Binding of AP Endonuclease-1 to G-Quadruplex DNA Depends on the N-Terminal Domain, Mg2+, and Ionic Strength

The base excisionrepair enzyme apurinic/apyrimidinic endonuclease-1(APE1) is also engaged in transcriptional regulation. APE1 can functionin both pathways when the protein binds to a promoter G-quadruplex(G4) bearing an abasic site (modeled with tetrahydrofuran, F) thatleads to enzymatic stalling on the noncanonical fold to recruit activatingtranscription factors. Biochemical and biophysical studies to addressAPE1's binding and catalytic activity with the vascular endothelialgrowth factor (VEGF) promoter G4 are lacking, andthe present work provides insight on this topic. Herein, the nativeAPE1 was used for cleavage assays, and the catalytically inactivemutant D210A was used for binding assays with double-stranded DNA(dsDNA) versus the native G4 or the G4 with F at various positions,revealing dependencies of the interaction on the cation concentrationsK(+) and Mg2+ and the N-terminal domain of theprotein. Assays in 0, 1, or 10 mM Mg2+ found that dsDNAand G4 substrates required the cation for both binding and catalysis,in which the G4 binding increased with [Mg2+]. Studieswith 50 versus physiological 140 mM K+ ions showed thatF-containing dsDNA was bound and cleaved by APE1, whereas the G4swith F were poorly cleaved in low salt and not cleaved at all at highersalt while the binding remained robust. Using & UDelta;33 or & UDelta;61N-terminal truncated APE1 proteins, the binding and cleavage of dsDNAwith F was minimally impacted; in contrast, the G4s required the N-terminusfor binding, and catalysis is nearly abolished without the N-terminus.With this knowledge, we found that APE1 could remodel the F-containing VEGF promoter dsDNA & RARR; G4 folds in solution. Lastly,the addition of the G4 ligand pyridostatin inhibited APE1 bindingand cleavage of F-containing G4s but not dsDNA. The biological andmedicinal chemistry implications of the results are discussed.

Automated summary

APE1, a base excision repair enzyme, is involved in transcriptional regulation by binding to promoter G-quadruplex structures with abasic sites, leading to recruitment of activating transcription factors. This study found that APE1's binding and cleavage activity on VEGF promoter G4 structures is dependent on cation concentrations and the N-terminal domain of the protein. Furthermore, APE1 can remodel F-containing VEGF promoter DNA structures and is inhibited by the G4 ligand pyridostatin.