DNA oxidation

Process of oxidative damage of deoxyribonucleic acid

DNA oxidation is the process of oxidative damage of deoxyribonucleic acid. As described in detail by Burrows et al., 8 oxo 2' deoxyguanosine (8 oxo dG) is the most common oxidative lesion observed in duplex DNA because guanine has a lower one electron reduction potential than the other nucleosides in DNA. The one electron reduction potentials of the nucleosides (in volts versus NHE) are guanine 1.29, adenine 1.42, cytosine 1.6 and thymine 1.7. About 1 in 40,000 guanines in the genome are present as 8 oxo dG under normal conditions. This means that >30,000 8 oxo dGs may exist at any given time in the genome of a human cell. Another product of DNA oxidation is 8 oxo dA. 8 oxo dA occurs at about 1/10 the frequency of 8 oxo dG. The reduction potential of guanine may be reduced by as much as 50%, depending on the particular neighboring nucleosides stacked next to it within DNA. Excess DNA oxidation is linked to certain diseases and cancers, while normal levels of oxidized nucleotides, due to normal levels of ROS, may be necessary for memory and learning.

More than 20 oxidatively damaged DNA base lesions were identified in 2003 by Cooke et al. and these overlap the 12 oxidized bases reported in 1992 by Dizdaroglu. Two of the most frequently oxidized bases found by Dizdaroglu after ionizing radiation (causing oxidative stress) were the two oxidation products of guanine shown in the figure. One of these products was 8 OH Gua (8 hydroxyguanine). (The article 8 oxo 2' deoxyguanosine refers to the same damaged base since the keto form 8 oxo Gua described there may undergo a tautomeric shift to the enol form 8 OH Gua shown here.) The other product was FapyGua (2,6 diamino 4 hydroxy 5 formamidopyrimidine). Another frequent oxidation product was 5 OH Hyd (5 hydroxyhydantoin) derived from cytosine.

Most oxidized bases are removed from DNA by enzymes operating within the base excision repair pathway.

Steady state levels of endogenous DNA damages represent the balance between formation and repair. Swenberg et al. measured average amounts of steady state endogenous DNA damages in mammalian cells. The seven most common damages they found are shown in Table 1. Only one directly oxidized base, 8 hydroxyguanine, at about 2,400 8 OH G per cell, was among the most frequent DNA damages present in the steady state. + Table 1. Steady state amounts of endogenous DNA damages Endogenous lesions Number per cellAbasic sites 30,000N7 (2 hydroxethyl)guanine (7HEG) 3,0008 hydroxyguanine 2,4007 (2 oxoethyl)guanine 1,500Formaldehyde adducts 960Acrolein deoxyguanine120Malondialdehyde deoxyguanine60

As reviewed by Valavanidis et al. increased levels of 8 oxo dG in a tissue can serve as a biomarker of oxidative stress. They also noted that increased levels of 8 oxo dG are frequently found associated with carcinogenesis and disease. In the figure shown in this section, the colonic epithelium from a mouse on a normal diet has a low level of 8 oxo dG in its colonic crypts (panel A). However, a mouse likely undergoing colonic tumorigenesis (due to deoxycholate added to its diet and this may contribute to tumorigenesis and carcinogenesis. Of 22 mice fed the diet supplemented with deoxycholate, 20 (91%) developed colonic tumors after 10 months on the diet, and the tumors in 10 of these mice (45% of mice) included an adenocarcinoma (cancer). the repair of various types of DNA damages can, with low frequency, leave remnants of the different repair processes and thereby cause epigenetic alterations. 8 oxo dG is primarily repaired by base excision repair (BER). Li et al. reviewed studies indicating that one or more BER proteins also participate(s) in epigenetic alterations involving DNA methylation, demethylation or reactions coupled to histone modification. Nishida et al. examined 8 oxo dG levels and also evaluated promoter methylation of 11 tumor suppressor genes (TSGs) in 128 liver biopsy samples. These biopsies were taken from patients with chronic hepatitis C, a condition causing oxidative damages in the liver. Among 5 factors evaluated, only increased levels of 8 oxo dG was highly correlated with promoter methylation of TSGs (p<0.0001). This promoter methylation could have reduced expression of these tumor suppressor genes and contributed to carcinogenesis.

Yasui et al. examined the fate of 8 oxo dG when this oxidized derivative of deoxyguanosine was inserted into the thymidine kinase gene in a chromosome within human lymphoblastoid cells in culture. They inserted 8 oxo dG into about 800 cells, and could detect the products that occurred after the insertion of this altered base, as determined from the clones produced after growth of the cells. 8 oxo dG was restored to G in 86% of the clones, probably reflecting accurate base excision repair or translesion synthesis without mutation. G:C to T:A transversions occurred in 5.9% of the clones, single base deletions in 2.1% and G:C to C:G transversions in 1.2%. Together, these more common mutations totaled 9.2% of the 14% of mutations generated at the site of the 8 oxo dG insertion. Among the other mutations in the 800 clones analyzed, there were also 3 larger deletions, of sizes 6, 33 and 135 base pairs. Thus 8 oxo dG, if not repaired, can directly cause frequent mutations, some of which may contribute to carcinogenesis.

As reviewed by Wang et al., oxidized guanine appears to have multiple regulatory roles in gene expression. As noted by Wang et al.,

A second mode of gene regulation by DNA oxidation at a guanine, occurs when an 8 oxo dG is formed in a guanine rich, potential G quadruplex forming sequence (PQS) in the coding strand of a promoter, after which active OGG1 excises the 8 oxo dG and generates an apurinic/apyrimidinic site (AP site). The AP site enables melting of the duplex to unmask the PQS, adopting a G quadruplex fold (G4 structure/motif) that has a regulatory role in transcription activation. A third mode of gene regulation by DNA oxidation at a guanine, showed that the lysine specific histone demethylase LSD1 generates a local burst of reactive oxygen species (ROS) that induces oxidation of nearby nucleotides when carrying out its function. As a specific example, after treatment of cells with an estrogen, LSD1 produced H2O2 as a by product of its enzymatic activity. The oxidation of DNA by LSD1 in the course of the demethylation of histone H3 at lysine 9 was shown to be required for the recruitment of OGG1 and also topoisomerase IIβ to the promoter region of bcl 2, an estrogen responsive gene, and subsequent transcription initiation. 8 oxo dG does not occur randomly in the genome. In mouse embryonic fibroblasts, a 2 to 5 fold enrichment of 8 oxo dG was found in genetic control regions, including promoters, 5' untranslated regions and 3' untranslated regions compared to 8 oxo dG levels found in gene bodies and in intergenic regions. In rat pulmonary artery endothelial cells, when 22,414 protein coding genes were examined for locations of 8 oxo dG, the majority of 8 oxo dGs (when present) were found in promoter regions rather than within gene bodies. Among hundreds of genes whose expression levels were affected by hypoxia, those with newly acquired promoter 8 oxo dGs were upregulated, and those genes whose promoters lost 8 oxo dGs were almost all downregulated. In the mammalian brain, ~62% of CpGs are methylated. More than 500 of these CpG sites are de methylated in neuron DNA during memory formation and memory consolidation in the hippocampus and cingulate cortex]]

The first figure in this section shows a CpG site where the cytosine is methylated to form 5 methylcytosine (5mC) and the guanine is oxidized to form 8 oxo 2' deoxyguanosine (in the figure this is shown in the tautomeric form 8 OHdG). When this structure is formed, the base excision repair enzyme OGG1 targets 8 OHdG and binds to the lesion without immediate excision. OGG1, present at a 5mCp 8 OHdG site recruits TET1, and TET1 oxidizes the 5mC adjacent to the 8 OHdG. This initiates de methylation of 5mC.

Evidence that oxidative stress induced DNA damage plays a role in bipolar disorder has been reviewed by Raza et al. Bipolar patients have elevated levels of oxidatively induced DNA base damages even during periods of stable mental state. The level of the base excision repair enzyme OGG1 that removes certain oxidized bases from DNA is also reduced compared to healthy individuals.

Postmortem studies of elderly patients with chronic schizophrenia showed that oxidative DNA damage is increased in the hippocampus region of the brain. The mean proportion of neurons with the oxidized DNA base 8 oxo dG was 10 fold higher in patients with schizophrenia than in comparison individuals. Evidence indicating a role of oxidative DNA damage in schizophrenia has been reviewed by Raza et al.

RNAs in native milieu are exposed to various insults. Among these threats, oxidative stress is one of the major causes of damage to RNAs. The level of oxidative stress that a cell endures is reflected by the quantity of reactive oxygen species (ROS). ROS are generated from normal oxygen metabolism in cells and are recognized as a list of active molecules, such as O2•−, 1O2, H2O2 and, •OH A nucleic acid can be oxidized by ROS through a Fenton reaction. To date, around 20 oxidative lesions have been discovered in DNA. RNAs are likely to be more sensitive to ROS for the following reasons: i) the basically single stranded structure exposes more sites to ROS; ii) compared with nuclear DNA, RNAs are less compartmentalized; iii) RNAs distribute broadly in cells not only in the nucleus as DNAs do, but also in large portions in the cytoplasm. This theory has been supported by a series of discoveries from rat livers, human leukocytes, etc. Actually, monitoring a system by applying the isotopical label [18O] H2O2 shows greater oxidation in cellular RNA than in DNA. Oxidation randomly damages RNAs, and each attack bring problems to the normal cellular metabolism. Although alteration of genetic information on mRNA is relatively rare, oxidation on mRNAs in vitro and in vivo results in low translation efficiency and aberrant protein products. Though the oxidation strikes the nucleic strands randomly, particular residues are more susceptible to ROS, such hotspot sites being hit by ROS at a high rate. Among all the lesions discovered thus far, one of the most abundant in DNA and RNA is the 8 hydroxyguanine. Moreover, 8 hydroxyguanine is the only one measurable among all the RNA lesions. Besides its abundance, 8 hydroxydeoxyguanosine (8 oxodG) and 8 hydroxyguanosine (8 oxoG) are identified as the most detrimental oxidation lesions for their mutagenic effect, in which this non canonical counterpart can faultily pair with both adenine and cytosine at the same efficiency. This mis pairing brings about the alteration of genetic information through the synthesis of DNA and RNA. In RNA, oxidation levels are mainly estimated through 8 oxoG based assays. So far, approaches developed to directly measure 8 oxoG level include HPLC based analysis and assays employing monoclonal anti 8 oxoG antibody. The HPLC based method measures 8 oxoG with an electrochemical detector (ECD) and total G with a UV detector. The ratio that results from comparing the two numbers provides the extent that the total G is oxidized. Monoclonal anti 8 oxoG mouse antibody is broadly applied to directly detect this residue on either tissue sections or membrane, offering a more visual way to study its distribution in tissues and in discrete subsets of DNA or RNA. The established indirect techniques are mainly grounded on this lesion’s mutagenic aftermath, such as the lacZ assay. This method was first set up and described by Taddei and was a potentially powerful tool to understand the oxidation situation at both the RNA sequence level and single nucleotide level. Another source of oxidized RNAs is mis incorporation of oxidized counterpart of single nucleotides. Indeed, the RNA precursor pool size is hundreds of sizes bigger than DNA’s.

There have been furious debates on whether the issue of RNA quality control does exist. However, with the concern of various lengths of half lives of diverse RNA species ranging from several minutes to hours, degradation of defective RNA can not easily be attributed to its transient character anymore. Indeed, reaction with ROS takes only few minutes, which is even shorter than the average life span of the most unstable RNAs. Some potential factors include ribonucleases, which are suspected to selectively degrade damaged RNAs under stresses. Also enzymes working at RNA precursor pool level, are known to control quality of RNA sequence by changing error precursor to the form that can't be included directly into nascent strand.