Christner D F, Lakshman M K, Sayer J M, Jerina D M, Dipple A
Chemistry of Carcinogenesis Laboratory, NCI-Frederick Cancer Research and Development Center, Maryland 21702.
Biochemistry. 1994 Nov 29;33(47):14297-305. doi: 10.1021/bi00251a043.
Four isomeric benzo[a]pyrene-deoxyadenosine adducts, corresponding to the products of trans opening of the epoxide ring in the four configurationally isomeric benzo[a]pyrene dihydrodiol epoxides by the amino group of deoxyadenosine, were separately introduced into each of two 16-mer sequence contexts. The sequences were from the supF gene, and the site of the adducted adenine was known, for some hydrocarbon dihydrodiol epoxides, to be a hotspot for mutation in Context I and a coldspot for mutation in Context II. Using primers complementary to the 3' ends of these oligonucleotides, the abilities of several polymerases to replicate these templates in vitro were investigated. Each adduct proved to be an effective block to primer extension such that only with high concentrations of exo- Klenow fragment was any bypass of adducts seen. DNA polymerase alpha and HIV-1 reverse transcriptase were blocked 3' to the adduct when the configuration at C10 of the hdyrocarbon was S, and some introduction of thymine opposite the adenine adduct was seen with the R configuration. Incorporation of a nucleotide opposite the adduct occurred more readily with Sequenase and the Klenow fragment, and the mutagenic introduction of adenine was apparent in most cases. This corresponded to the A-->T transversions frequently seen in mutation studies with hydrocarbon dihydrodiol epoxides that react extensively with adenine in DNA. Overall, it was clear that sequence context, adduct stereochemistry, and the choice of polymerase all influenced the polymerization reaction. With these in vitro systems, no major differences correlating with the differing tumorigenicities of the isomeric dihydrodiol epoxides or with the hotspot or coldspot nature of the sequences were detected.
四种异构体苯并[a]芘 - 脱氧腺苷加合物，对应于脱氧腺苷的氨基对四种构型异构体苯并[a]芘二氢二醇环氧化物的环氧化物环进行反式开环的产物，被分别引入到两个16聚体序列环境中。这些序列来自supF基因，并且已知对于某些烃二氢二醇环氧化物，加合腺嘌呤的位点在环境I中是突变热点，而在环境II中是突变冷点。使用与这些寡核苷酸3'末端互补的引物，研究了几种聚合酶在体外复制这些模板的能力。每种加合物都被证明是引物延伸的有效阻碍，以至于只有在高浓度的外切酶Klenow片段存在时才能观察到加合物的任何跨越。当烃类化合物C10位的构型为S时，DNA聚合酶α和HIV - 1逆转录酶在加合物的3'端被阻断，并且在R构型时可以看到一些与腺嘌呤加合物相对的胸腺嘧啶的掺入。与加合物相对的核苷酸掺入在Sequenase和Klenow片段的作用下更容易发生，并且在大多数情况下腺嘌呤的诱变掺入是明显的。这与在与DNA中的腺嘌呤广泛反应的烃二氢二醇环氧化物的突变研究中经常看到的A→T颠换相对应。总体而言，很明显序列环境、加合物立体化学和聚合酶的选择都影响聚合反应。在这些体外系统中，未检测到与异构体二氢二醇环氧化物的不同致瘤性或与序列的热点或冷点性质相关的主要差异。
