Kohda K, Tsunomoto H, Kasamatsu T, Sawamura F, Terashima I, Shibutani S
Faculty of Pharmaceutical Sciences, Nagoya City University, Japan.
Chem Res Toxicol. 1997 Dec;10(12):1351-8. doi: 10.1021/tx970111k.
Aryl radicals and arenediazonium ions are suspected to react with cellular DNA, resulting in C8-arylguanine adducts. 8-Phenyl-2'-deoxyguanosine (8-PhdG) was synthesized as a model adduct by reacting dG with benzenediazonium chloride and incorporated into oligodeoxynucleotides using phosphoramidite techniques. A site-specifically modified oligodeoxynucleotide containing a single 8-PhdG was then used as a template for primer extension reactions catalyzed by the intact (exo+) or 3'-->5' exonuclease-free (exo-) Klenow fragment of Escherichia coli DNA polymerase I and mammalian DNA polymerase alpha (pol alpha). Although primer extensions catalyzed by the Klenow fragments were retarded at the position of 8-PhdG, most of the primer extension passed the lesion to form the fully extended products. In contrast, primer extensions catalyzed by pol alpha were strongly blocked opposite the lesion. The fully extended products formed during DNA synthesis were analyzed to quantify the miscoding specificities of 8-PhdG. The exo- Klenow fragment incorporated primarily dCMP, the correct base, opposite 8-PhdG, along with small amounts of incorporation of dAMP. Two-base deletions were also observed. In contrast, the exo+ Klenow fragment incorporated dCMP opposite the lesion. When pol alpha was used, 8-PhdG promoted small amounts of misincorporation of dAMP and dGMP as well as one- and two-base deletions. The duplex containing 8-PhdG.dG was thermally and thermodynamically more stable than dG.dG. The duplex containing 8-PhdG.dA was thermodynamically more stable than dG.dA. We conclude that 8-PhdG is a weak miscoding lesion, capable of generating G-->T and G-->C transversions and deletions in cells.
芳基自由基和芳基重氮离子被怀疑会与细胞DNA发生反应，从而产生C8-芳基鸟嘌呤加合物。通过使dG与苯重氮氯化物反应，合成了8-苯基-2'-脱氧鸟苷（8-PhdG）作为模型加合物，并使用亚磷酰胺技术将其掺入寡脱氧核苷酸中。然后，将含有单个8-PhdG的位点特异性修饰寡脱氧核苷酸用作模板，用于由大肠杆菌DNA聚合酶I和哺乳动物DNA聚合酶α（polα）的完整（exo +）或无3'→5'核酸外切酶（exo-）的Klenow片段催化的引物延伸反应。尽管由Klenow片段催化的引物延伸在8-PhdG的位置处受到阻碍，但大多数引物延伸都越过损伤形成了完全延伸的产物。相比之下，由polα催化的引物延伸在损伤对面被强烈阻断。对DNA合成过程中形成的完全延伸产物进行分析，以量化8-PhdG的错配特异性。exo-Klenow片段在8-PhdG对面主要掺入正确的碱基dCMP，同时也有少量dAMP的掺入。还观察到了两个碱基的缺失。相比之下，exo + Klenow片段在损伤对面掺入dCMP。当使用polα时，8-PhdG会促进少量dAMP和dGMP的错掺入以及一个和两个碱基的缺失。含有8-PhdG.dG的双链体在热稳定性和热力学稳定性上比dG.dG更高。含有8-PhdG.dA的双链体在热力学上比dG.dA更稳定。我们得出结论，8-PhdG是一种弱错配损伤，能够在细胞中产生G→T和G→C的颠换以及缺失。
