The RAD17 promoter sequence contains a potential tail-dependent G-quadruplex that downregulates gene expression upon oxidative modification

摘要

Our laboratory has recently proposed that the oxidation of guanine (G) to 8-oxo-7,8-dihydroguanine (OG) in G-rich promoter regions of DNA repair genes can serve as a regulatory mechanism of gene transcription. These regions also have the potential to fold into G-quadruplexes (G4). The human RAD17 promoter sequence has such a region in the template strand of the gene. In this work, the potential G-quadruplex sequence (PQS) of the RAD17 gene promoter was interrogated in different sequence contexts. With two extra nucleotides of the native sequence on either side of the G4, the structure was found to fold into a hybrid-like G4, similar to the hybrid-1 fold that the human telomere sequence can adopt. With only one nucleotide on either side of the PQS, the topology of the structure was observed to be mixed, and without extra nucleotides on the ends, the sequence adopted a parallel fold. Next, the sequence was studied with synthetic incorporation of the oxidative modification OG into specific sites and installed into the promoter of plasmids with a luciferase gene. These plasmids were transfected into a human cell line to observe the effect of the G4s on transcription. The RAD17 PQS was found to decrease luciferase expression with the presence of OG that is consistent with RAD17 expression under oxidative stress. This serves as an example of how oxidative modification could affect transcription in the context of a G4.