Oxidative Modification of the Potential G-Quadruplex Sequence in the PCNA Gene Promoter Can Turn on Transcription

Because of its low redox potential, guanine (G) is the most frequent site of oxidation in the genome. Metabolic processes generate reactive oxygen species (ROS) that can oxidize G to yield 8-oxo-7,8-dihydroguanine (OG) as a key two-electron oxidation product. In a genome, G-rich sites including many gene promoters are sensitive to oxidative modification, and some of these regions have the propensity to form G-quadruplexes (G4s). Recently, OG formation in G-rich gene promoters was demonstrated to regulate mRNA expression via the base excision repair (BER) pathway. The proliferating cell nuclear antigen ( PCNA) gene was previously found to be activated by metabolic ROS, and the gene has a five G-track potential G4 in the coding strand of its promoter. Herein, we demonstrated the ability for four G runs of the PCNA promoter sequence to adopt a parallel-stranded G4. Next, we identified G nucleotides in the PCNA G4 sequence sensitive to oxidative modification. The G oxidation product OG and its initial BER product, an abasic site, were synthetically incorporated into the four- and five-track PCNA sequences at the sensitive sites followed by interrogation of G4 folding by five methods. We found the modifications impacted the G4 folds with positional dependency. Additionally, the fifth G track maintained the stability of the modified G4s by extrusion of the oxidatively modified G run. Finally, we synthetically inserted a portion of the promoter into a reporter plasmid with OG at select oxidation-prone positions to monitor expression in human glioblastoma cells. Our results demonstrate that OG formation in the context of the PCNA G4 can lead to increased gene expression consistent with the previous studies identifying that metabolic ROS activates transcription of the gene. This study provides another example of a G4 with the potential to serve as a regulatory agent for gene expression upon G oxidation.