# APE1 Activity is Controlled by Non‐G‐Quadruplex Conformations in Single‐ and Double‐Stranded G‐Quadruplex Constructs

## TL;DR

APE1, a DNA repair enzyme, is less active on fully folded G-quadruplex DNA structures but efficiently cleaves partially folded or non-G4 conformations.

## Contribution

The study reveals that APE1 activity is controlled by non-G-quadruplex conformations in G-rich DNA structures.

## Key findings

- APE1 efficiently cleaves AP sites in non-G4 conformations but shows reduced activity with increasing G4 folding.
- Non-G4 duplex-embedded G4 scaffolds show positional dependency in APE1 cleavage yield.
- CD spectra confirm noncanonical structures in sequences that do not fully adopt a G4 fold.

## Abstract

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.

G‐quadruplex (G4) folding in DNA is inhibitory to AP endonuclease, although the protein still binds well to G4s. Curiously, partially folded G‐rich sequences, possibly transient hairpins, containing AP are very good substrates for cleavage by APE1. Herein, the enzyme activity and G4 folding are studied in single‐stranded vs. duplex contexts.