Characterization of cDNA encoding mouse DNA repair protein O sup 6 -methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli

Journal Article · Tue Feb 25 00:00:00 EST 1992 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00122a001· OSTI ID:5562711

Shiota, Susumu; Tano, Keizo ^[1]; von Wronski, M A; Brent, T P ^[2]; Bigner, D D ^[3]; Mitra, S ^[4]

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1. Univ. of Tennessee Graduate School of Biomedical Sciences, Oak Ridge (United States)
2. St. Jude Children's Research Hospital, Memphis, TN (United States)
3. Duke Univ. Medical Center, Durham, NC (United States)
4. Oak Ridge National Lab., TN (United States)

A mouse cDNA clone encoding O{sup 6}-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O{sup 6}-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.

• DOE Contract Number: AC05-84OR21400
• OSTI ID: 5562711
• Journal Information: Biochemistry; (United States), Vol. 31:7; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA ADDUCTS
DNA REPAIR
METHYL TRANSFERASES
AMINO ACID SEQUENCE
RECOMBINANT DNA
DNA SEQUENCING
ESCHERICHIA COLI
GUANINE
MAN
MOLECULAR BIOLOGY
ADDUCTS
AMINES
ANIMALS
AROMATICS
AZAARENES
BACTERIA
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CARBON-GROUP TRANSFERASES
DNA
ENZYMES
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
MAMMALS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PRIMATES
PROTEINS
PURINES
RECOVERY
REPAIR
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
VERTEBRATES
550200* - Biochemistry