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.
The 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.
Wilkinson
M C
MC
Department of Carcinogenesis, Christie Hospital and Holt Radium Institute, Manchester, UK.
Potter
P M
PM
Cawkwell
L
L
Georgiadis
P
P
Patel
D
D
Swann
P F
PF
Margison
G P
GP
D000477:Alkylating Agents; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004260:DNA Repair; D003854:Deoxyribonucleotides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013941:Thymine / Q000031:analogs & derivatives* / Q000378:metabolism
D000477:Alkylating Agents; D000596:Amino Acids; D003854:Deoxyribonucleotides; C021223:O-4-methylthymine; C007673:6-ethylguanine; D006147:Guanine; C008449:O-(6)-methylguanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013941:Thymine