Binding of the EcoRII methyltransferase to 5-fluorocytosine-containing DNA. Isolation of a bound peptide.
The properties of the interaction of 5-fluorocytosine-containing DNA with the EcoRII methyltransferase were studied. The DNA used was either a polymer synthesized in vitro, or a 20-mer containing one CCA/TGG sequence. The DNA could be methylated by the enzyme. In the process the enzyme formed a tight binding adduct with the DNA that could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzyme activity was inhibited by this interaction. The 20-mer could be used to titrate the active site of the enzyme. The DNA polymer formed a tight binding complex that could be identified following digestion of the DNA with pancreatic deoxyribonuclease or micrococcal nuclease. A peptide-DNA adduct could be isolated after digestion of the EcoRII-DNA adduct with staphylococcal protease V8 by high pressure liquid chromatography and polyacrylamide gel electrophoresis. Sequencing of the peptide indicated the DNA bound to a region of the protein that is conserved in all procaryotic DNA(cytosine-5)-methyltransferases. We have previously shown that this region contains a cysteine that can be photomethylated with adenosylmethionine. This region, in addition to forming part of, or being adjacent to, the AdoMet binding site, also forms part of the DNA binding site.
Friedman
S
S
Department of Pharmacology, SUNY Health Science Center, Brooklyn 11203.
Ansari
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005437:Flucytosine / Q000378:metabolism*; D008745:Methylation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D004247:DNA; D005437:Flucytosine; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases