DNA binding properties in vivo and target recognition domain sequence alignment analyses of wild-type and mutant RsrI [N6-adenine] DNA methyltransferases

A genetic selection method, the P22 challenge-phage assay, was used to char acterize DNA binding in vivo by the prokaryotic beta class [N6-adenine] DNA methyltransferase M . Rsrl. M . Rsrl mutants with altered binding affiniti es in vivo were isolated, Unlike the wild-type enzyme, a catalytically comp romised mutant, M . Rsrl (L72P), demonstrated site-specific DNA binding in vivo. The L72P mutation is located near the highly conserved catalytic moti f IV, DPPY (residues 65-68), A double mutant, M . Rsrl(L72P/D173A), showed less binding in vivo than did M . Rsrl (L72P), Thus, introduction of the D1 73A mutation deleteriously affected DNA binding. D173 is located in the put ative target recognition domain (TRD) of the enzyme. Sequence alignment ana lyses of several beta class MTases revealed a TRD sequence element that con tains the D173 residue, Phylogenetic analysis suggested that divergence in the amino acid sequences of these methyltransferases correlated with differ ences in their DNA target recognition sequences. Furthermore, MTases of oth er classes (alpha and gamma) having the same DNA recognition sequence as th e beta class MTases share related regions of amino acid sequences in their TRDs.