Purification of the KpnI DNA methyltransferase and photolabeling of the enzyme with S-adenosyl-L-methionine.
An Escherichia coli strain overproducing the KpnI DNA methyltransferase (M.KpnI) was constructed by cloning the kpnIM gene downstream from the inducible T7 phage luminal diameter 10 promoter. A method involving three chromatographic steps has been developed to purify M.KpnI to homogeneity. The purified enzyme has a pH optimum around 7.3 and is inhibited by salts. M.KpnI can be photolabeled by UV-irradiation of the enzyme in the presence of S-adenosyl-L-[methyl-3H]methionine ([methyl-3H]AdoMet). Photolabeling results from a specific interaction between M.KpnI and AdoMet, as indicated by the dependence of photolabeling on native enzyme conformation and by the inhibitory effect of the AdoMet analogs, sinefungin and S-adenosyl-L-homocysteine (AdoHcy).
Finta
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C
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.
Sulima
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U
Venetianer
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P
Kiss
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A
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012435:S-Adenosylhomocysteine / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000528:radiation effects; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays
D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; C072049:DNA modification methylase KpnI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D000241:Adenosine; C006235:sinefungin