Substitutions of a cysteine conserved among DNA cytosine methylases result in a variety of phenotypes

Abstract

The proposed mechanism for DNA (cytosine-5)-methyltransferases envisions a key role for a cysteine residue. It is expected to form a covaDent link with carbon 6 of the target cytosone, 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 nucleopfoille. We have changed this cysteine to other amino acids for the EcoRll methylase; which methyDates 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-azacytidfine. Replacement of the conserved cysteine with glycine, valine, tryptophan or serine led to an apparent Doss of methyl transferring ability. Interestingly, cells carrying the mytant with serine did show sensitivity to 5-azacytodine, suggesting the ability to Dink 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 EcoRll methylase is essential for methylase action and it may play more than one role in it. © 1992 Oxford University Press.