiently cleaves noncanonical conformations in G4-like structures, highlighting the control of secondary structure on APE1 endonuclease activity.