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Chemical methods for decoding cytosine modifications in DNA.

Booth MJ, Raiber EA, Balasubramanian S - Chem. Rev. (2014)

View Article: PubMed Central - PubMed

Affiliation: †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW United Kingdom.

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Quantification by massspectrometry of DNA digested into nucleosides showed that the genomicDNA of ES cells contained 5fC at levels of around 0.2% relative toG and 5caC at 10-fold lower levels than 5fC... In mammalian brain tissues, levels of 5fC were found to be 2–3and 5caC 3–4 orders of magnitude lower than 5hmC... There are several proposedpathways for demethylation; one mechanismsuggests the iterative oxidation of 5mC by the TET family enzymes,followed by base excision repair or deformylation/decarboxylation.A potential mechanism for active demethylation is through the thymineDNA glycosylase (TDG) enzyme, which can excise both 5fC and 5caC fromDNA but does not remove 5mC or 5hmC... The reaction ofhydrazine (which leads to cleavage at C and T) with 5mC is inefficientand therefore does not introduce a strand cleavage... This results ina gap in the sequencing pattern... Münzel et al. describeda chemical method to discriminatebetween C and 5mC... The chemical reagentO-allylhydroxylamine, in contrast to bisulfite, does not exploit reactivitydifferences but gives different reaction products with cytosine and5mC (Figure 11)... The deamination ratealso decreases above pH 5.3 as the N3 unprotonated bisulfite adductdeaminates at 1% of the rate of the N3 protonated adduct... However,deamination of the bisulfite adduct is base-catalyzed, so the ratealso decreases at pH values below 5.0 due to the protonation of themost effective catalytic species, sulphite... Furthermore, it is possible to detect thenatural cytosine CpG methylation status of DNA around each nucleosome.It would be of great interest to combine ChIP-BS-Seq/BisChiP-Seq withNOMe-Seq to generate a joint map of 5mC with specific histone modificationsalong with the exact position of each nucleosome... The realization that5hmC exists in mammalian DNA has revealed animportant shortcoming of BS-Seq treatment of 5hmC with bisulfite resultsin a stable cytosine-5-methylsulfonate adduct (CMS) that, like 5mC,does not undergo deamination and is therefore read as C during sequencingdata... The kinetics of nucleotide incorporationis dependent on the nature of the bases and typically the polymeraseincorporation rate at the modified base position is slower. 5mC and5hmC have a similar low kinetic signature, which makes it is difficultto distinguish between them and nonmodified C... However, 5fC and 5caC have a greater signal than 5mC and5hmC and, through oxidiation of 5mC with the TET enzymes, have beenused to detect 5mC... It is now possible to decode 5mC, 5hmC,5fC, and 5caC in addition to G, C, A, and T in DNA at single baseresolution.

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Mechanism of sodium bisulfite deamination of cytosineto uracilthrough the addition of sodium bisulfite across the C5–6 doublebond of C, followed by deamination of the cytosine bisulfite adduct.The uracil bisulfite adduct can then be worked up using alkaline conditions.
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fig12: Mechanism of sodium bisulfite deamination of cytosineto uracilthrough the addition of sodium bisulfite across the C5–6 doublebond of C, followed by deamination of the cytosine bisulfite adduct.The uracil bisulfite adduct can then be worked up using alkaline conditions.

Mentions: The deamination reaction of C to U with bisulfitewas first observed in 1970.47 The bisulfiteanion adds across the C5–6 double bond of C at acidic pH, togenerate an adduct, which has lost aromaticity of the base and undergoeshydroysis with loss of ammonia. The resulting uracil bisulfite adductrearomatises to form uracil upon an increase in pH (Figure 12). This reaction requires the single stranded formof DNA (ssDNA), owing to the inaccessibility of the C5–6 doublebond to the bisulfite anion in the double helix.48


Chemical methods for decoding cytosine modifications in DNA.

Booth MJ, Raiber EA, Balasubramanian S - Chem. Rev. (2014)

Mechanism of sodium bisulfite deamination of cytosineto uracilthrough the addition of sodium bisulfite across the C5–6 doublebond of C, followed by deamination of the cytosine bisulfite adduct.The uracil bisulfite adduct can then be worked up using alkaline conditions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4378238&req=5

fig12: Mechanism of sodium bisulfite deamination of cytosineto uracilthrough the addition of sodium bisulfite across the C5–6 doublebond of C, followed by deamination of the cytosine bisulfite adduct.The uracil bisulfite adduct can then be worked up using alkaline conditions.
Mentions: The deamination reaction of C to U with bisulfitewas first observed in 1970.47 The bisulfiteanion adds across the C5–6 double bond of C at acidic pH, togenerate an adduct, which has lost aromaticity of the base and undergoeshydroysis with loss of ammonia. The resulting uracil bisulfite adductrearomatises to form uracil upon an increase in pH (Figure 12). This reaction requires the single stranded formof DNA (ssDNA), owing to the inaccessibility of the C5–6 doublebond to the bisulfite anion in the double helix.48

View Article: PubMed Central - PubMed

Affiliation: †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW United Kingdom.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Quantification by massspectrometry of DNA digested into nucleosides showed that the genomicDNA of ES cells contained 5fC at levels of around 0.2% relative toG and 5caC at 10-fold lower levels than 5fC... In mammalian brain tissues, levels of 5fC were found to be 2–3and 5caC 3–4 orders of magnitude lower than 5hmC... There are several proposedpathways for demethylation; one mechanismsuggests the iterative oxidation of 5mC by the TET family enzymes,followed by base excision repair or deformylation/decarboxylation.A potential mechanism for active demethylation is through the thymineDNA glycosylase (TDG) enzyme, which can excise both 5fC and 5caC fromDNA but does not remove 5mC or 5hmC... The reaction ofhydrazine (which leads to cleavage at C and T) with 5mC is inefficientand therefore does not introduce a strand cleavage... This results ina gap in the sequencing pattern... Münzel et al. describeda chemical method to discriminatebetween C and 5mC... The chemical reagentO-allylhydroxylamine, in contrast to bisulfite, does not exploit reactivitydifferences but gives different reaction products with cytosine and5mC (Figure 11)... The deamination ratealso decreases above pH 5.3 as the N3 unprotonated bisulfite adductdeaminates at 1% of the rate of the N3 protonated adduct... However,deamination of the bisulfite adduct is base-catalyzed, so the ratealso decreases at pH values below 5.0 due to the protonation of themost effective catalytic species, sulphite... Furthermore, it is possible to detect thenatural cytosine CpG methylation status of DNA around each nucleosome.It would be of great interest to combine ChIP-BS-Seq/BisChiP-Seq withNOMe-Seq to generate a joint map of 5mC with specific histone modificationsalong with the exact position of each nucleosome... The realization that5hmC exists in mammalian DNA has revealed animportant shortcoming of BS-Seq treatment of 5hmC with bisulfite resultsin a stable cytosine-5-methylsulfonate adduct (CMS) that, like 5mC,does not undergo deamination and is therefore read as C during sequencingdata... The kinetics of nucleotide incorporationis dependent on the nature of the bases and typically the polymeraseincorporation rate at the modified base position is slower. 5mC and5hmC have a similar low kinetic signature, which makes it is difficultto distinguish between them and nonmodified C... However, 5fC and 5caC have a greater signal than 5mC and5hmC and, through oxidiation of 5mC with the TET enzymes, have beenused to detect 5mC... It is now possible to decode 5mC, 5hmC,5fC, and 5caC in addition to G, C, A, and T in DNA at single baseresolution.

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