Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA

Kuo, Kenneth C.; McCune, Roy A.; Gehrke, Charles W.; Midgett, Rosemarie; Ehrlich, Melanie

doi:10.1093/nar/8.20.4763

Cited by 293 publications

(108 citation statements)

References 42 publications

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“…For the determination of 5-MedC in DNA by HPLC-UV detection the analyses are performed at the 2′-deoxynucleoside [25][26][27][28][29][30] or 2′-deoxynucleotide [31,32] level following enzymatic hydrolysis or at the nucleobase level following acid hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

Determination of 5-methyl-2′-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet detection

Sandhu

Kaur

Armstrong

et al. 2009

Journal of Chromatography B

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2 Abbreviations: 5-MedC, 5-methyl-2′-deoxycytidine; dC, 2′-deoxycytidine; dG, 2′-deoxyguanosine; T, thymidine; dA, 2′-deoxyadenosine; G, guanosine 3 AbstractThe formation of 5-methyl-2′-deoxycytidine (5-MedC) following methylation of the C-5 position of cytosine in genomic DNA provides an epigenetic mechanism for the regulation of gene expression and cellular differentiation. We describe the development of a method using HPLC-ultraviolet (UV) detection for the accurate determination of 5-MedC in DNA. Genomic DNA was obtained from HeLa cells and rat liver tissue using an optimised anion-exchange column DNA extraction procedure incorporating a ribonuclease incubation step to remove any potential interference from RNA. Following extraction the DNA samples were enzymatically hydrolysed to 2′-deoxynucleosides using a combination of an endo-exonuclease plus 5′-exonuclease together with a 3′-nucleotidase. The hydrolysed DNA samples (10 µg on column)were analysed using narrow-bore reverse phase HPLC-UV detection. The level of 5-MedC in the DNA samples was expressed as a percentage of the level of 2′-deoxycytidine (dC) determined from calibration lines constructed using authentic standards for 5-MedC and dC. The percentage 5-MedC level determined for commercially available calf thymus DNA was 6.26%, for HeLa cell DNA was 3.02% and for rat liver DNA was 3.55%.4

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Section: Introductionmentioning

confidence: 99%

Determination of 5-methyl-2′-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet detection

Sandhu

Kaur

Armstrong

et al. 2009

Journal of Chromatography B

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“…Labeled DNA was precipitated with 10% trichloroacetic acid at 0°C and radioactivity was determined as described (16). Products of the DNA MeTase assay were analyzed by HPLC after enzymatic degradation of the 3H-methylated copolymer to deoxyribonucleosides (22,23).…”

Section: Methodsmentioning

confidence: 99%

Ethylation of poly(dC-dG).poly(dC-dG) by ethyl methanesulfonate stimulates the activity of mammalian DNA methyltransferase in vitro.

Farrance¹,

Ivarie²

1985

Proc. Natl. Acad. Sci. U.S.A.

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Ethylation of poly(dC-dG) poly(dC-dG) with ethyl methanesulfonate (EtMes), a known carcinogen, at increasing molar ratios of EtMes/C-G base pairs progressively stimulated the methyl-accepting ability of the DNA during in vitro methylation by partially purified rat DNA (cytosine-5)-methyltransferase (EC 2.1.1.37). Maximum stimulation was 2-fold over mock-treated DNA when 2.7% of the guanines were modified at the N-7 position, the major site of ethylation by EtMes in DNA. If a CpG site "hemiethylated" at guanine N-7 mimics a hemimethylated CpG site, we calculate that the enzyme has a relative affinity for hemiethylated CpG 18-fold above unmodified CpG. If ethylation of a dioxyphosphate oxygen of the phosphodiester bond is responsible for stimulation, the relative affinity could be much higher, up to 370-fold.DNA methylation at cytosine C-5 in the rare CpG dinucleotide appears to play an important role in regulating the expression of many mammalian genes (reviewed in refs. 1-7) and is being extensively studied as a potential target for chemical and radiation-induced carcinogenesis. A large body of evidence now exists showing that chemical carcinogens and UV irradiation, both of which chemically modify DNA, significantly decrease enzymatic methylation of DNA in vivo and in vitro (8)(9)(10)(11)(12)(13)(14). Demethylation may, therefore, play a role in activation of the cancer cell phenotype as proposed by Holliday (15).By contrast, however, we recently found that one widely used chemical carcinogen, ethyl methanesulfonate (EtMes), inactivated specific gene expression (16) apparently by promoting an increase in enzymatic methylation of cultured cell DNA (17). Methyl methanesulfonate has also been reported to increase the level of 5-methylcytidine in DNA in regenerating rat liver (18). Because EtMes introduces ethyl groups primarily at guanine N-7 and secondarily at the phosphodiester bond (19), it was proposed that EtMes might promote enzymatic methylation (17) by creating a "fraudulent" hemimethylated site by rotation of an ethyl group at guanine N-7 or the phosphodiester in close proximity to cytosine C-5 in CpG sequences in B-DNA. This ethylated site might be recognized as hemimethylated by DNA (cytosine-5)-methyltransferase (MeTase; EC 2.1.1.37) and subsequently undergo methylation. We have tested a prediction of the model that unmethylated CpG sites modified by EtMes will be more efficient substrates for DNA methyltransferase in vitro than unmodified sites, and we report the results here. MATERIALS AND METHODSDNA MeTase. The enzyme was extracted with 0.8 M KCl from purified rat liver and spleen nuclei as described (20,21). The crude nuclear extract in 20 mM NaCi in buffer A (50 mM Tris HCl, pH 7.8/1 mM dithiothreitol) was loaded onto a S-adenosyl-L-homocysteine agarose column (3 ml; Bethesda Research Laboratories); the column was washed with 30 ml of 60 mM KCI/buffer A/1 mM EDTA and 15 ml of the same buffer with 20 ,uM S-adenosylmethionine (AdoMet). Enzyme was eluted with 0.3 M KCI in buffer A/1 mM EDTA a...

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“…The methylated DNA immunoprecipitation (MeDIP) assay (Weber et al, 2005;Weber et al, 2007) coupled with methylation-specific PCR and bisulfate sequencing (Noonan et al, 1990;Herman et al, 1996) represents the most promising experimental approach for future work. In parallel, the global level of DNA methylation could also be established by reversed-phase highperformance liquid chromatography (Kuo et al, 1980) or the cytosine extension assay previously used by the Pogribny group (Pogribny et al, 2006). …”

Section: Future Workmentioning

confidence: 99%

The effects of methyl-donor deficiency on mutation induction and transgenerational instability in mice

Voutounou

Glen

Dubrova

2012

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA

Cited by 293 publications

References 42 publications

Determination of 5-methyl-2′-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet detection

Determination of 5-methyl-2′-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet detection

Ethylation of poly(dC-dG).poly(dC-dG) by ethyl methanesulfonate stimulates the activity of mammalian DNA methyltransferase in vitro.

The effects of methyl-donor deficiency on mutation induction and transgenerational instability in mice

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