The core element of the EcoRII methylase as defined by protease digestion and deletion analysis.
Binding of the EcoRII DNA methyltransferase to azacytosine-containing DNA protects the enzyme from digestion by proteases. The limit digest yields a product having a Mr on SDS-PAGE 20% less than the intact protein. The N terminus of the tryptic digestion product was sequenced and found to be missing the N terminal 82 amino acids. Under the conditions used unbound enzyme was digested to small peptides. Protection of the enzyme from protease digestion implies that the enzyme undergoes major conformational changes when bound to DNA. The trypsin sensitive region of the EcoRII methyltransferase occurs prior to the first constant region shared with other procaryotic DNA(cytosine-5)methyltransferases. To determine if this region played a role in substrate binding or specificity, N-terminal deletion mutants were studied. Deletion of 97 amino acids resulted in a decrease of enzyme activity. Further deletions caused a complete loss of activity. Enzyme deleted through amino acid 85 was purified and found to have the same specificity as wild type however there was an increase in Km for both S-adenosylmethionine (AdoMet) and DNA of 27 and 18 fold respectively. The N-terminus of the EcoRII methylase, although a variable region present in many procaryotic DNA(cytosine-5)methylases, plays no role in determining enzyme specificity, although it does contribute to the interaction with both AdoMet and DNA.
Friedman
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Department of Pharmacology, SUNY Health Science Center, Brooklyn 11203.
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Yang
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002872:Chromosome Deletion; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D015183:Restriction Mapping; D012436:S-Adenosylmethionine / Q000378:metabolism
D004269:DNA, Bacterial; D046911:Macromolecular Substances; C039854:5-azacytosine; D012436:S-Adenosylmethionine; D003596:Cytosine; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases; D010450:Endopeptidases