Why the ROS matters: One-electron oxidants focus DNA damage and repair on G-quadruplexes for gene regulation

Abstract

Hydrogen peroxide is a precursor to reactive oxygen species (ROS) in cells because of its high reactivity with iron(II) carbonate complexes formed in the labile iron pool due to a high concentration of intracellular bicarbonate (25-100 mM). This chemistry leads to the formation of carbonate radical anion rather than hydroxyl radical, and unlike the latter ROS, CO₃^•- is a milder one-electron oxidant with high specificity for guanine oxidation in DNA and RNA. In addition to metabolism, another major source of DNA oxidation is inflammation which generates peroxynitrite, another precursor to CO₃^•- via reaction with dissolved CO₂. The identity of the ROS is important because not all radicals react with DNA in the same way. Whereas hydroxyl radical forms adducts at all four bases and reacts with multiple positions on ribose leading to base loss and strand breaks, carbonate radical anion is focused on guanosine oxidation to yield 8-oxo-7,8-dihydroguanosine in nucleic acids and the nucleotide pool, a modification that can function epigenetically in the context of a G-quadruplex. DNA sequences of multiple adjacent guanines, as found in G-quadruplex-forming sequences of gene promoters, are particularly susceptible to oxidative damage, and the focusing of CO₃^•- chemistry on these sites can lead to a transcriptional response during base excision repair. In this pathway, AP-endonuclease 1 plays a key role in accelerating G-quadruplex folding as well as recruiting activating transcription factors to impact gene expression.

Keywords: 8-oxoguanine; AP-endonuclease 1; G-quadruplexes; Oxidative stress; base excision repair; hydroxyl radical; reactive oxygen species.

Figure 1.

DNA oxidation products by HO^• and the subset of those generated by CO₃^•− (yellow circle).

Figure 2.

Graphical representation of the change in ROS concentration formed from the iron-Fenton reaction as a function of bicarbonate concentration. A typical cytosolic concentration for HCO₃⁻ is 25 mM; Hank’s buffer contains 4 mM HCO₃⁻ which is near the crossover point, such that both radical species are likely formed.

Figure 3.

Carbonate radical anion oxidizes duplex DNA to yield an electron hole, which migrates by charge transport, to react at the lowest energy site. The IP values provided in the chart were previously reported [55].

Figure 4.

(A) Potential G-quadruplex-forming sequences are enriched around the TSS in gene promoters. (B) A duplex → G4 shift must occur to reveal the non-canonical folds held together by Hoogsteen hydrogen bonds and coordination to K⁺ ions. The PQS distribution plot was previously reported [64].

Figure 5.

Oxidative modification of a G in a gene promoter activates transcription. The DNA oxidizing agent and key protein for gene activation are (A) LSD1-generated H₂O₂ for generation of CO₃^•− by the Meyerstein-Fenton reaction with OGG1 and TOP2B activation, (B) CO₃^•− with OGG1 and NF-κB activation, or (C) H₂O₂ for generation of CO₃^•− by the Meyerstein-Fenton reaction and APE1 binding to an AP in the loop of a hairpin (or G4).

Figure 6.

The proposal from our laboratory for activation of gene expression by CO₃^•− oxidation of a G to yield OG in a promoter PQS for targeting by the BER pathway. Removal of OG by OGG1 yields a duplex destabilizing AP with the possible assistance by APE1 for initiation of the duplex → G4 transition. The G4 fold is bound by APE1 for the recruitment of activating transcription factors to induce gene expression.

Figure 7.

The BER pathway with intermediates in the process for the removal of OG to return the strand to a G nucleotide.

Figure 8.

(A) Structure for the c-MYC-DGD (PDB: 8DUT) [123], and (B) the sequence of the VEGF-DGD studied to address the impact of BER repair intermediates (see Fig. 6) on G4 folding kinetics.

Scheme 1.. Reaction of G^•+ with H₂O to yield OG.