Highly mutagenic bypass synthesis by T7 RNA polymerase of site-specific benzo[a]pyrene diol epoxide-adducted template DNA.
We have previously developed an in vitro system that allows quantitative evaluation of the fidelity of transcription during synthesis on a natural template in the presence of all four nucleotides. Here, we have employed this system using a TAA ochre codon reversion assay to examine the fidelity of transcription by T7 RNA polymerase past an adenine residue adducted at the N6-position with (-)-anti-trans- or (+)-anti-trans-benzo[a]pyrene diol epoxide (BPDE). T7 RNAP was capable of transcribing past either BPDE isomer to generate full-length run-off transcripts. The extent of bypass was found to be 32% for the (-)-anti-trans-isomer and 18% for the (+)-anti-trans-isomer. Transcription past both adducts was highly mutagenic. The reversion frequency of bypass synthesis of the (-)-anti-trans-isomer was elevated 11,000-fold and that of the (+)-anti-trans-isomer 6000-fold, relative to the reversion frequency of transcription on unadducted template. Adenine was misinserted preferentially, followed by guanine, opposite the adenine adducted with either BPDE isomer. Although base substitution errors were by far the most frequent mutation on the adducted template, three- and six-base deletions were also observed. These results suggest that transcriptional errors, particularly with regard to damage bypass, may contribute to the mutational burden of the cell.
Remington
K M
KM
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Bennett
S E
SE
Harris
C M
CM
Harris
T M
TM
Bebenek
K
K
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000138:chemical synthesis* / Q000737:chemistry; D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D018736:DNA Adducts / Q000138:chemical synthesis* / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D007536:Isomerism; D009153:Mutagens / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins
D018736:DNA Adducts; D009153:Mutagens; D014764:Viral Proteins; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases