Mutation induced in vitro on a C-8 guanine aminofluorene containing template by a modified T7 DNA polymerase.
We reacted uracil-containing M13mp2 DNA with N-hydroxy-2-aminofluorene to produce a template with N-(deoxyguanosin-8-yl)-2-aminofluorene adducts. This template was hybridized to a non-uracil-containing linear fragment from which the lac z complementing insert had been removed to produce a gapped substrate. DNA synthesis using this substrate with the modified T7 DNA polymerase Sequenase led to an increase in the number and frequency of lac- mutations observed. Escherichia coli DNA polymerase I (Kf) did not yield a comparable increase in mutation frequency or number even though both Sequenase and the E. coli polymerase had similar, low, 3'----5' exonuclease activities as compared to T4 DNA polymerase. We did not observe an increase in mutations when synthesis was attempted on a template reacted with N-acetoxy-2-(acetylamino)fluorene to give N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene adducts. Both E. coli and T7 enzymes terminate synthesis before all (acetylamino)fluorene lesions. Only some of the putative aminofluorene adducts produced strong termination bands, and there was a difference in the pattern generated by Sequenase and E. coli pol I (Kf) using the same substrate. Analysis of the mutations obtained from Sequenase synthesis on the aminofluorene-containing templates indicated a preponderance of -1 deletions at G's and of G----T transversions.
Sahm
J
J
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.
Turkington
E
E
LaPointe
D
D
Strauss
B
B
D001483:Base Sequence; D004256:DNA Polymerase I / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives*; D004926:Escherichia coli / Q000201:enzymology*; D005449:Fluorenes / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009153:Mutagens / Q000494:pharmacology*; D009154:Mutation; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D013698:Templates, Genetic; D014162:Transfection; D014498:Uracil
D004279:DNA, Viral; D005449:Fluorenes; D009153:Mutagens; C031838:N-(deoxyguanosin-8-yl)-2-aminofluorene; D014498:Uracil; C001368:N-hydroxy-2-aminofluorene; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine