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1.
Nucleic Acids Res ; 21(2): 295-301, 1993 Jan 25.
Article in English | MEDLINE | ID: mdl-8441637

ABSTRACT

All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position. We show here that substitutions of this cysteine in the E. coli methylase M. EcoRII with either serine or tryptophan results in a complete loss of ability to transfer methyl groups to DNA. Interestingly, mutants with either serine or glycine substitution bind tightly to substrate DNA. These mutants resemble the wild-type enzyme in that their binding to substrate is not eliminated by the presence of non-specific DNA in the reaction, it is sensitive to methylation status of the substrate and is stimulated by an analog of the methyl donor. Hence the conserved cysteine is not essential for the specific stable binding of the enzyme to its substrate. However, substitution of the cysteine with the bulkier tryptophan does reduce DNA binding. We also report here a novel procedure for the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA substrate for M. EcoRII in which the target cytosine is replaced by 5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms an irreversible complex with the enzyme. As expected, this modified substrate does not form irreversible complexes with the mutants.


Subject(s)
Cysteine/metabolism , DNA-Cytosine Methylases/metabolism , DNA/metabolism , Base Sequence , DNA/biosynthesis , DNA-Cytosine Methylases/chemistry , DNA-Cytosine Methylases/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Flucytosine/metabolism , Methylation , Molecular Sequence Data , Mutation , Protein Binding , Substrate Specificity
2.
Nucleic Acids Res ; 20(2): 319-26, 1992 Jan 25.
Article in English | MEDLINE | ID: mdl-1371346

ABSTRACT

The proposed mechanism for DNA (cytosine-5)-methyltransferases envisions a key role for a cysteine residue. It is expected to form a covalent link with carbon 6 of the target cytosine, activating the normally inactive carbon 5 for methyl transfer. There is a single conserved cysteine among all DNA (cytosine-5)-methyltransferases making it the candidate nucleophile. We have changed this cysteine to other amino acids for the EcoRII methylase; which methylates the second cytosine in the sequence 5'-CCWGG-3'. Mutants were tested for their methyl transferring ability and for their ability to form covalent complexes with DNA. The latter property was tested indirectly with the use of a genetic assay involving sensitivity of cells to 5-azacytidine. Replacement of the conserved cysteine with glycine, valine, tryptophan or serine led to an apparent loss of methyl transferring ability. Interestingly, cells carrying the mutant with serine did show sensitivity to 5-azacytidine, suggesting the ability to link to DNA. Unexpectedly, substitution of the cysteine with glycine results in the inhibition of cell growth and the mutant allele can be maintained in the cells only when it is poorly expressed. These results suggest that the conserved cysteine in the EcoRII methylase is essential for methylase action and it may play more than one role in it.


Subject(s)
Cysteine/metabolism , DNA-Cytosine Methylases/metabolism , Azacitidine/pharmacology , Cloning, Molecular , Codon/genetics , Cysteine/genetics , DNA Mutational Analysis , DNA-Cytosine Methylases/drug effects , DNA-Cytosine Methylases/genetics , Mutagenesis, Site-Directed , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
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