Dimerization of the bacterial RsrI N6-adenine DNA methyltransferase.
Dimeric restriction endonucleases and monomeric modification methyltransferases were long accepted as the structural paradigm for Type II restriction systems. Recent studies, however, have revealed an increasing number of apparently dimeric DNA methyltransferases. Our initial characterization of RsrI methyltransferase (M.RsrI) was consistent with the enzyme functioning as a monomer, but, subsequently, the enzyme crystallized as a dimer with 1500 A2 of buried surface area. This result led us to re-examine the biochemical properties of M.RsrI. Gel-shift studies of M.RsrI binding to DNA suggested that binding cooperativity targets hemimethylated DNA preferentially over unmethylated DNA. Size-exclusion chromatography indicated that the M.RsrI-DNA complex had a size and stoichiometry consistent with a dimeric enzyme binding to the DNA. Kinetic measurements revealed a quadratic relationship between enzyme velocity and concentration. Site-directed mutagenesis at the dimer interface affected the kinetics and DNA-binding of the enzyme, providing support for a model proposing an active enzyme dimer. We also identified a conserved motif in the dimer interfaces of the beta-class methyltransferases M.RsrI, M.MboIIA and M2.DpnII. Taken together, these data suggest that M.RsrI may be part of a sub-class of MTases that function as dimers.
Thomas
Chad B
CB
Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Gumport
Richard I
RI
D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)