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

Abstract

A genetic selection method, the P22 challenge-phage assay, was used to characterize DNA binding in vivo by the prokaryotic β class [N6-adenine] DNA methyltransferase M·Rsrl. M·Rsrl mutants with altered binding affinities in vivo were isolated. Unlike the wild-type enzyme, a catalytically compromised mutant, M·Rsrl (L72P), demonstrated site-specific DNA binding in vivo. The L72P mutation is located near the highly conserved catalytic motif 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 D173A mutation deleteriously affected DNA binding. D173 is located in the putative target recognition domain (TRD) of the enzyme. Sequence alignment analyses of several β class MTases revealed a TRD sequence element that contains the D173 residue. Phylogenetic analysis suggested that divergence in the amino acid sequences of these methyltransferases correlated with differences in their DNA target recognition sequences. Furthermore, MTases of other classes (α and γ) having the same DNA recognition sequence as the β class MTases share related regions of amino acid sequences in their TRDs.