Nucleic Acids Research, 2000, Vol. 28, No. 20 3972-3981
© 2000 Oxford University Press
DNA binding properties in vivo and target recognition domain sequence alignment analyses of wild-type and mutant RsrI [N6-adenine] DNA methyltransferases
Department of Biochemistry and College of Medicine, 600 South Mathews Avenue, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
A genetic selection method, the P22 challenge-phage assay, was used to characterize DNA binding in vivo by the prokaryotic ß class [N6-adenine] DNA methyltransferase M·RsrI. M·RsrI mutants with altered binding affinities in vivo were isolated. Unlike the wild-type enzyme, a catalytically compromised mutant, M·RsrI (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·RsrI (L72P/D173A), showed less binding in vivo than did M·RsrI (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 ß 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 (
and 
) having the same DNA recognition sequence as the ß class MTases share related regions of amino acid sequences in their TRDs.
* To whom correspondence should be addressed. Tel: +1 217 333 2852; Fax: +1 217 333 6461; Email: gumport@uiuc.edu Present address: Sandra S. Szegedi, Department of Chemistry and Biochemistry, University of California at Santa Barbara, Santa Barbara, CA 93106, USA
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