Abstract:A 32P-postlabeling assay has been developed for monitoring the formation within DNA of adenine N-1-oxide, the specific H2O2-mediated oxidation product under nonradical conditions. This has required the chemical synthesis of both 2'-deoxyadenosine N-1-oxide 3'-monophosphate and 2'-deoxyadenosine N-1-oxide 5'-monophosphate, the substrate and the product of polynucleotide kinase mediated phosphorylation. Isolation of the substrate from the other nucleotides was found to be necessary in order to improve the rate o… Show more
“…This is in agreement with previous measurements of oligonucleotide strand breaks when an alkaline elution assay was performed after extraction of the DNA from mammalian cells and its subsequent incubation with the E. coli FPG protein (72)(73)(74). The level of 8- [32P]-postlabeling approach was already applied for the determination of 2'deoxyadenosine N-1 oxide and 5-(hydroxymethyl)-2'-deoxyuridine (79,80). Another example of overestimation of the measurement level of 8-oxodGuo is provided by an immunoslot blot assay (81).…”
In this paper we review recent aspects of the measurement of oxidized DNA bases, currently a matter of debate. There has long been an interest in the determination of the level of oxidized bases in cellular DNA under both normal and oxidative stress conditions. In this respect, the situation is confusing because variations that may be as large as two orders of magnitude have been reported for the yield of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) in similar DNA samples. However, recent findings clearly show that application of several assays like gas chromatography-mass spectrometry (GC-MS) and -32P--postlabeling may lead to a significant overestimation of the level of oxidized bases in cellular DNA. In particular, the silylation step, which is required to make the samples volatile for the GC-MS analysis, has been shown to induce oxidation of normal bases at the level of about one oxidized base per 10(4) normal bases. This has been found to be a general process that applies in particular to 8-oxoGua, 8-oxo-7, 8-dihydroadenine,5-hydroxycytosine, 5-(hydroxymethyl)uracil, and 5-formyluracil. Interestingly, prepurification of the oxidized bases from DNA hydrolysate prior to the derivatization reaction prevents artefactual oxidation. Under these conditions, the level of oxidized bases measured by GC-MS is similar to that obtained by HPLC associated with electrochemical detection (HPLC-EC). It should be added that the level of 8-oxo-7,8-dihydro-2;-deoxyguanosine in control cellular DNA has been found to be about fivefold lower than in earlier HPLC-EC measurements by using appropriate conditions of extraction and enzymatic digestion of DNA. Similar conclusions were reached by measuring formamidopyrimidine-DNA glycosylase sensitive sites as revealed by the single cell gel electrophoresis (comet) assay.
“…This is in agreement with previous measurements of oligonucleotide strand breaks when an alkaline elution assay was performed after extraction of the DNA from mammalian cells and its subsequent incubation with the E. coli FPG protein (72)(73)(74). The level of 8- [32P]-postlabeling approach was already applied for the determination of 2'deoxyadenosine N-1 oxide and 5-(hydroxymethyl)-2'-deoxyuridine (79,80). Another example of overestimation of the measurement level of 8-oxodGuo is provided by an immunoslot blot assay (81).…”
In this paper we review recent aspects of the measurement of oxidized DNA bases, currently a matter of debate. There has long been an interest in the determination of the level of oxidized bases in cellular DNA under both normal and oxidative stress conditions. In this respect, the situation is confusing because variations that may be as large as two orders of magnitude have been reported for the yield of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) in similar DNA samples. However, recent findings clearly show that application of several assays like gas chromatography-mass spectrometry (GC-MS) and -32P--postlabeling may lead to a significant overestimation of the level of oxidized bases in cellular DNA. In particular, the silylation step, which is required to make the samples volatile for the GC-MS analysis, has been shown to induce oxidation of normal bases at the level of about one oxidized base per 10(4) normal bases. This has been found to be a general process that applies in particular to 8-oxoGua, 8-oxo-7, 8-dihydroadenine,5-hydroxycytosine, 5-(hydroxymethyl)uracil, and 5-formyluracil. Interestingly, prepurification of the oxidized bases from DNA hydrolysate prior to the derivatization reaction prevents artefactual oxidation. Under these conditions, the level of oxidized bases measured by GC-MS is similar to that obtained by HPLC associated with electrochemical detection (HPLC-EC). It should be added that the level of 8-oxo-7,8-dihydro-2;-deoxyguanosine in control cellular DNA has been found to be about fivefold lower than in earlier HPLC-EC measurements by using appropriate conditions of extraction and enzymatic digestion of DNA. Similar conclusions were reached by measuring formamidopyrimidine-DNA glycosylase sensitive sites as revealed by the single cell gel electrophoresis (comet) assay.
“…3 C and D). The conditions (16). Moreover, the lesion was not removed by treatment of '-yirradiated DNA with alkali or E. coli endonuclease IV, demonstrating that it was not an abasic site (data not shown).…”
Plasmid DNA was rirradiated or treated with H202 in the presence of Cu2+ to generate oxygen free radical-induced lesions. Open circular DNA molecules were removed by ethidium bromide/CsCI density gradient centrifugation. The closed circular DNA fraction was treated with the Escherichia coli reagent enzymes endonuclease IH (Nth protein) and Fpg protein. This treatment converted DNA molecules containing the major base lesions pyrimidine hydrates and 8-hydroxyguanine to a nicked form. Remaining closed circular DNA containing other oxygen radical-induced base lesions was used as a substrate for nudeotide excision-repair in a cell-free system. Extracts from normal human cells, but not extracts from xeroderma pigmentosum cells, catalyzed repair synthesis in this DNA. The repair defect in the latter extracts could be specifically corrected by in vitro complementation. The data suggest that accumulation of endogenous oxidative damage in cellular DNA from xeroderma pigmentosum patients contributes to the increased frequency ofinternal cancers and the neural degeneration occurring in serious cases of the syndrome.UV light and ionizing radiation induce the formation of different and diverse types of DNA lesions. After exposure of DNA to UV light, pyrimidine dimers are the main products, whereas oxygen free radicals produced by ionizing radiation generate single-and double-strand breaks as well as a variety of ring-saturated, ring-fragmented, and oxidized base derivatives. Different DNA repair processes are used by cells to correct the damage: pyrimidine dimers are removed as part of oligonucleotides in a nucleotide excision-repair pathway initiated by a multisubunit incision nuclease, while several oxidized bases are liberated in free form by DNA glycosylases in a base excision-repair process. In cells from patients with the human inherited disease xeroderma pigmentosum (XP), incision at pyrimidine dimers is defective. As a consequence, the main symptoms of XP involve skin and eye lesions occurring after exposure to sunlight, including multiple skin cancers. However, other symptoms of XP are not readily explained as being due to UV-induced DNA damage. In severe cases, sometimes termed de SanctisCacchione syndrome, patients exhibit progressive neurological deterioration with massive loss of neurons and a 10-to 20-fold increase in frequency of several types of internal cancers (1). The origins of these symptoms have been unclear, but they could arise because of accumulation of DNA damage generated by exposure to environmental chemical mutagens or reactive cellular metabolites (2). In the latter regard, DNA is known to undergo endogenous damage due to hydrolysis, reaction with active oxygen, and nonenzymatic methylation. However, the main known lesions introduced in this fashion are corrected by base excision-repair (3).Reagent Enzymes and DNA Substrates. The following proteins were purified from Escherichia coli overproducer strains as described: E. coli endonuclease III (Nth protein) (4), E. coli Fpg protein (5), a...
“…It is a major non-radical species produced by a series of metabolic processes of non-reactive superoxide anions, and is widely present in living cells ( 19–21 ). The main products obtained by the oxidation of cytosine and adenine bases with hydrogen peroxide are cytosine N -oxide (C o ) and adenine N -oxide (A o ) derivatives, respectively ( Figure 1 ) ( 22–32 ). Figure 1.…”
Section: Introductionmentioning
confidence: 99%
“…Cadet reported that adenine N 1 -oxide is formed using hydrogen peroxide as a predominant ionic DNA base damage under non-radical conditions ( 29 ). 2′-Deoxyadenosine N 1 -oxide (3.2/10 6 bases) was detected to be present in the DNA of the untreated wild-type Proteus mirabilis cells ( 28 ). When they were exposed to nonlethal level of 10 mM hydrogen peroxide, the amount of this modified nucleoside was found to be 8.3 times higher than in the untreated cells ( 28 ).…”
Section: Introductionmentioning
confidence: 99%
“…2′-Deoxyadenosine N 1 -oxide (3.2/10 6 bases) was detected to be present in the DNA of the untreated wild-type Proteus mirabilis cells ( 28 ). When they were exposed to nonlethal level of 10 mM hydrogen peroxide, the amount of this modified nucleoside was found to be 8.3 times higher than in the untreated cells ( 28 ). Upon exposure of calf thymus DNA to 10 mM hydrogen peroxide for 30 min, the number of the oxidized adenine residues was reported to be 0.7/10 4 bases.…”
To clarify the biochemical behavior of 2′-deoxyribonucleoside 5′-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (Co) and adenine N-oxide (Ao), we examined their base recognition ability in DNA duplex formation using melting temperature (Tm) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the Tm values of modified DNA–DNA duplexes incorporating 2′-deoxyribonucleoside N-oxide derivatives significantly decreased compared with those of the unmodified duplexes. However, single insertion reactions by DNA polymerases of Klenow fragment (KF) (exo−) and Vent (exo−) suggested that Co and Ao selectively recognized G and T, respectively. Meanwhile, the kinetic study showed that the incorporation efficiencies of the modified bases were lower than those of natural bases. Ab initio calculations suggest that these modified bases can form the stable base pairs with the original complementary bases. These results indicate that the modified bases usually recognize the original bases as partners for base pairing, except for misrecognition of dATP by the action of KF (exo−) toward Ao on the template, and the primers could be extended on the template DNA. When they misrecognized wrong bases, the chain could not be elongated so that the modified base served as the chain terminator.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
