title: DNA binding properties in vivo and target recognition domain sequence alignment analyses of wild-type and mutant RsrI [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 beta class [N:6-adenine] DNA methyltransferase M.RSR:I. M.RSR:I mutants with altered binding affinities in vivo were isolated. Unlike the wild-type enzyme, a catalytically compromised mutant, M.RSR:I (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.RSR:I (L72P/D173A), showed less binding in vivo than did M.RSR:I (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 beta 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 (alpha and gamma) having the same DNA recognition sequence as the beta class MTases share related regions of amino acid sequences in their TRDs.
authors.affiliation: Department of Biochemistry and College of Medicine, 600 South Mathews Avenue, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: S S
authors.initials: SS
authors.lastname: Szegedi
authors.forename: R I
authors.initials: RI
authors.lastname: Gumport
mesh_terms: D000225:Adenine / Q000378:metabolism; D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001435:Bacteriophages / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000821:virology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008242:Lysogeny / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D004268:DNA-Binding Proteins; D007544:Isopropyl Thiogalactoside; D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D000225:Adenine; D000241:Adenosine; C006235:sinefungin