Purification of the E. coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethylguanine and O4-methylthymine in dodecadeoxyribonucleotides.
AffiliationDepartment of Carcinogenesis, Christie Hospital and Holt Radium Institute, Manchester, UK.
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AbstractThe E. coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (approximately 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG greater than O6-EtG greater than O4-MeT, however, the ogt protein was found to repair O6-MeG, O6-EtG and O4-Met, 1.1, 173 and 84 times, respectively, faster than the ada protein.
CitationPurification of the E. coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethylguanine and O4-methylthymine in dodecadeoxyribonucleotides. 1989, 17 (21):8475-84 Nucleic Acids Res.
JournalNucleic Acids Research
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