Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli.
A mouse cDNA clone encoding O6-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O6-alkylguanine in DNA, was cloned from a lambda gt11 library. On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa. The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally. The deduced amino acid sequence of the mouse MGMT is 70% homologous to that of the human MGMT. Cysteine-149 was shown to be the only alkyl acceptor residue in the mouse protein, in confirmation of the prediction based on conserved sequences of different MGMTs. Mouse MGMT protein is recognized by some monoclonal antibodies specific for human MGMT. Site-directed mutagenesis was utilized to reclone the mouse cDNA in a T7 promoter-based vector for overexpression of the native repair protein in Escherichia coli. The mouse protein has a tetrapeptide sequence, Pro-Glu-Gly-Val at positions 56-59, absent in the human protein. Neither deletion of this tetrapeptide nor substitution of valine-169 with alanine affected the activity of the mutant proteins.
University of Tennessee, Oak Ridge Graduate School of Biomedical Sciences 37831.
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Shiota
M A
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von Wronski
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Tano
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Bigner
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Brent
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Mitra
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D008780:Methyltransferases / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D019853:O(6)-Methylguanine-DNA Methyltransferase; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D012313:RNA; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase