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Mapping structurally defined guanine oxidation products along DNA duplexes: influence of local sequence context and endogenous cytosine methylation.

ABSTRACT: DNA oxidation by reactive oxygen species is nonrandom, potentially leading to accumulation of nucleobase damage and mutations at specific sites within the genome. We now present the first quantitative data for sequence-dependent formation of structurally defined oxidative nucleobase adducts along p53 gene-derived DNA duplexes using a novel isotope labeling-based approach. Our results reveal that local nucleobase sequence context differentially alters the yields of 2,2,4-triamino-2H-oxal-5-one (Z) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) in double stranded DNA. While both lesions are overproduced within endogenously methylated (Me)CG dinucleotides and at 5' Gs in runs of several guanines, the formation of Z (but not OG) is strongly preferred at solvent-exposed guanine nucleobases at duplex ends. Targeted oxidation of (Me)CG sequences may be caused by a lowered ionization potential of guanine bases paired with (Me)C and the preferential intercalation of riboflavin photosensitizer adjacent to (Me)C:G base pairs. Importantly, some of the most frequently oxidized positions coincide with the known p53 lung cancer mutational "hotspots" at codons 245 (GGC), 248 (CGG), and 158 (CGC) respectively, supporting a possible role of oxidative degradation of DNA in the initiation of lung cancer.

SUBMITTER: Ming X

PROVIDER: S-EPMC3985951 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Mapping three guanine oxidation products along DNA following exposure to three types of reactive oxygen species.

Project description:Reactive oxygen and nitrogen species generated during respiration, inflammation, and immune response can damage cellular DNA, contributing to aging, cancer, and neurodegeneration. The ability of oxidized DNA bases to interfere with DNA replication and transcription is strongly influenced by their chemical structures and locations within the genome. In the present work, we examined the influence of local DNA sequence context, DNA secondary structure, and oxidant identity on the efficiency and the chemistry of guanine oxidation in the context of the Kras protooncogene. A novel isotope labeling strategy developed in our laboratory was used to accurately map the formation of 2,2-diamino-4-[(2-deoxy-β-D-erythropentofuranosyl)amino]- 5(2 H)-oxazolone (Z), 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG), and 8-nitroguanine (8-NO2-G) lesions along DNA duplexes following photooxidation in the presence of riboflavin, treatment with nitrosoperoxycarbonate, and oxidation in the presence of hydroxyl radicals. Riboflavin-mediated photooxidation preferentially induced OG lesions at 5' guanines within GG repeats, while treatment with nitrosoperoxycarbonate targeted 3'-guanines within GG and AG dinucleotides. Little sequence selectivity was observed following hydroxyl radical-mediated oxidation. However, Z and 8-NO2-G adducts were overproduced at duplex ends, irrespective of oxidant identity. Overall, our results indicate that the patterns of Z, OG, and 8-NO2-G adduct formation in the genome are distinct and are influenced by oxidant identity and the secondary structure of DNA.

| S-EPMC6858621 | biostudies-literature

Generation, repair and replication of guanine oxidation products.

Project description:Guanine is the most readily oxidized of the four DNA bases, and guanine oxidation products cause G:C-T:A and G:C-C:G transversions through DNA replication. 8-Oxo-7,8-dihydroguanine (8-oxoG) causes G:C-T:A transversions but not G:C-C:G transversions, and is more readily oxidized than guanine. This review covers four major findings. (i) 2,2,4-Triamino-5(2H)-oxazolone (Oz) is produced from guanine and 8-oxoG under various oxidative conditions. Guanine is incorporated opposite Oz by DNA polymerases, except REV1. (ii) Several enzymes exhibit incision activity towards Oz. (iii) Since the redox potential of GG is lower than that of G, contiguous GG sequences are more readily oxidized by a one-electron oxidant than a single guanine, and OzOz is produced from GG in double-stranded DNA. Unlike most DNA polymerases, DNA polymerase ζ efficiently extends the primer up to full-length across OzOz. (iv) In quadruplex DNA, 3'-guanine is mainly damaged by one-electron oxidation in quadruplex DNA, and this damage depends on the highest occupied molecular orbital (HOMO). The oxidation products in quadruplex DNA are different from those in single-stranded or double-stranded DNA.

| S-EPMC5537945 | biostudies-other

Rates of chemical cleavage of DNA and RNA oligomers containing guanine oxidation products.

Project description:The nucleobase guanine in DNA (dG) and RNA (rG) has the lowest standard reduction potential of the bases, rendering it a major site of oxidative damage in these polymers. Mapping the sites at which oxidation occurs in an oligomer via chemical reagents utilizes hot piperidine for cleaving oxidized DNA and aniline (pH 4.5) for cleaving oxidized RNA. In the present studies, a series of time-dependent cleavages of DNA and RNA strands containing various guanine lesions were examined to determine the strand scission rate constants. The guanine base lesions 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), 5-guanidinohydantoin (Gh), 2,2,4-triamino-2H-oxazol-5-one (Z), and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) were evaluated in piperidine-treated DNA and aniline-treated RNA. These data identified wide variability in the chemical lability of the lesions studied in both DNA and RNA. Further, the rate constants for cleaving lesions in RNA were generally found to be significantly smaller than for lesions in DNA. The OG nucleotides were poorly cleaved in DNA and RNA; Sp nucleotides were slowly cleaved in DNA and did not cleave significantly in RNA; Gh and Z nucleotides cleaved in both DNA and RNA at intermediate rates; and 2Ih oligonucleotides cleaved relatively quickly in both DNA and RNA. The data are compared and contrasted with respect to future experimental design.

| S-EPMC4482417 | biostudies-literature

APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure.

Project description:Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (i.e., APOBEC3G or A3G) is an evolutionarily conserved cytosine deaminase that potently restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons and other viruses. A3G has a nucleotide target site specificity for cytosine dinucleotides, though only certain cytosine dinucleotides are 'hotspots' for cytosine deamination, and others experience little or no editing by A3G. The factors that define these critical A3G hotspots are not fully understood. To investigate how A3G hotspots are defined, we used an in vitro fluorescence resonance energy transfer-based oligonucleotide assay to probe the site specificity of A3G. Our findings strongly suggest that the target single-stranded DNA (ssDNA) secondary structure as well as the bases directly 3' and 5' of the cytosine dinucleotide are critically important A3G recognition. For instance, A3G cannot readily deaminate a cytosine dinucleotide in ssDNA stem structures or in nucleotide base loops composed of three bases. Single-stranded nucleotide loops up to seven bases in length were poor targets for A3G activity unless cytosine residues flanked the cytosine dinucleotide. Furthermore, we observed that A3G favors adenines, cytosines and thymines flanking the cytosine dinucleotide target in unstructured regions of ssDNA. Low cytosine deaminase activity was detected when guanines flanked the cytosine dinucleotide. Taken together, our findings provide the first demonstration that A3G cytosine deamination hotspots are defined by both the sequence context of the cytosine dinucleotide target as well as the ssDNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on processes such as HIV-1 genetic variation.

| S-EPMC3695494 | biostudies-literature

Reversible phenol oxidation and reduction in the structurally well-defined 2-Mercaptophenol-α₃C protein.

Project description:2-Mercaptophenol-α₃C serves as a biomimetic model for enzymes that use tyrosine residues in redox catalysis and multistep electron transfer. This model protein was tailored for electrochemical studies of phenol oxidation and reduction with specific emphasis on the redox-driven protonic reactions occurring at the phenol oxygen. This protein contains a covalently modified 2-mercaptophenol-cysteine residue. The radical site and the phenol compound were specifically chosen to bury the phenol OH group inside the protein. A solution nuclear magnetic resonance structural analysis (i) demonstrates that the synthetic 2-mercaptophenol-α₃C model protein behaves structurally as a natural protein, (ii) confirms the design of the radical site, (iii) reveals that the ligated phenol forms an interhelical hydrogen bond to glutamate 13 (phenol oxygen-carboxyl oxygen distance of 3.2 ± 0.5 Å), and (iv) suggests a proton-transfer pathway from the buried phenol OH (average solvent accessible surface area of 3 ± 5%) via glutamate 13 (average solvent accessible surface area of the carboxyl oxygens of 37 ± 18%) to the bulk solvent. A square-wave voltammetry analysis of 2-mercaptophenol-α₃C further demonstrates that (v) the phenol oxidation-reduction cycle is reversible, (vi) formal phenol reduction potentials can be obtained, and (vii) the phenol-O(•) state is long-lived with an estimated lifetime of ≥180 millisecond. These properties make 2-mercaptophenol-α₃C a unique system for characterizing phenol-based proton-coupled electron transfer in a low-dielectric and structured protein environment.

| S-EPMC3848601 | biostudies-literature

Quantitation of four guanine oxidation products from reaction of DNA with varying doses of peroxynitrite.

Project description:The oxidation products obtained from the reaction of peroxynitrite (ONOO-) with dG include-among others-8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 2,2-diamino-4[(2-deoxy-beta-d-erythro-pentafuranosyl)amino]-5(2H)-oxazolone (oxazolone), spiroiminodihydantoin, and N1-(beta-d-erythro-pentofuranosyl)-5-guanidinohydantoin (guanidinohydantoin). In the present work, the formation of these products from the treatment of calf thymus DNA with varying amounts of ONOO- was studied quantitatively in vitro. 13C-, 15N-labeled standards were synthesized for the nucleosides of interest, and calf thymus DNA was reacted with ONOO- and digested enzymatically down to the nucleoside level. Specific modifications in the DNA were measured by HPLC separation followed by electrospray ionization tandem mass spectrometric analysis in the selected reaction-monitoring mode. Artifacts of the above four oxidation products, arising from oxidation of dG and/or 8-oxodG during DNA digestion and subsequent workup, were evaluated with 7-15N-dG and/or stable-isotope-labeled 8-oxodG as internal standards. Levels of artifactual 8-oxodG were about 5/10(6) nucleosides. The artifacts of spiroiminodihydantoin and guanidinohydantoin, arising from 8-oxodG, were 3.7% and 0.6% of the measured 8-oxodG values, respectively. No artifacts of oxazolone were detected. 8-OxodG and oxazolone were formed dose-dependently in DNA treated with ONOO-, while the levels of spiroiminodihydantoin and guanidinohydantoin increased significantly at low ONOO- doses, and then dropped off at higher ONOO- doses. The complexity of these dose-response relationships is likely due to the dual role of peroxynitrite as both an oxidant and a nucleophile in competition with water.

| S-EPMC2526347 | biostudies-literature

Effects of Tet-induced oxidation products of 5-methylcytosine on Dnmt1- and DNMT3a-mediated cytosine methylation.

Project description:We investigated systematically the effects of Tet-induced oxidation products of 5-methylcytosine on Dnmt1- and DNMT3a-mediated cytosine methylation in synthetic duplex DNA. We found that the replacement of 5-methylcytosine at a CpG site with a 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxylcytosine or 5-hydroxymethyluracil resulted in altered methylation of cytosine at both the opposite and the neighboring CpG sites. Our results provided important new knowledge about the implications of the 5-methylcytosine oxidation products in maintenance cytosine methylation.

| S-EPMC4052722 | biostudies-literature

Calculating distortions of short DNA duplexes with base pairing between an oxidatively damaged guanine and a guanine.

Project description:DNA is constantly being oxidized, and oxidized DNA is prone to mutation; moreover, guanine is highly sensitive to several oxidative stressors. Several oxidatively damaged forms of guanine-including 2,2,4-triamino-5(2H)-oxazolone (Oz), iminoallantoin (Ia), and spiroiminodihydantoin (Sp)-can be paired with guanine, and cause G:C-C:G transversions. Previous findings indicate that guanine is incorporated more efficiently opposite Oz than opposite Ia or Sp, and that these differences in efficiency cannot be explained by differences in the stabilities of G:Oz, G:Ia, and G:Sp base pairs calculated ab initio. Here, to explain previous experimental result, we used a 3-base-pair model DNA duplex to calculate the difference in the stability and the distortion of DNA containing a G:Oz, G:Ia, or G:Sp base pair. We found that the stability of the structure containing 5' and 3' base pairs adjacent to G:Oz was more stable than that containing the respective base pairs adjacent to G:Ia or G:Sp. Moreover, the distortion of the structure in the DNA model duplex that contained a G:Oz was smaller than that containing a G:Ia or G:Sp. Therefore, our discussion can explain the previous results involving translesion synthesis past an oxidatively damaged guanine.

| S-EPMC6271426 | biostudies-literature

Chasing Particularities of Guanine- and Cytosine-Rich DNA Strands.

Project description:By substitution of natural nucleotides by their abasic analogs (i.e., 1',2'-dideoxyribose phosphate residue) at critically chosen positions within 27-bp DNA constructs originating from the first intron of N-myc gene, we hindered hybridization within the guanine- and cytosine-rich central region and followed formation of non-canonical structures. The impeded hybridization between the complementary strands leads to time-dependent structural transformations of guanine-rich strand that are herein characterized with the use of solution-state NMR, CD spectroscopy, and native polyacrylamide gel electrophoresis. Moreover, the DNA structural changes involve transformation of intra- into inter-molecular G-quadruplex structures that are thermodynamically favored. Intriguingly, the transition occurs in the presence of complementary cytosine-rich strands highlighting the inability of Watson-Crick base-pairing to preclude the transformation between G-quadruplex structures that occurs via intertwining mechanism and corroborates a role of G-quadruplex structures in DNA recombination processes.

| S-EPMC7037129 | biostudies-literature

Phylogenetic and Guanine-Cytosine Content Analysis of Symbiobacterium thermophilum Genes.

Project description:Although the bacterium Symbiobacterium thermophilum has a genome with a high guanine-cytosine (GC) content (69%), it belongs to a low GC content bacterial group. We detected only 18 low GC content regions with 5 or more consecutive genes whose GC contents were below 65% in the genome of this organism. S. thermophilum has 66 transposase genes, which are markers of transposable genetic elements, and 38 (58%) of them were located in the low GC content regions, suggesting that Symbiobacterium has a similar gene silencing system as Salmonella. The top hit (best match) analyses for each Symbiobacterium protein showed that putative horizontally transferred genes and vertically inherited genes are scattered across the genome. Approximately 25% of the 3338 Symbiobacterium proteins have the highest similarity with the protein of a phylogenetically distant organism. The putative horizontally transferred genes also have a high GC content, suggesting that Symbiobacterium has gained many DNA fragments from phylogenetically distant organisms during the early stage of Firmicutes evolution. After acquiring genes, Symbiobacterium increased the GC content of the horizontally transferred genes and thereby maintained a genome with a high GC content.

| S-EPMC3039409 | biostudies-literature

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