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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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Detection of 5mC and5hmC with restriction endonucleases. 5mC and5hmC can be distinguished at an HpaII/MspI restriction digestion site by comparing digestion reactions. HpaII only digests DNA that does not contain 5mC or 5hmC. MspI digests DNA that contains 5mC and 5hmC. Glucosylationof 5hmC (5gC) inhibits digestion with MspI. 5hmCcan be distinguished by comparing glucosylation/MspI digestion and MspI only digestion. 5mC can bedistinguished by comparing glucosylatin/MspI digestionand HpaII digestion.
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fig6: Detection of 5mC and5hmC with restriction endonucleases. 5mC and5hmC can be distinguished at an HpaII/MspI restriction digestion site by comparing digestion reactions. HpaII only digests DNA that does not contain 5mC or 5hmC. MspI digests DNA that contains 5mC and 5hmC. Glucosylationof 5hmC (5gC) inhibits digestion with MspI. 5hmCcan be distinguished by comparing glucosylation/MspI digestion and MspI only digestion. 5mC can bedistinguished by comparing glucosylatin/MspI digestionand HpaII digestion.

Mentions: The two restriction enzymes regularlyused to detect 5mC are HpaII and MspI, which both cut at the sameDNA sequence; CCGG. This sequence is ideal as it contains a CpG dinucleotide,which is where the majority of 5mC resides in mammals.27HpaII is methylation-sensitiveand will only cut a CCGG sequence that does not contain 5mC, whereas MspI is methylation-insensitive and will cut a CCGG sequencewith or without 5mC (Figure 6).


Chemical methods for decoding cytosine modifications in DNA.

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

Detection of 5mC and5hmC with restriction endonucleases. 5mC and5hmC can be distinguished at an HpaII/MspI restriction digestion site by comparing digestion reactions. HpaII only digests DNA that does not contain 5mC or 5hmC. MspI digests DNA that contains 5mC and 5hmC. Glucosylationof 5hmC (5gC) inhibits digestion with MspI. 5hmCcan be distinguished by comparing glucosylation/MspI digestion and MspI only digestion. 5mC can bedistinguished by comparing glucosylatin/MspI digestionand HpaII digestion.
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Related In: Results  -  Collection

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

fig6: Detection of 5mC and5hmC with restriction endonucleases. 5mC and5hmC can be distinguished at an HpaII/MspI restriction digestion site by comparing digestion reactions. HpaII only digests DNA that does not contain 5mC or 5hmC. MspI digests DNA that contains 5mC and 5hmC. Glucosylationof 5hmC (5gC) inhibits digestion with MspI. 5hmCcan be distinguished by comparing glucosylation/MspI digestion and MspI only digestion. 5mC can bedistinguished by comparing glucosylatin/MspI digestionand HpaII digestion.
Mentions: The two restriction enzymes regularlyused to detect 5mC are HpaII and MspI, which both cut at the sameDNA sequence; CCGG. This sequence is ideal as it contains a CpG dinucleotide,which is where the majority of 5mC resides in mammals.27HpaII is methylation-sensitiveand will only cut a CCGG sequence that does not contain 5mC, whereas MspI is methylation-insensitive and will cut a CCGG sequencewith or without 5mC (Figure 6).

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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