Quantitation of base substitutions and deletions induced by chemical mutagens during DNA synthesis in vitro.
An experimental system has been developed by which base substitutions and frameshift deletions can be quantitated in vitro, using two-phase 20% polyacrylamide gel electrophoresis. Oligodeoxynucleotides, modified site-specifically, were used as templates in primer extension reactions catalyzed by DNA polymerase alpha, polymerase beta, and the Klenow fragment of Escherichia coli DNA polymerase I, with and without 3'-->5' exonuclease activity. Lesions studied included 7,8-dihydro-8-oxodeoxyguanosine, 7,8-dihydro-8-oxodeoxyadenosine, O6-methyldeoxyguanosine, N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene, and N-(deoxyguanosin-8-yl)-2- aminofluorene. Products of translesional synthesis contained dC, dA, dG, or dT opposite the lesion or one- and two-base deletions and were separated using a two-phase polyacrylamide gel system. When a template containing 8-oxoguanine was used, dAMP and/or dCAMP was incorporated opposite the lesion, the relative amounts depending on the DNA polymerase used. In contrast, the nonmutagenic base, dTMP, was incorporated exclusively opposite 8-oxodA in reactions catalyzed by Klenow fragment and pol alpha. The improved resolution provided by the two-phase gel system revealed misincorporation of dGMP opposite 8-oxodA in reactions catalyzed by pol beta. dTMP and small amounts of dCMP were incorporated opposite the lesion on an O6MedG-modified template. The bulky adduct, dG-C8-AAF, principally produced deletions; in contrast, dG-C8-AF promoted incorporation of dCMP, a nonmutagenic base. This experimental system should prove useful for establishing the miscoding potential of defined lesions in DNA templates and in correlating this information with the mutagenic properties of DNA adducts observed in cells.
Shibutani
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Department of Pharmacological Sciences, State University of New York at Stony Brook 11794-8651.
D000477:Alkylating Agents / Q000494:pharmacology; D001483:Base Sequence; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000187:drug effects*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D016368:Frameshift Mutation / Q000187:drug effects; D008969:Molecular Sequence Data; D009153:Mutagens / Q000633:toxicity*; D009841:Oligonucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D017354:Point Mutation / Q000187:drug effects; D013698:Templates, Genetic
D000477:Alkylating Agents; D004269:DNA, Bacterial; D009153:Mutagens; D009841:Oligonucleotides; D004259:DNA-Directed DNA Polymerase