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A simple method for locating methylated bases in DNA using class-IIS restriction enzymes
Gene, 74 (1988) 179-181 Elsevier
179
GEN 02628
A simple method for locating methylated bases in DNA using class-IIS restriction enzymes * (Recombinant DNA; F&I endonuclease; methyladenine; 5-methylcytosine; M - BspRI and M. FokIA DNAmethyltransferases; nucleotide sequence data base; DNA replication)
Gytirgy P6sfai * * and Waclaw Szybalski McArdle Laboratory for Cancer Research, Universityof Wisconsin, Madison, WI 53706 (U.S.A.) Tel. (608)262-2047 Received 21 May 1988 Accepted 20 June 1988 Received by publisher 6 July 1988
(a) Introduction
Sequence-specific DNA modification methyltransferases have been shown to produce three kinds of methylated nucleotides: 5-methylcytosine (m5C), N4-methylcytosine (m*C), and N6-methyladenine (mA) (Nelson and McClelland, 1987). The methylated nucleotide and its position in the recognition sequence of the methyltransferase are usually determined by chemical or enzymatic hydrolysis of methylated DNA molecules, followed by chromatographic analysis. In some cases the methylation specificity can be deduced from methylationmediated interference with restriction enzyme cleavage of overlapping sites. Also, the reduced reactivity Correspondenceto: Dr. W. Szybalski, McArdle Laboratory, of Wisconsin, (U.S.A.) University Madison, WI Tel. (608)262-1259. * Presented at the New England Biolabs Workshop on Biological DNA Modification, Gloucester, MA (U.S.A.) 20-23 May 1988. ** Permanent address: Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged (Hungary) Tel. (62)23-022. Abbreviations: AdoMet, S-adenosylmethionine; bp, base pair(s); class-IIS restriction enzymes, subclass ofclass- restriction enzymes for which the cut points are not located within the recognition sequence but are at precisely defined location(s) in respect of the recognition site; M. , methyltransferase; mA, N6methyladenine; m4C, N4-methylcytosine; m5C, 5-methylcytosine; nt, nucleotide(s). 0378-1119/88/$03.50
0 1988 Elsevler
Science Publishers
B.V. (Biomedical
of m5C with hydrazine can be used for identification. These methods are either laborious or provide limited information about the methylation pattern. We present here a simple and rapid method, which directly shows the position of the methylated base in the recognition sequence of the methyltransferase. (b) Outline of the method The method is based on our observation that class-IIS restriction enzymes (which cut the DNA at a fixed distance away from their recognition site) are not inhibited by methylation at the cleavage site. Thus, one could locate the methylated base by the following steps : (1) Isolate a DNA fragment which contains the recognition site of the methyltransferase under study, appropriately overlapping with a class-IIS restriction enzyme cleavage site. Such a sequence could be found by searching various nucleotide data bases by a computer program. (2) Methylate the DNA fragment with the methyltransferase to be characterized, using [ 3H]AdoMet methyl donor. (3) Cleave the methylated site with the class-IIS enzyme. (4) Separate the asymmetric cleavage products by gel electrophoresis. (5) Identify the 3H-labeled fragment. In the following we illustrate the use of this method by determining the methylation specificity of a cytoDivision)
180
sine methyltransferase with a symmetric recognition sequence, and of an adenine methyltransferase with asymmetric recognition sequence. (c) The methylation specificity of M - BspRI is GGmVC We isolated a 169-bp DdeI-FokI fragment of pBR322 DNA (nt 4292-98) containing a single M * BspRI site, GGCC (nt 4344-4347) (Koncz et al., 1978), and at an appropriate distance a Mb011 recognition site (nt 4354-4358). The DNA fragment was methylated by M - BspRI (kindly provided by Dr. A. Kiss) using [ 3H]AdoMet methyl donor. Subsequent digestion by Mb011 cuts the fragment inside the M *BspRI site in the following way: G G C C. CCGG r’ The two fragments were separated by agarose gel electrophoresis, run into pieces of Whatman DE81 paper, and counted in liquid scintillation cocktail. The experimental details are similar to those described by Posfai and Szybalski (1988a). We found that essentially all counts were associated with the leftward fragment (leftward fragment: 3 166 cpm, rightward fragment: 172 cpm, background: 50 cpm); thus we can exclude the guanines and the external cytosine as targets of methylation. To determine whether the internal cytosine is m4C or m5C, we sequenced through a methylated GGCC site (data not shown) by the chemical sequencing method (Maxam and Gilbert, 1980). The virtual lack of reaction with hydrazine (Ohmori et al., 1978; Butkus et al., 1985) indicated that the internal cytosine must be m5C. (d) M * FokIA methylates only one strand of its recognition site The cleavage specificity of FokI is (Sugisaki and Kanazawa, 1981). The corresponding methyltransferase, M +FokIA has been kindly supplied by Dr. I. Schildkraut of the New England Biolabs, Beverly, MA. Methylation by M *FokIA protected several DNA fragments tested from cleavage by FokI (see also Posfai and Szybalski, 1988b). With the aid of the method described we found that the two kinds of cleavage of M *FokIA placed the methyl
group on the leftward or on the rightward sequence, respectively (Posfai and Szybalski, 1988a). These results indicate that M *FokIA methylates only one strand of its recognition sequence, resulting in CX$nAAz sequence. The asymmetric methylation by M *FokIA implies that either another methyltransferase that methylates the bottom strand is present in vivo, or some mechanisms must protect from restriction activity the totally unmethylated DNA created during replication from the originally unmethylated strand. It was reported at this meeting (I. Schildkraut, R. Feehery, D. Wise, and D. Landry, personal communication) that there might be another methyltransferase (M *FokIB), which methylates both DNA strands. (e) Comments on the method (1) Since a relatively large number of class-IIS enzymes are presently available, a phage I DNA data base is statistically extensive enough to find proper overlaps for any 4- or 5-bp sequences. For longer recognition sequences one might need a larger data base. Also, other techniques (e.g., cloning, creating sequential deletions, use of synthetic oligodeoxynucleotides) could be used to derive the required sequence. (2) Usually two different cuts (sometimes even one) are sufficient to locate the methylated nucleotide. (3) Our method does not distinguish between m4C or m5C. However, it would greatly aid the chemical analysis to know the location of the methylated base. Since m4C reacts with hydrazine at the same rate as cytosine (Butkus et al., 1985), while m5C reacts much more slowly (Ohmori et al., 1978), by combining our method with chemical nucleotide sequencing we can identify the two types of cytosine methylation. (4) The validity of this method is based on the assumption that the cuts are very precise in relation to the enzyme recognition site, as was the case with our experiments. However, it was reported for two class-IIS enzymes that the cuts might depend either on the particular nucleotide sequence (F&AI; 8/7 or 9/8; Kleid et al., 1976) or may vary even for the same sequence (BcefI; 1l-13/12-14; Venetianer and Orosi, 1988). Thus, these two enzymes could be used only in very special cases.
181 REFERENCES Butkus, V., Klimafiauskas, S., KerSulyte, D., Vaitkevicius, D., Lebionka, A. and Janulaitis, A.: Investigation of restrictionmodification enzymes from M. variunr RFL19 with a new type of specificity toward modification of substrate. Nucleic Acids Res. 13 (1985) 5727-5746. Kleid, D., Humayun, Z., JetTrey, A. and Ptashne, M.: Novel properties of a restriction endonuclease isolated from HaemophiIusparahaemoIyticus. Proc. Natl. Acad. Sci. USA 73 (1976) 293-297. Koncz, C., Kiss, A. and Venetianer, P.: Biochemical characterization of the restriction-modification system of Bacillus sphaericus. Eur. J. Biochem. 89 (1978) 523-529. Mazam, A.M. and Gilbert, W.: Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65 (1980) 499-560. Nelson, M. and McClelland, M.: The effect of site-specific
methylation on restriction-modification enzymes. Nucleic Acids Res. 15 (1987) r219-r230. Ohmori, H., Tomizawa, J.-i. and Mazam, A.M.: Detection of 5-methylcytosine in DNA sequences. Nucleic Acids Res. 5 (1978) 1479-1485. P6sfai, G. and Szybalski, W.: A simple method for locating methylated bases in DNA, as applied to detect asymmetric methylation by M. FokIA. Gene (1988a) in press. P6sfai, G. and Szybalski, W.: Increasing the FokI cleavage speciticity from 5 to 7 base pairs by two-step methylation. Nucleic Acids Res. 16 (1988b) 6245. Sugisaki, H. and Kanazawa, S.: New restriction endonucleases from Flavobacterium okeanokoites (FokI) and Micrococcus luteus (MuI). Gene 16 (1981) 73-78. Venetianer, P. and Orosz, A.: BcefI, a new type IIS restriction endonuclease. Nucleic Acids Res. 16 (1988) 3053-3060. Edited by R.M. Blumenthal.
179
GEN 02628
A simple method for locating methylated bases in DNA using class-IIS restriction enzymes * (Recombinant DNA; F&I endonuclease; methyladenine; 5-methylcytosine; M - BspRI and M. FokIA DNAmethyltransferases; nucleotide sequence data base; DNA replication)
Gytirgy P6sfai * * and Waclaw Szybalski McArdle Laboratory for Cancer Research, Universityof Wisconsin, Madison, WI 53706 (U.S.A.) Tel. (608)262-2047 Received 21 May 1988 Accepted 20 June 1988 Received by publisher 6 July 1988
(a) Introduction
Sequence-specific DNA modification methyltransferases have been shown to produce three kinds of methylated nucleotides: 5-methylcytosine (m5C), N4-methylcytosine (m*C), and N6-methyladenine (mA) (Nelson and McClelland, 1987). The methylated nucleotide and its position in the recognition sequence of the methyltransferase are usually determined by chemical or enzymatic hydrolysis of methylated DNA molecules, followed by chromatographic analysis. In some cases the methylation specificity can be deduced from methylationmediated interference with restriction enzyme cleavage of overlapping sites. Also, the reduced reactivity Correspondenceto: Dr. W. Szybalski, McArdle Laboratory, of Wisconsin, (U.S.A.) University Madison, WI Tel. (608)262-1259. * Presented at the New England Biolabs Workshop on Biological DNA Modification, Gloucester, MA (U.S.A.) 20-23 May 1988. ** Permanent address: Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged (Hungary) Tel. (62)23-022. Abbreviations: AdoMet, S-adenosylmethionine; bp, base pair(s); class-IIS restriction enzymes, subclass ofclass- restriction enzymes for which the cut points are not located within the recognition sequence but are at precisely defined location(s) in respect of the recognition site; M. , methyltransferase; mA, N6methyladenine; m4C, N4-methylcytosine; m5C, 5-methylcytosine; nt, nucleotide(s). 0378-1119/88/$03.50
0 1988 Elsevler
Science Publishers
B.V. (Biomedical
of m5C with hydrazine can be used for identification. These methods are either laborious or provide limited information about the methylation pattern. We present here a simple and rapid method, which directly shows the position of the methylated base in the recognition sequence of the methyltransferase. (b) Outline of the method The method is based on our observation that class-IIS restriction enzymes (which cut the DNA at a fixed distance away from their recognition site) are not inhibited by methylation at the cleavage site. Thus, one could locate the methylated base by the following steps : (1) Isolate a DNA fragment which contains the recognition site of the methyltransferase under study, appropriately overlapping with a class-IIS restriction enzyme cleavage site. Such a sequence could be found by searching various nucleotide data bases by a computer program. (2) Methylate the DNA fragment with the methyltransferase to be characterized, using [ 3H]AdoMet methyl donor. (3) Cleave the methylated site with the class-IIS enzyme. (4) Separate the asymmetric cleavage products by gel electrophoresis. (5) Identify the 3H-labeled fragment. In the following we illustrate the use of this method by determining the methylation specificity of a cytoDivision)
180
sine methyltransferase with a symmetric recognition sequence, and of an adenine methyltransferase with asymmetric recognition sequence. (c) The methylation specificity of M - BspRI is GGmVC We isolated a 169-bp DdeI-FokI fragment of pBR322 DNA (nt 4292-98) containing a single M * BspRI site, GGCC (nt 4344-4347) (Koncz et al., 1978), and at an appropriate distance a Mb011 recognition site (nt 4354-4358). The DNA fragment was methylated by M - BspRI (kindly provided by Dr. A. Kiss) using [ 3H]AdoMet methyl donor. Subsequent digestion by Mb011 cuts the fragment inside the M *BspRI site in the following way: G G C C. CCGG r’ The two fragments were separated by agarose gel electrophoresis, run into pieces of Whatman DE81 paper, and counted in liquid scintillation cocktail. The experimental details are similar to those described by Posfai and Szybalski (1988a). We found that essentially all counts were associated with the leftward fragment (leftward fragment: 3 166 cpm, rightward fragment: 172 cpm, background: 50 cpm); thus we can exclude the guanines and the external cytosine as targets of methylation. To determine whether the internal cytosine is m4C or m5C, we sequenced through a methylated GGCC site (data not shown) by the chemical sequencing method (Maxam and Gilbert, 1980). The virtual lack of reaction with hydrazine (Ohmori et al., 1978; Butkus et al., 1985) indicated that the internal cytosine must be m5C. (d) M * FokIA methylates only one strand of its recognition site The cleavage specificity of FokI is (Sugisaki and Kanazawa, 1981). The corresponding methyltransferase, M +FokIA has been kindly supplied by Dr. I. Schildkraut of the New England Biolabs, Beverly, MA. Methylation by M *FokIA protected several DNA fragments tested from cleavage by FokI (see also Posfai and Szybalski, 1988b). With the aid of the method described we found that the two kinds of cleavage of M *FokIA placed the methyl
group on the leftward or on the rightward sequence, respectively (Posfai and Szybalski, 1988a). These results indicate that M *FokIA methylates only one strand of its recognition sequence, resulting in CX$nAAz sequence. The asymmetric methylation by M *FokIA implies that either another methyltransferase that methylates the bottom strand is present in vivo, or some mechanisms must protect from restriction activity the totally unmethylated DNA created during replication from the originally unmethylated strand. It was reported at this meeting (I. Schildkraut, R. Feehery, D. Wise, and D. Landry, personal communication) that there might be another methyltransferase (M *FokIB), which methylates both DNA strands. (e) Comments on the method (1) Since a relatively large number of class-IIS enzymes are presently available, a phage I DNA data base is statistically extensive enough to find proper overlaps for any 4- or 5-bp sequences. For longer recognition sequences one might need a larger data base. Also, other techniques (e.g., cloning, creating sequential deletions, use of synthetic oligodeoxynucleotides) could be used to derive the required sequence. (2) Usually two different cuts (sometimes even one) are sufficient to locate the methylated nucleotide. (3) Our method does not distinguish between m4C or m5C. However, it would greatly aid the chemical analysis to know the location of the methylated base. Since m4C reacts with hydrazine at the same rate as cytosine (Butkus et al., 1985), while m5C reacts much more slowly (Ohmori et al., 1978), by combining our method with chemical nucleotide sequencing we can identify the two types of cytosine methylation. (4) The validity of this method is based on the assumption that the cuts are very precise in relation to the enzyme recognition site, as was the case with our experiments. However, it was reported for two class-IIS enzymes that the cuts might depend either on the particular nucleotide sequence (F&AI; 8/7 or 9/8; Kleid et al., 1976) or may vary even for the same sequence (BcefI; 1l-13/12-14; Venetianer and Orosi, 1988). Thus, these two enzymes could be used only in very special cases.
181 REFERENCES Butkus, V., Klimafiauskas, S., KerSulyte, D., Vaitkevicius, D., Lebionka, A. and Janulaitis, A.: Investigation of restrictionmodification enzymes from M. variunr RFL19 with a new type of specificity toward modification of substrate. Nucleic Acids Res. 13 (1985) 5727-5746. Kleid, D., Humayun, Z., JetTrey, A. and Ptashne, M.: Novel properties of a restriction endonuclease isolated from HaemophiIusparahaemoIyticus. Proc. Natl. Acad. Sci. USA 73 (1976) 293-297. Koncz, C., Kiss, A. and Venetianer, P.: Biochemical characterization of the restriction-modification system of Bacillus sphaericus. Eur. J. Biochem. 89 (1978) 523-529. Mazam, A.M. and Gilbert, W.: Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65 (1980) 499-560. Nelson, M. and McClelland, M.: The effect of site-specific
methylation on restriction-modification enzymes. Nucleic Acids Res. 15 (1987) r219-r230. Ohmori, H., Tomizawa, J.-i. and Mazam, A.M.: Detection of 5-methylcytosine in DNA sequences. Nucleic Acids Res. 5 (1978) 1479-1485. P6sfai, G. and Szybalski, W.: A simple method for locating methylated bases in DNA, as applied to detect asymmetric methylation by M. FokIA. Gene (1988a) in press. P6sfai, G. and Szybalski, W.: Increasing the FokI cleavage speciticity from 5 to 7 base pairs by two-step methylation. Nucleic Acids Res. 16 (1988b) 6245. Sugisaki, H. and Kanazawa, S.: New restriction endonucleases from Flavobacterium okeanokoites (FokI) and Micrococcus luteus (MuI). Gene 16 (1981) 73-78. Venetianer, P. and Orosz, A.: BcefI, a new type IIS restriction endonuclease. Nucleic Acids Res. 16 (1988) 3053-3060. Edited by R.M. Blumenthal.