NEIL3 promoter G-quadruplex with oxidatively modified bases shows magnesium-dependent folding that stalls polymerase bypass.

Oxidative stress unleashes reactive species capable of oxidizing 2'-deoxyguanosine (G) nucleotides in G-rich sequences of the genome, such as the potential G-quadruplex forming sequencing (PQS) in the NEIL3 gene promoter. Oxidative modification of G yields 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) that can be further oxidized to hydantoin products. Herein, OG was synthesized into the NEIL3 PQS that was allowed to fold to a G-quadruplex (G4) in K ion solutions with varying amounts of Mg in the physiological range. The Mg dependency in the oxidatively modified NEIL3 G4 to stall a replicative DNA polymerase was evaluated. The polymerase was found to stall at the G4 or OG, as well as continue to full-length extension with dependency on the location of the modification and the concentration of Mg. To provide some clarity on these findings, OG or the hydantoins were synthesized in model NEIL3 G4 folding sequences at the positions of the polymerase study. The model G4 sequences were allowed to fold in K ion solutions with varying levels of Mg to identify how the presence of the divalent metal impacted G4 folding depending on the location of the modification. The presence of Mg either caused the transition of the NEIL3 G4 folds from an antiparallel to parallel orientation of the strands or had no impact. Structural models are proposed to understand the findings using the literature as a guide. The biological significance of the results is discussed.