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Single amino acid changes which alter the sequence specificity of the T4 and T2 (Dam) DNA-adenine methyltransferases
275
Gene, 74 (1988) 275-276 Elsevier GEN 02614
Single amino acid changes which alter the sequence specificity of the T4 and T2 (Dam) DNA-adenine methyltransferases * (Amber mutant; nonglucosylating mutant; regions of homology; suppressor gene)
Zoe Miner, Samuel Schlagman and Stanley Hattman Department of Biology. University of Rochester, Rochester, NY 14627 (U.S.A.) Received 22 May 1988 Accepted 16 June 1988 Received by publisher 1 July 1988
Many enteric bacteria produce a Dam methyltransferase that methylates the A residue in the sequence, GATC. The related T-even phages, T2 and T4 (but not T6), also encode Dam methyltransferases (Gold et al., 1964; Fujimoto et al., 1965); however, the normal substrate for the phage enzymes is hrnC-containing DNA, since these viruses contain this base in place of C (the hmC is modified further by glucosylation). Nonglucosylating mutants (gt-) (Revel et al., 1965) are different from their gt’ parents in that they are unable to grow on certain strains, such as Pl lysogenic hosts. Derivatives of T2 gt - and T4 gt - phage capable of growth on Pl lysogens were isolated and designated damh, because they exhibit hypermethylation of their DNA (for reviews see Revel and Luria, 1970; Hattman, 1983). Thus, Damh, but not Dam+ methylation protects against Pl restriction of the asymmetric sequence, AGACC. Correspondencefoe:Dr. 2. Miner, Department ofBiology, University of Rochester, Rochester, NY 14627 (U.S.A.) Tel. (716)275-3846. * Presented at the New England Biolabs Workshop on Biological DNA Modification, Gloucester, MA (U.S.A.) 20-23 May 1988. Abbreviations: aa, amino acid(s); am, amber mutation; Dam, DNA (N6-adenine)methyltransferase; dam, gene encoding Dam; dam’, damh, dam-x denote mutational alterations in the T4 dam gene; gr’ , gt- refer to the (u@ and Bgt) glucosyltransferase-encoding genes of E. coli phage T4 which add glucose residues to hmC; hmC, 5-hydroxymethylcytosine; Py, pyrimidine. 0378-l 119/88/$03.50 0 1988 Elsevier Science Publishers B.V.
To begin to understand the nature of Dam+ and Darnh methylation, we cloned a functional T4 dam + gene (Schlagman and Hattman, 1983) and its nucleotide sequence was established (Macdonald and Mosig, 1984). To date, attempts to clone the T4 damh gene directly have been unsuccessful. However, we have isolated second-site mutants of damh, designated damh dam-x, which are unable to methylate phage DNA. We have cloned and sequenced two independent T4 damh dam-x variants. They share a common amino acid substitution, Pro-126 to Ser-126; in addition, they each have a frameshift mutation (+ 1 or -1) at different sites farther downstream. Furthermore, a portion of the dam gene from a T2 damh dam-x mutant was cloned and sequenced; this mutant exhibits the same Pro-126 to Ser-126 substitution. Therefore, we propose that this alteration is a consequence of the dam+ to damh mutation. It is interesting that this residue is located within one of three aa sequence homology regions common to several prokaryotic Dam methyltransferases (Hattman et al., 1985). Finally, a spontaneous T4 dam mutant, designated dam’, was found that methylates C-DNA, but not hmC-DNA. This mutant has a single base substitution altering Phe-127 to Val-127. Thus, two different single amino acid substitutions at adjacent sites alter the methylation capability of the Dam enzyme. This implicates homology region III in nucleotide sequence recognition. Recently, we succeeded in creating a nonsense
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276
UAG at codon 126 in a cloned T4 dam gene. Introducing another plasmid with a cloned, temperaturedependent supD suppressor gene (inserts Ser at am codons), results in tem~rat~~dependent production of DNA methylase activity. Having such an am mutant will allow us to take advantage of a series of vectors with different cloned sup genes. By separately introducing each sap vector, whose suppression at UAG is known, the effect on DNA methylation of known amino acids at aa residue 126 can be evaluated. Initial experiments have shown that an extract from cells containing the am mutant and the supD suppressor, grown at 3O”C, is able to add additional methyi groups to purified T4 dam + DNA in vitro. T2 Dam appears to have a lower sequence specificity than T4 Dam since it methylates various DNAs to higher extents (Hattman, 1970); both enzymes methylate some subset of GAPy. We have cloned and sequenced the T2 dam gene and compared it to the known sequence of T4 dam. They are ahnost identical except for three aa differences: T2(Pro-20) vs. T4(Ser-20); T2(Asp-26) vs. T4(Asn-26) (both of these codons are located in Dam homology region I); and T2(Glu- 188) vs. T4(Asp- 188). We do not know yet if one or more of these amino acid changes is responsible for the different specificity of the two Dam enzymes, In addition to the three coding changes, there are 18 silent mutations differences between T2 dam and T4 dam. A startling finding is that our original T2 dam subelone produces a functional hybrid polypeptide; viz. it has a reading frame encoding the first 163 aa of T2 Dam followed by 84 aa encoded by the pUC 18 vector. This hybrid enzyme methylates GATC in C-DNA, but is unable to methylate hmC-DNA, nor can it methylate all the sequences that wild-type T2 Dam recognizes in C-DNA. Although there seems to be little amino acid sequence homology between the C-terminal end provided by the vector to the missing portion of T2 Dam, they both exhibit very similar distributions of hydrophilic and hydrophobic resi-
dues. In addition, there is a Pro-Pro pair present in the hybrid Dam and in the normal Dam polypeptide, as well as in several other DNA-adenine methyltransferases (Hattm~ et al., 1985; Lauster et al., 1987).
REFERENCES Fujimoto, D., Srinivasan, P.R. and Borek, E.: On the nature of deoxyribonucleic acid methylases. Biological evidence for the multiple nature of the enzymes. Biochemistry 4 (1965) 2849-2855. Gold, M., Hausmann, R., Maitra, U. and Hurwitz, J.: The enzymatic methylation of RNA and DNA, VIII. Effects of bacteriophage infection on the methylation enzymes. Proc. Natl. Acad. Sci. USA 52 (1964) 292-297. Hattman, S.: DNA methylation of T-even bacteriophages and of their non-~ucosylat~ mut~ts: its role in Pl directed restriction. Virology 42 (1970) 359-370. Hattman, S.: DNA modification: methylation. In Mathews, C.K., Kutter, E.M., Mosig, G. and Berget, P.B. (Eds.), Bacteriophage T4. American Society for Microbiolo~, Washington, DC, 1983, pp. 152-155. Hattman, S., Wilkinson, J., Swinton, D., Schlagman, S., Macdonald, P.M. and Mosig, G.: Common evolutionary origin of the phage T4 dam and host Es&e&&a coii dnm DNA-adenine methyltransferase genes. J. Bacterial 164 (1985) 932-937. Lauster, R., Kriebardis, A. and Guschlbauer, W.: The GATATG modification enzyme EcoRV is closely related to the GATCrecognizing methyltr~sferases DpuII and dam from E. coli and phage T4. FEBS Lett. 220 (1987) 167-175. Macdonald, P.M. and Mosig, G.: Regulation of a new bacteriophage T4 gene, 69, that spans an origin of DNA replication. EMBO J. 3 (1984) 2863-2871. Revel, H.R. and Luria, S.: DNA-glucosylation in T-even phage: genetic determination and role in phage-host interaction. Annu. Rev. Genet. 4 (1970) 177-192. Revel, H.R., Hattman, S. and Luria, S.: Mutants of bacteriophage T2 and T6 defective in alpha-glucosyl trausferase. Biochem. Biophys. Res. Commun. 18 (1965) 545-550. Schlagman, S.L. and Hattman, S.: Molecular cloning of a functional dam’ gene coding for phage T4 DNA adenine methylase. Gene 22 ( 1983) 139-l 56. Edited by J.E. Brooks.
Gene, 74 (1988) 275-276 Elsevier GEN 02614
Single amino acid changes which alter the sequence specificity of the T4 and T2 (Dam) DNA-adenine methyltransferases * (Amber mutant; nonglucosylating mutant; regions of homology; suppressor gene)
Zoe Miner, Samuel Schlagman and Stanley Hattman Department of Biology. University of Rochester, Rochester, NY 14627 (U.S.A.) Received 22 May 1988 Accepted 16 June 1988 Received by publisher 1 July 1988
Many enteric bacteria produce a Dam methyltransferase that methylates the A residue in the sequence, GATC. The related T-even phages, T2 and T4 (but not T6), also encode Dam methyltransferases (Gold et al., 1964; Fujimoto et al., 1965); however, the normal substrate for the phage enzymes is hrnC-containing DNA, since these viruses contain this base in place of C (the hmC is modified further by glucosylation). Nonglucosylating mutants (gt-) (Revel et al., 1965) are different from their gt’ parents in that they are unable to grow on certain strains, such as Pl lysogenic hosts. Derivatives of T2 gt - and T4 gt - phage capable of growth on Pl lysogens were isolated and designated damh, because they exhibit hypermethylation of their DNA (for reviews see Revel and Luria, 1970; Hattman, 1983). Thus, Damh, but not Dam+ methylation protects against Pl restriction of the asymmetric sequence, AGACC. Correspondencefoe:Dr. 2. Miner, Department ofBiology, University of Rochester, Rochester, NY 14627 (U.S.A.) Tel. (716)275-3846. * Presented at the New England Biolabs Workshop on Biological DNA Modification, Gloucester, MA (U.S.A.) 20-23 May 1988. Abbreviations: aa, amino acid(s); am, amber mutation; Dam, DNA (N6-adenine)methyltransferase; dam, gene encoding Dam; dam’, damh, dam-x denote mutational alterations in the T4 dam gene; gr’ , gt- refer to the (u@ and Bgt) glucosyltransferase-encoding genes of E. coli phage T4 which add glucose residues to hmC; hmC, 5-hydroxymethylcytosine; Py, pyrimidine. 0378-l 119/88/$03.50 0 1988 Elsevier Science Publishers B.V.
To begin to understand the nature of Dam+ and Darnh methylation, we cloned a functional T4 dam + gene (Schlagman and Hattman, 1983) and its nucleotide sequence was established (Macdonald and Mosig, 1984). To date, attempts to clone the T4 damh gene directly have been unsuccessful. However, we have isolated second-site mutants of damh, designated damh dam-x, which are unable to methylate phage DNA. We have cloned and sequenced two independent T4 damh dam-x variants. They share a common amino acid substitution, Pro-126 to Ser-126; in addition, they each have a frameshift mutation (+ 1 or -1) at different sites farther downstream. Furthermore, a portion of the dam gene from a T2 damh dam-x mutant was cloned and sequenced; this mutant exhibits the same Pro-126 to Ser-126 substitution. Therefore, we propose that this alteration is a consequence of the dam+ to damh mutation. It is interesting that this residue is located within one of three aa sequence homology regions common to several prokaryotic Dam methyltransferases (Hattman et al., 1985). Finally, a spontaneous T4 dam mutant, designated dam’, was found that methylates C-DNA, but not hmC-DNA. This mutant has a single base substitution altering Phe-127 to Val-127. Thus, two different single amino acid substitutions at adjacent sites alter the methylation capability of the Dam enzyme. This implicates homology region III in nucleotide sequence recognition. Recently, we succeeded in creating a nonsense
(BiomedicalDivision)
276
UAG at codon 126 in a cloned T4 dam gene. Introducing another plasmid with a cloned, temperaturedependent supD suppressor gene (inserts Ser at am codons), results in tem~rat~~dependent production of DNA methylase activity. Having such an am mutant will allow us to take advantage of a series of vectors with different cloned sup genes. By separately introducing each sap vector, whose suppression at UAG is known, the effect on DNA methylation of known amino acids at aa residue 126 can be evaluated. Initial experiments have shown that an extract from cells containing the am mutant and the supD suppressor, grown at 3O”C, is able to add additional methyi groups to purified T4 dam + DNA in vitro. T2 Dam appears to have a lower sequence specificity than T4 Dam since it methylates various DNAs to higher extents (Hattman, 1970); both enzymes methylate some subset of GAPy. We have cloned and sequenced the T2 dam gene and compared it to the known sequence of T4 dam. They are ahnost identical except for three aa differences: T2(Pro-20) vs. T4(Ser-20); T2(Asp-26) vs. T4(Asn-26) (both of these codons are located in Dam homology region I); and T2(Glu- 188) vs. T4(Asp- 188). We do not know yet if one or more of these amino acid changes is responsible for the different specificity of the two Dam enzymes, In addition to the three coding changes, there are 18 silent mutations differences between T2 dam and T4 dam. A startling finding is that our original T2 dam subelone produces a functional hybrid polypeptide; viz. it has a reading frame encoding the first 163 aa of T2 Dam followed by 84 aa encoded by the pUC 18 vector. This hybrid enzyme methylates GATC in C-DNA, but is unable to methylate hmC-DNA, nor can it methylate all the sequences that wild-type T2 Dam recognizes in C-DNA. Although there seems to be little amino acid sequence homology between the C-terminal end provided by the vector to the missing portion of T2 Dam, they both exhibit very similar distributions of hydrophilic and hydrophobic resi-
dues. In addition, there is a Pro-Pro pair present in the hybrid Dam and in the normal Dam polypeptide, as well as in several other DNA-adenine methyltransferases (Hattm~ et al., 1985; Lauster et al., 1987).
REFERENCES Fujimoto, D., Srinivasan, P.R. and Borek, E.: On the nature of deoxyribonucleic acid methylases. Biological evidence for the multiple nature of the enzymes. Biochemistry 4 (1965) 2849-2855. Gold, M., Hausmann, R., Maitra, U. and Hurwitz, J.: The enzymatic methylation of RNA and DNA, VIII. Effects of bacteriophage infection on the methylation enzymes. Proc. Natl. Acad. Sci. USA 52 (1964) 292-297. Hattman, S.: DNA methylation of T-even bacteriophages and of their non-~ucosylat~ mut~ts: its role in Pl directed restriction. Virology 42 (1970) 359-370. Hattman, S.: DNA modification: methylation. In Mathews, C.K., Kutter, E.M., Mosig, G. and Berget, P.B. (Eds.), Bacteriophage T4. American Society for Microbiolo~, Washington, DC, 1983, pp. 152-155. Hattman, S., Wilkinson, J., Swinton, D., Schlagman, S., Macdonald, P.M. and Mosig, G.: Common evolutionary origin of the phage T4 dam and host Es&e&&a coii dnm DNA-adenine methyltransferase genes. J. Bacterial 164 (1985) 932-937. Lauster, R., Kriebardis, A. and Guschlbauer, W.: The GATATG modification enzyme EcoRV is closely related to the GATCrecognizing methyltr~sferases DpuII and dam from E. coli and phage T4. FEBS Lett. 220 (1987) 167-175. Macdonald, P.M. and Mosig, G.: Regulation of a new bacteriophage T4 gene, 69, that spans an origin of DNA replication. EMBO J. 3 (1984) 2863-2871. Revel, H.R. and Luria, S.: DNA-glucosylation in T-even phage: genetic determination and role in phage-host interaction. Annu. Rev. Genet. 4 (1970) 177-192. Revel, H.R., Hattman, S. and Luria, S.: Mutants of bacteriophage T2 and T6 defective in alpha-glucosyl trausferase. Biochem. Biophys. Res. Commun. 18 (1965) 545-550. Schlagman, S.L. and Hattman, S.: Molecular cloning of a functional dam’ gene coding for phage T4 DNA adenine methylase. Gene 22 ( 1983) 139-l 56. Edited by J.E. Brooks.