Site-specific gap-misrepair mutagenesis by O4-ethylthymine.
The mutagenicity of the DNA base O-alkylation adduct, O4-ethylthymine, specifically incorporated into the plasmid vector pUC8 at the unique SalI and HincII recognition sites, was studied in vivo. Escherichia coli, Micrococcus luteus and AMV DNA polymerases catalyze the incorporation of O4-ethylTMP against template adenine and guanine residues, resulting in DNA sequence alteration during subsequent replication in the host E. coli K-12 strain JM83. The greatest mutation frequency was observed with error-prone AMV DNA polymerase. High levels of cognate restriction endonuclease-resistant mutant plasmid isolates were obtained by gap replication repair in the presence of O4-ethylTTP. The yields of mutant isolates were dependent upon the relative concentration of the competing pyrimidine deoxynucleoside triphosphates, TTP and dCTP, in the misreplication reaction. Repair of incorporated O4-ethylTMP of plasmid DNA by in vitro treatment with specific alkyltransferase, prior to transformation in the host, effectively increases the mutagenic efficiency of the adduct. The results obtained are consistent with the high miscoding potential O4-ethylthymine observed in in vitro studies and its ability to base-pair with noncomplementary guanine residues in DNA.
D019883:Alkyl and Aryl Transferases; D004260:DNA Repair / Q000187:drug effects*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation / Q000187:drug effects*; D010957:Plasmids; D013941:Thymine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013942:Thymine Nucleotides / Q000378:metabolism; D014166:Transferases / Q000378:metabolism
D003845:Deoxycytosine Nucleotides; D013942:Thymine Nucleotides; C040384:4-ethylthymine; C024107:2'-deoxycytidine 5'-triphosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C050956:DNA alkyltransferase; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes; C024157:thymidine 5'-triphosphate; D013941:Thymine