Purification and characterization of the M.RsrI DNA methyltransferase from Escherichia coli.
The gene (rsrIM) encoding the RsrI DNA methyltransferase (M.RsrI) from Rhodobacter sphaeroides was cloned and expressed in Escherichia coli. Under the control of a bacteriophage T7 promoter, 2% of the total protein in a crude extract was M.RsrI. This level of expression represents an approximately 50-fold increase over that present in the natural host. Chromatography using DNA cellulose and the S-adenosylmethionine analogue, sinefungin, was useful in purifying the enzyme to homogeneity. The purification yielded 100 times more enzyme than was obtained from the same quantity of R. sphaeroides cell paste. M.RsrI deposits one methyl group per productive DNA-binding event, as does its functional but sequence-nonhomologous analogue, M.EcoRI. Unlike M.EcoRI, the R. sphaeroides enzyme is a dimer at micromolar concentrations.
Kaszubska
W
W
Department of Biochemistry, College of Medicine, University of Illinois, Urbana 61801-3792.
Webb
H K
HK
Gumport
R I
RI
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008745:Methylation; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; D011994:Recombinant Proteins; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)