The irreversible binding of azacytosine-containing DNA fragments to bacterial DNA(cytosine-5)methyltransferases.
DNA containing 5-azacytosine is an irreversible inhibitor of DNA(cytosine-5)methyltransferase. This paper describes the binding of DNA methyltransferase to 32P-labeled fragments of DNA containing 5-azacytosine. The complexes were identified by gel electrophoresis. The EcoRII methyltransferase specified by the R15 plasmid was purified from Escherichia coli B(R15). This enzyme methylates the second C in the sequence CCAGG and has a molecular mass of 60,000 Da. Specific binding of enzyme to DNA fragments could be detected if either excess unlabeled DNA or 0.8% sodium dodecyl sulfate was added to the reaction mixture prior to electrophoresis. Binding was dependent upon the presence of both the CCAGG sequence and azacytosine in the DNA fragment. S-Adenosylmethionine stimulated the formation of the complex. The complex was stable to 6 M urea but could be digested with pronase. These DNA fragments could be used to detect the presence of several different methyltransferases in crude extracts of E. coli. No DNA protein complexes could be detected in E. coli B extracts, a strain that contains no DNA(cytosine-5)methyltransferases. The chromosomally determined methylase with the same specificity as the purified EcoRII methylase could be detected in crude extracts of E. coli K12 strains. The MspI methylase cloned in E. coli HB101 could also be detected in crude extracts. These enzymes are the only proteins that bind azacytosine-containing DNA in crude extracts of E. coli.
Friedman
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D003596:Cytosine / Q000031:analogs & derivatives*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D015257:DNA-Cytosine Methylases; D019003:Deoxyribonuclease HpaII; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011189:Potassium Chloride / Q000494:pharmacology; D011402:Pronase / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000494:pharmacology
D004269:DNA, Bacterial; C039854:5-azacytosine; D011189:Potassium Chloride; D012436:S-Adenosylmethionine; D003596:Cytosine; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases; D008780:Methyltransferases; D004262:DNA Restriction Enzymes; D019003:Deoxyribonuclease HpaII; D011402:Pronase