A simple method for locating methylated bases in DNA, as applied to detect asymmetric methylation by M FokIA

G. Pósfai, Waclaw Szybalski

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Class-IIS restriction enzymes, which cut the DNA outside their recognition sequence, could be used for locating the bases methylated by a DNA-modification methylase. This is possible because methylation of the class-IIS cut sites does not interfere with the cleavage. The method consists of (i) selection of a nucleotide sequence with appropriate overlap between the methylase recognition site and the class-IIS enzyme cut site, (ii) methylation using S-adenosylmethionine as [3H]methyl donor, (iii) cleavage of the methylated sequence with the class-IIS enzyme, (iv) separation of the cleavage products and identification of the 3H-labelled fragment. Using this simple and straightforward method, we have shown that M · FokIA is an adenine methylase and methylates asymmetrically one strand of the FokI recognition site, resulting in the 5'-GGmATG(N)9 sequence. In addition, it was observed that another class-IIS restriction enzyme, SfaNI, CC TAC(N)13 is completely inhibited by methylation of its recognition site, 5'-GC A TC, by M. FokIA. CGTmAG.

Original languageEnglish
Pages (from-to)147-151
Number of pages5
JournalGene
Volume69
Issue number1
DOIs
Publication statusPublished - Sep 15 1988

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Methylation
DNA
Enzymes
DNA Modification Methylases
S-Adenosylmethionine
Adenine

Keywords

  • class-IIS restriction enzymes
  • DNA methylase
  • DNA replication
  • nucleotide sequence data base
  • Recombinant DNA

ASJC Scopus subject areas

  • Genetics

Cite this

A simple method for locating methylated bases in DNA, as applied to detect asymmetric methylation by M FokIA. / Pósfai, G.; Szybalski, Waclaw.

In: Gene, Vol. 69, No. 1, 15.09.1988, p. 147-151.

Research output: Contribution to journalArticle

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abstract = "Class-IIS restriction enzymes, which cut the DNA outside their recognition sequence, could be used for locating the bases methylated by a DNA-modification methylase. This is possible because methylation of the class-IIS cut sites does not interfere with the cleavage. The method consists of (i) selection of a nucleotide sequence with appropriate overlap between the methylase recognition site and the class-IIS enzyme cut site, (ii) methylation using S-adenosylmethionine as [3H]methyl donor, (iii) cleavage of the methylated sequence with the class-IIS enzyme, (iv) separation of the cleavage products and identification of the 3H-labelled fragment. Using this simple and straightforward method, we have shown that M · FokIA is an adenine methylase and methylates asymmetrically one strand of the FokI recognition site, resulting in the 5'-GGmATG(N)9 sequence. In addition, it was observed that another class-IIS restriction enzyme, SfaNI, CC TAC(N)13 is completely inhibited by methylation of its recognition site, 5'-GC A TC, by M. FokIA. CGTmAG.",
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N2 - Class-IIS restriction enzymes, which cut the DNA outside their recognition sequence, could be used for locating the bases methylated by a DNA-modification methylase. This is possible because methylation of the class-IIS cut sites does not interfere with the cleavage. The method consists of (i) selection of a nucleotide sequence with appropriate overlap between the methylase recognition site and the class-IIS enzyme cut site, (ii) methylation using S-adenosylmethionine as [3H]methyl donor, (iii) cleavage of the methylated sequence with the class-IIS enzyme, (iv) separation of the cleavage products and identification of the 3H-labelled fragment. Using this simple and straightforward method, we have shown that M · FokIA is an adenine methylase and methylates asymmetrically one strand of the FokI recognition site, resulting in the 5'-GGmATG(N)9 sequence. In addition, it was observed that another class-IIS restriction enzyme, SfaNI, CC TAC(N)13 is completely inhibited by methylation of its recognition site, 5'-GC A TC, by M. FokIA. CGTmAG.

AB - Class-IIS restriction enzymes, which cut the DNA outside their recognition sequence, could be used for locating the bases methylated by a DNA-modification methylase. This is possible because methylation of the class-IIS cut sites does not interfere with the cleavage. The method consists of (i) selection of a nucleotide sequence with appropriate overlap between the methylase recognition site and the class-IIS enzyme cut site, (ii) methylation using S-adenosylmethionine as [3H]methyl donor, (iii) cleavage of the methylated sequence with the class-IIS enzyme, (iv) separation of the cleavage products and identification of the 3H-labelled fragment. Using this simple and straightforward method, we have shown that M · FokIA is an adenine methylase and methylates asymmetrically one strand of the FokI recognition site, resulting in the 5'-GGmATG(N)9 sequence. In addition, it was observed that another class-IIS restriction enzyme, SfaNI, CC TAC(N)13 is completely inhibited by methylation of its recognition site, 5'-GC A TC, by M. FokIA. CGTmAG.

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