Michaels M L, Reid T M, King C M, Romano L J
Department of Chemistry, Wayne State University, Detroit, MI.
Carcinogenesis. 1991 Sep;12(9):1641-6. doi: 10.1093/carcin/12.9.1641.
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.
我们已经测量了DNA聚合酶I（大片段）在跨损伤合成过程中越过氨基芴（AF）加合物时体外合成的准确性。这些研究是使用含有单个AF加合物的位点特异性修饰模板进行的。模板的制备方法是先修饰一个17个碱基长的寡核苷酸中的唯一鸟嘌呤，然后对该产物进行广泛纯化和表征。接着将修饰后的17聚体连接到一个合成双链体上，产生一条31个核苷酸长的模板链，该链含有与一个14聚体退火的AF加合物，使得3'-羟基引物末端在修饰的鸟嘌呤之前四个核苷酸处。DNA聚合酶I在该模板上的合成有效地越过了AF加合物，并产生了全长双链31聚体。另一方面，T7 DNA聚合酶虽然在相同的未修饰模板上有活性，但却无法利用AF修饰的模板。然后将DNA聚合酶I合成的链与修饰链分离，与一个互补寡核苷酸退火，并将得到的异源双链体克隆到M13中。分离得到的49个克隆中的每一个的序列都表明，胞嘧啶已掺入到与AF修饰的鸟嘌呤相对的位置。
