Second Harmonic Generation Interrogation of the Endonuclease APE1 Binding Interaction with G-Quadruplex DNA

The binding interaction between the DNA repair enzyme apurinic/ apyrimidinic endonuclease-1 (APE1) with promoter G-quadruplex (G4) folds bearing an abasic site (AP) can serve as a gene regulatory switch during oxidative stress. Prior fluorescence-based analysis in solution suggested APE1 binds the VEGF promoter G4 but whether this interaction was specific or not remained an open question. Second harmonic generation (SHG) was used in this work to measure the noncanonical DNA-protein binding interaction in a label-free assay with high sensitivity to demonstrate the interaction is ordered and specific. The binding of APE1 to the VEGF promoter G4 with AP sites modeled by a tetrahydrofuran analogue produced dissociation constants of similar to 100 nM that differed from duplex and single-stranded DNA control studies. The SHG measurements confirmed APE1 binds the VEGF G4 folds in a specific manner resolving a remaining question regarding how this endonuclease with gene regulatory features engages G4 folds. The studies demonstrate the power of SHG to interrogate noncanonical DNA-protein interactions providing a foundational example for the use of this analytical method in future biochemical analyses.

Automated summary

This study used SHG to measure the binding interaction between the DNA repair enzyme APE1 and the VEGF promoter G4 folds with AP sites, demonstrating that the interaction is ordered and specific. This resolves the question of how APE1 engages G4 folds with gene regulatory features.