Substitutions of a cysteine conserved among DNA cytosine methylases result in a variety of phenotypes.
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.
Wyszynski
M W
MW
Department of Chemistry, Wayne State University, Detroit, MI 48202.
Gabbara
S
S
Bhagwat
A S
AS
D001374:Azacitidine / Q000494:pharmacology; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D015257:DNA-Cytosine Methylases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
D003062:Codon; D011993:Recombinant Fusion Proteins; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases; D003545:Cysteine; D001374:Azacitidine