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© 1991 Oxford University Press

research-article

Accurate in vitro, translesion synthesis by Escherichia coli DNA polymerase I (large fragment) on a site-specific, aminofluorenemodified oligonucleotide

Mark Leo Michales, Thomas M. Reid 1, Charles M. King 1 and Louis J. Romano 2

Department of Chemistry, Wayne State University , Michigan Cancer Foundation Detroit, MI 48202, USA
1Departmnent of Chemical Carcinogenesis, Michigan Caner Foundation Detroit, MI 48202, USA

2To whom correspondence should be addressed

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 3lmers. T7 DNA plymerase, 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-modlfled guanine.


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