Site-directed mutation of the Escherichia coli ada gene: effects of substitution of methyl acceptor cysteine-321 by histidine in Ada protein.
Oligodeoxynucleotide-mediated mutagenesis of the ada gene of Escherichia coli was used to produce two mutant Ada proteins. In mutant I the methyl acceptor Cys-321 for O6-methylguanine was replaced by histidine; and in mutant II the positions of Cys-321 and His-322 of the wild-type protein were inverted. Neither mutant protein had O6-methylguanine-DNA methyltransferase activity, but both retained the phosphotriester-DNA methyltransferase activity involving methyl group transfer to Cys-69. Under the control of the endogenous promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with N-methyl-N'-nitro-N-nitrosoguanidine. This appeared to be due to both inhibition of transcription of the mutant gene and degradation of the synthesized protein. On the other hand, mutant II protein was inducible by N-methyl-N'-nitro-N-nitrosoguanidine, although to a smaller extent than the wild-type protein was, and the phosphotriester-DNA methyltransferase activity appeared to reside in 24- to 30-kilodalton cleavage products. Mutant I protein could be produced under lac promoter control, and its cleavage products, unlike those of mutant II protein, tended to aggregate. These results indicate that (i) Cys-321 cannot be replaced or transposed with the nucleophilic amino acid histidine for O6-methylguanine-DNA methyltransferase function, (ii) single amino acid replacement or transposition at the O6-methylguanine methyl acceptor site can have a profound effect on the in vivo stability and regulatory function of the Ada protein, and (iii) the integrity of the protein may not be absolutely needed for its transcription-activation function.
Tano
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School of Biomedical Sciences, Oak Ridge National Laboratory, Tennessee 37831.
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D003545:Cysteine; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005796:Genes; D005798:Genes, Bacterial; D006639:Histidine; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010957:Plasmids; D015183:Restriction Mapping; D014157:Transcription Factors; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D014157:Transcription Factors; D006639:Histidine; D008780:Methyltransferases; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; D001616:beta-Galactosidase; D003545:Cysteine