The regulatory G4 motif of the Kirsten ras (KRAS) gene is sensitive to guanine oxidation: implications on transcription

Abstract

KRAS is one of the most mutated genes in human cancer. It is controlled by a G4 motif located upstream of the transcription start site. In this paper, we demonstrate that 8-oxoguanine (8-oxoG), being more abundant in G4 than in non-G4 regions, is a new player in the regulation of this oncogene. We designed oligonucleotides mimicking the KRAS G4-motif and found that 8-oxoG impacts folding and stability of the G-quadruplex. Dimethylsulphate-footprinting showed that the G-run carrying 8-oxoG is excluded from the G-tetrads and replaced by a redundant G-run in the KRAS G4-motif. Chromatin immunoprecipitation revealed that the base-excision repair protein OGG1 is recruited to the KRAS promoter when the level of 8-oxoG in the G4 region is raised by H2O2. Polyacrylamide gel electrophoresis evidenced that OGG1 removes 8-oxoG from the G4-motif in duplex, but when folded it binds to the G-quadruplex in a non-productive way. We also found that 8-oxoG enhances the recruitment to the KRAS promoter of MAZ and hnRNP A1, two nuclear factors essential for transcription. All this suggests that 8-oxoG in the promoter G4 region could have an epigenetic potential for the control of gene expression.

The roles of base excision repair enzyme OGG1 in gene expression

“…Proto-oncogenes in human genome [such as MYC and Kirsten Ras (K-RAS)] have PQS in their promoters, whose activation is influenced by oxidative stress [ 59 ]. A recent study presented another role of OGG1 in G-quadruplex-based transcription regulation, during which OGG1 behaves as a bona fide BER enzyme.…”

“…In the promoter of the KRAS oncogene, there is a G-rich region, which able to fold into a G-quadruplex structure (G4 motif). This unusual DNA conformation is recognized by nuclear proteins including MYC-associated zinc-finger protein (MAZ) and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) [ 59 , 60 ]. MAZ recognizes runs of guanines, unfolds the G-quadruplex, and leads to the transformation of G-quadruplex into duplexes [ 60 ].…”

“…MAZ recognizes runs of guanines, unfolds the G-quadruplex, and leads to the transformation of G-quadruplex into duplexes [ 60 ]. Guanine oxidation is shown to be higher in a sequence able to fold into G4 motif compared with other G-rich regions [ 59 ]. The incorporation of 8-oxoG in a G-tetrad enhances the recruitment of MAZ and hnRNP A1, and destabilizes the G-quadruplex [ 59 ].…”

“…Guanine oxidation is shown to be higher in a sequence able to fold into G4 motif compared with other G-rich regions [ 59 ]. The incorporation of 8-oxoG in a G-tetrad enhances the recruitment of MAZ and hnRNP A1, and destabilizes the G-quadruplex [ 59 ]. OGG1 is recruited to the KRAS G4 motif region that carries 8-oxoG more than non-G4 regions when the cells are treated with hydrogen peroxide (H 2 O 2 ) or nuclear factor (erythroid-derived 2)-like 2 (NRF2) inhibitor that causes increased cellular ROS [ 59 ].…”

An oxidatively damaged G-quadruplex/hemin DNAzyme

“…In living organisms, H2O2 is known to oxidize DNA, notably converting Gs into 8-oxoguanine (8-oxoG, Fig. 1) [31][32][33][34] . It was thus of interest to study the possible side-effect of H2O2 on the structural integrity of the G4 pre-catalyst.…”