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Discovery of bisulfite-mediated cytosine conversion to uracil, the key reaction for DNA methylation analysis--a personal account.

Hayatsu H - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2008)

Bottom Line: At the same time, Shapiro and his coworkers in New York University found the same reaction independently.We also reported that 5-methylcytosine was deaminated by bisulfite only very slowly.The author's recent work that has resulted in an improvement of the procedure of analysis by use of a newly devised high concentration bisulfite solution is also described.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan. hayatsuh@cc.okayama-u.ac.jp

ABSTRACT
Methylation at position 5 of cytosine in DNA is being intensively studied in many areas of biological sciences, as the methylation is intimately associated with the control of gene functions. The principal analytical method for determining the sites of 5-methylcytosine in genome at the sequence level involves bisulfite modification of DNA. The utility of this chemical treatment is based on the property of bisulfite to selectively deaminate cytosine residues. The bisulfite-mediated cytosine deamination was discovered in 1970 by us in the University of Tokyo. At the same time, Shapiro and his coworkers in New York University found the same reaction independently. We also reported that 5-methylcytosine was deaminated by bisulfite only very slowly. These findings were later utilized by a group of Australian scientists to devise a means to analyze 5-methylcytosine in DNA; thus, a method called 'bisulfite genomic sequencing' was invented by these researchers in 1992. This review describes the author's reflection of the discovery of bisulfite reactions with pyrimidine bases. The author's recent work that has resulted in an improvement of the procedure of analysis by use of a newly devised high concentration bisulfite solution is also described.

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Relationship between bisulfite concentration and rate of deamination of cytidine 5′-phosphate at pH 5.0 and 37 °C. The dotted line represents rates with no participation of bisulfite in hydrolyzing [C-SO3−], and the solid line those predicted in case of participation of bisulfite in the process of hydrolysis.
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f8-321: Relationship between bisulfite concentration and rate of deamination of cytidine 5′-phosphate at pH 5.0 and 37 °C. The dotted line represents rates with no participation of bisulfite in hydrolyzing [C-SO3−], and the solid line those predicted in case of participation of bisulfite in the process of hydrolysis.

Mentions: The mechanism of bisulfite-mediated deamination of cytosine was then shown to involve a mysterious action of bisulfite. The key process, [C-SO3−] to [U-SO3−] (Step 2 in Fig. 5), was revealed to be not a simple hydrolysis. This hydrolysis seemed to involve an additional role of bisulfite. If the hydrolysis is a simple attack of OH− to position 4 of [C-SO3−] to replace –NH2 with –OH, a theoretical consideration indicated that the relationship between the rate of the conversion [C] to [U-SO3−] and the concentration of bisulfite should be represented by a dotted line as shown in Fig. 8. However, the fact is that the observed rates of the reaction, as shown by the filled circles in the Figure, were far apart from the expected line. We found that by assuming an active participation of bisulfite in the process of this hydrolysis, a dose-response curve can be drawn fitting with those data points (the solid line in Fig. 8). How bisulfite catalyzes the hydrolysis, however, remains unknown until today.19) Shapiro’s team also made the same observation and considered various possibilities for the role of bisulfite in the hydrolysis, but they too encountered difficulty to uncover it.20)


Discovery of bisulfite-mediated cytosine conversion to uracil, the key reaction for DNA methylation analysis--a personal account.

Hayatsu H - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2008)

Relationship between bisulfite concentration and rate of deamination of cytidine 5′-phosphate at pH 5.0 and 37 °C. The dotted line represents rates with no participation of bisulfite in hydrolyzing [C-SO3−], and the solid line those predicted in case of participation of bisulfite in the process of hydrolysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3722019&req=5

f8-321: Relationship between bisulfite concentration and rate of deamination of cytidine 5′-phosphate at pH 5.0 and 37 °C. The dotted line represents rates with no participation of bisulfite in hydrolyzing [C-SO3−], and the solid line those predicted in case of participation of bisulfite in the process of hydrolysis.
Mentions: The mechanism of bisulfite-mediated deamination of cytosine was then shown to involve a mysterious action of bisulfite. The key process, [C-SO3−] to [U-SO3−] (Step 2 in Fig. 5), was revealed to be not a simple hydrolysis. This hydrolysis seemed to involve an additional role of bisulfite. If the hydrolysis is a simple attack of OH− to position 4 of [C-SO3−] to replace –NH2 with –OH, a theoretical consideration indicated that the relationship between the rate of the conversion [C] to [U-SO3−] and the concentration of bisulfite should be represented by a dotted line as shown in Fig. 8. However, the fact is that the observed rates of the reaction, as shown by the filled circles in the Figure, were far apart from the expected line. We found that by assuming an active participation of bisulfite in the process of this hydrolysis, a dose-response curve can be drawn fitting with those data points (the solid line in Fig. 8). How bisulfite catalyzes the hydrolysis, however, remains unknown until today.19) Shapiro’s team also made the same observation and considered various possibilities for the role of bisulfite in the hydrolysis, but they too encountered difficulty to uncover it.20)

Bottom Line: At the same time, Shapiro and his coworkers in New York University found the same reaction independently.We also reported that 5-methylcytosine was deaminated by bisulfite only very slowly.The author's recent work that has resulted in an improvement of the procedure of analysis by use of a newly devised high concentration bisulfite solution is also described.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan. hayatsuh@cc.okayama-u.ac.jp

ABSTRACT
Methylation at position 5 of cytosine in DNA is being intensively studied in many areas of biological sciences, as the methylation is intimately associated with the control of gene functions. The principal analytical method for determining the sites of 5-methylcytosine in genome at the sequence level involves bisulfite modification of DNA. The utility of this chemical treatment is based on the property of bisulfite to selectively deaminate cytosine residues. The bisulfite-mediated cytosine deamination was discovered in 1970 by us in the University of Tokyo. At the same time, Shapiro and his coworkers in New York University found the same reaction independently. We also reported that 5-methylcytosine was deaminated by bisulfite only very slowly. These findings were later utilized by a group of Australian scientists to devise a means to analyze 5-methylcytosine in DNA; thus, a method called 'bisulfite genomic sequencing' was invented by these researchers in 1992. This review describes the author's reflection of the discovery of bisulfite reactions with pyrimidine bases. The author's recent work that has resulted in an improvement of the procedure of analysis by use of a newly devised high concentration bisulfite solution is also described.

Show MeSH