Oxidative Damage Fine-Tunes G-Quadruplex Structures in Human Gene Promoters

Oxidative damage can convert guanine (G) into 8-oxoguanine (^O8G), resulting in altered gene expression and genome instability. However, the underlying molecular mechanisms remain poorly understood. Herein, we show that the NEIL3 gene proximal promoter sequence forms a mixture of parallel and hybrid G-quadruplex structures (NEIL3-G4s), exhibiting intrinsic structural polymorphism. Strikingly, site-specific ^O8G modifications significantly reduce this polymorphism, promoting the stabilization of either the parallel or the (3+1) hybrid-1 G4 topology. A single G-to-^O8G substitution is sufficient to trigger a clear structural transition from the parallel to the (3+1) hybrid-1 G4, highlighting the profound impact of ^O8G on G4-mediated epigenetic regulation. We have determined the NMR solution structures of both native and ^O8G-modified NEIL3-G4s, providing mechanistic insights into how ^O8G induces specific G4 structural rearrangements. Functional analysis demonstrates that both forms of NEIL3-G4s can form in extended DNA contexts and inhibit DNA polymerase activity. Under oxidative stress, the formation of NEIL3-G4s correlates with elevated NEIL3 gene expression, suggesting that they play a role as sensors of oxidative damage and function as molecular switches for gene upregulation. Collectively, these findings underscore the crucial role of ^O8G-induced G4 structural plasticity in the cellular response to oxidative stress and in regulating gene expression.