Accurate in vitro translesion synthesis by Escherichia coli DNA polymerase I (large fragment) on a site-specific, aminofluorene-modified oligonucleotide.
We have measured the accuracy of in vitro synthesis by DNA polymerase I (large fragment) during translesion synthesis past an aminofluorene (AF) adduct. These studies were carried out using a site-specifically modified template which contained a single AF adduct. The template was prepared by first modifying the lone guanine in a 17 base long oligonucleotide and extensively purifying and characterizing this product. The modified 17mer was then ligated to a synthetic duplex to produce a 31 nucleotide long template strand containing the AF adduct annealed to a 14mer, such that the 3'-hydroxyl primer terminus was four nucleotides before the modified guanine. Synthesis on this template by DNA polymerase I efficiently bypassed the AF adduct and produced full-length duplex 31mers. T7 DNA polymerase, on the other hand, was unable to utilize the AF-modified template though it was active on an identical unmodified one. The strand synthesized by DNA polymerase I was then separated from the modified strand, annealed to a complementary oligonucleotide, and the resulting heteroduplex cloned into M13. Each of the 49 clones isolated had sequences which indicated that cytidine had been incorporated opposite the AF-modified guanine.
Michaels
M L
ML
Department of Chemistry, Wayne State University, Detroit, MI.
Reid
T M
TM
King
C M
CM
Romano
L J
LJ
D001483:Base Sequence; D002273:Carcinogens; D004247:DNA / Q000737:chemistry; D018736:DNA Adducts; D004256:DNA Polymerase I / Q000378:metabolism*; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005449:Fluorenes / Q000737:chemistry / Q000633:toxicity*; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides / Q000737:chemistry*; D013604:T-Phages / Q000235:genetics; D013698:Templates, Genetic
C052668:2-aminofluorene-DNA complex; D002273:Carcinogens; D018736:DNA Adducts; D005449:Fluorenes; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; C012177:2-aminofluorene; D004247:DNA; D004256:DNA Polymerase I