Oxidative Modification of Guanine in a Potential Z-DNA-Forming Sequence of a Gene Promoter Impacts Gene Expression.

Abstract

One response to oxidation of guanine (G) to 8-oxo-7,8-dihydroguanine (OG) in a gene promoter is regulation of mRNA expression suggesting an epigenetic-like role for OG. A proposed mechanism involves G oxidation within a potential G-quadruplex-forming sequence (PQS) in the promoter, enabling a structural shift from B-DNA to a G-quadruplex fold (G4). When OG was located in the coding vs template strand, base excision repair led to an on/off transcriptional switch. Herein, a G-rich, potential Z-DNA-forming sequence (PZS) comprised of a d(GC) n repeat was explored to determine whether oxidation in this motif was also a transcriptional switch. Bioinformatic analysis found 1650 PZSs of length >10 nts in the human genome that were overrepresented in promoters and 5'-UTRs. Studies in human cells transfected with a luciferase reporter plasmid in which OG was synthesized in a PZS context in the promoter found that a coding strand OG increased expression and a template strand OG decreased expression. The initial base excision repair product of OG, an abasic site (AP), was also found to yield similar expression changes as OG. Biophysical studies on model Z-DNA strands found OG favored a shift in the equilibrium to Z-DNA from B-DNA, while an AP disrupted Z-DNA to favor a hairpin, placing AP in the loop where it is a poor substrate for the endonuclease APE1. Overall, the impact of OG and AP in a PZS on gene expression was similar to that in a PQS but reduced in magnitude.