Site-directed mutagenesis of DNA-O/sup 6/-methylguanine methyltransferase of E. coli

Journal Article · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5144966

Mitra, S; Foote, R S

E. coli Ada protein (39kD) and its 19kD cleavage product act as DNA-O/sup 6/-methylguanine methyltransferase (MGMT) by stoichiometrically accepting the methyl group from promutagenic O/sup 6/-methylguanine residues (and phosphotriesters in the case of Ada protein) in DNA. Methylated Ada acts as a positive regulator for its own gene and some other DNA repair genes. The methyl group of 0/sup 6/-methylguanine is transferred to a specific cysteine residue (in a Pro-Cys-His sequence) in MGMT. The authors have substituted the cysteine with histidine in the Ada protein by oligodeoxynucleotide-directed mutagenesis of the cloned ada gene. Both wild type and an ada (noninducible) mutant of E. coli harboring plasmids containing either normal or mutant ada genes have comparable levels of MGMT. Furthermore, the level of induction of MGMT by N-methyl-N'-nitro-N-nitrosoguanidine is similar in all cases. Whether the mutant protein causes induction of host MGMT or is itself active as a methyl acceptor is being investigated. Other MGMT mutants in which the cysteine residue was replaced with alanine or aspartic acid or in which the cysteine-histidine sequence was transposed have been prepared, and their enzymatic activity and autoregulation are being studied.

• Research Organization: Oak Ridge National Lab., TN
• OSTI ID: 5144966
• Report Number(s): CONF-8606151-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
• Country of Publication: United States
• Language: English

Related Subjects

560302* -- Chemicals Metabolism & Toxicology-- Microorganisms-- (-1987)
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AMINO ACIDS
AZOLES
BACTERIA
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CARBOXYLIC ACIDS
CYSTEINE
DNA
DNA REPAIR
ENZYME INDUCTION
ENZYMES
ESCHERICHIA COLI
GENE REGULATION
GENES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HISTIDINE
IMIDAZOLES
MICROORGANISMS
MUTAGENESIS
MUTANTS
NITROSO COMPOUNDS
NUCLEIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
RECOVERY
REPAIR
STOICHIOMETRY
THIOLS
TRANSFERASES