Interplay of Guanine Oxidation and G-Quadruplex Folding in Gene Promoters

Abstract

Living in an oxygen atmosphere demands an ability to thrive in the presence of reactive oxygen species (ROS). Aerobic organisms have successfully found solutions to the oxidative threats imposed by ROS by evolving an elaborate detoxification system, upregulating ROS during inflammation, and utilizing ROS as messenger molecules. In this Perspective, recent studies are discussed that demonstrate ROS as signaling molecules for gene regulation by combining two emergent properties of the guanine (G) heterocycle in DNA, namely, oxidation sensitivity and a propensity for G-quadruplex (G4) folding, both of which depend upon sequence context. In human gene promoters, this results from an elevated 5'-GG-3' dinucleotide frequency and GC enrichment near transcription start sites. Oxidation of DNA by ROS drives conversion of G to 8-oxo-7,8-dihydroguanine (OG) to mark target promoters for base excision repair initiated by OG-glycosylase I (OGG1). Sequence-dependent mechanisms for gene activation are available to OGG1 to induce transcription. Either OGG1 releases OG to yield an abasic site driving formation of a non-canonical fold, such as a G4, to be displayed to apurinic/apyrimidinic 1 (APE1) and stalling on the fold to recruit activating factors, or OGG1 binds OG and facilitates activator protein recruitment. The mechanisms described drive induction of stress response, DNA repair, or estrogen-induced genes, and these pathways are novel potential anticancer targets for therapeutic intervention. Chemical concepts provide a framework to discuss the regulatory or possible epigenetic potential of the OG modification in DNA, in which DNA "damage" and non-canonical folds collaborate to turn on or off gene expression. The next steps for scientific discovery in this growing field are discussed.

Figure 1.

Sequences of DNA abundant in 5`-GG-3` dinucleotides are oxidation prone. (A) Comparison of dinucleotide frequencies between H. sapiens and C. elegans that have a similar G abundance but different dinucleotide frequencies. The data are replotted from the literature. Because of sequence complementarity in duplex DNA, not all possible dinucleotide combinations are shown (i.e., GG = CC and only GG is shown). (B) Plot of ionization potentials from Saito et al. illustrating G has the lowest potential, and runs of G render these sequences even more oxidation prone. (C) In duplex DNA, oxidation at a remote site can induce a chemical reaction at a distal 5`-GG-3` via generation of a migratory electron hole (h⁺).

Figure 2.

Metabolism and inflammation yield ROS that induce oxidative modification of the G heterocycle.

Figure 3.

Certain G-rich sequences can adopt G4 folds. (A) The consensus sequence for a PQS (2 examples with a 5^th G track on either the 5` or 3` side of the principal G4 folding sequence). (B) Hoogsteen base pairing in a G-tetrad. (C) Cartoon representations of the global G4 folds to depict the location of the Gs, syn (red) vs. anti (blue) conformations of the Gs, and loop nomenclature.

Figure 4.

Potential G-quadruplex forming sequences show enrichment in the human genome in regulatory elements. (A) Regions in which PQSs are found enriched in the human genome, and (B) histograms of PQS distributions around the TSSs of human genes in the template (left) or coding (right) strands. The complete distributions of PQSs around TSSs (gray left or red right) are also compared to PQSs specific to human DNA repair genes (blue lines). The data in panel B are replotted from our original report on these distributions.

Figure 5.

The structural plasticity of native G4 folds can accommodate the structurally destabilizing modification OG. (A) G-Tetrad hydrogen bonding is compromised by OG. (B) In the human VEGF promoter G4, the presence of OG engages the 5^th G run or “spare tire” to maintain the G4 fold.

Figure 6.

Oxidative modification of G to OG yields a Janus-faced purine that base pairs with C on the Watson-Crick face or A on the Hoogsteen face leading to a G→T transversion mutation in the absence of faithful DNA repair.

Figure 7.

The base excision repair of an OG:C base pair initiated by OGG1. *The proposed mechanism assumes OGG1 is a monofunctional glycosylase as supported by recent cellular studies.

Figure 8.

Proposed pathways by which G oxidation to OG in a gene promoter activates transcription. (A) Avvendimento’s proposed pathway by estrogen-receptor mediated gene activation for driving OG formation in a promoter and OGG1 activity leading to an entry point for TOPB2-mediated gene activation. (B) Boldogh/Ba and co-workers found Cys oxidation in OGG1 (OGG1^ox) attenuates the glycosylase activity to stall the protein for active recruitment of NF-κB to OG-bearing promoters for gene activation during inflammation. (C) Our laboratory found OGG1 release of OG in a promoter provides the drive for a duplex→G4 switch to display an AP to APE1 with stalled endonuclease activity on the G4 allowing recruitment of activating transcription factors via the Ref-1 properties during oxidative/inflammatory stress. (D) Xodo and co-workers found OG stabilized G4s were binding substrates for MAZ and A1 that led to resolving the non-canonical fold for BER-mediated activation during oxidative stress. (E) Tell and co-workers found oxidation in the nCaRE hairpin forming sequence provided a substrate for APE1 to interact with Ku70/80 and RNA pol II for gene activation under oxidative stress.

Figure 9.

Proposed relative energy-level diagram for gene regulation via G oxidation in a promoter PQS.

Figure 10.

DNA repair gene promoter oxidation is the stimulus for upregulated transcription.

Figure 11.

Structural comparisons of OG and 5mC. (A) The dominant tautomers of OG and 5mC favor Watson-Crick base pairing. (B) The OG:C and 5mC:G base pairs alter the major groove hydrogen bond donor and acceptor patterns.

Figure 12.

The BER process returns the oxidation products of G and 5mC back to the native state.

Figure 13.

The impact of G oxidation to OG in a gene promoter sequence that can switch structure to a non-canonical fold. (A) In the coding strand of a promoter, OG in sequences that can adopt G4s, i-motifs (iM), Z-DNA enhance gene expression; in contrast, OG in a sequence only capable of B-DNA formation has no impact on transcription. (B) In the template strand of the promoter, OG in all four sequence contexts caused a significant decrease in gene expression. Significance was determined by a Student’s t-test where *P < 0.05, **P < 0.01, and ***P <0.001. This figure is adapted from our prior publication on this topic that provides all the experimental details for collection of the data.