Kohda K, Tsunomoto H, Minoura Y, Tanabe K, Shibutani S
Faculty of Pharmaceutical Sciences, Nagoya City University, Japan.
Chem Res Toxicol. 1996 Dec;9(8):1278-84. doi: 10.1021/tx9601059.
8-Methyl-2'-deoxyguanosine (8-MedG) was synthesized by reacting dG under the methyl radical generating system and incorporated into oligodeoxynucleotides using phosphoramidite techniques. The site-specifically modified oligodeoxynucleotide containing a single 8-MedG was then used as a template for primer extension reactions catalyzed by the 3' --> 5' exonuclease-free (exo-) Klenow fragment of Escherichia Coli DNA polymerase I and mammalian DNA polymerase alpha. Primer extension catalyzed by the exo- Klenow fragment readily passed the 8-MedG lesion in the template while that catalyzed by pol alpha was retarded opposite the lesion. The fully extended products formed during DNA synthesis were analyzed to quantify the miscoding specificities of 8-MedG. Both DNA polymerases incorporated primarily dCMP, the correct base opposite the lesion, along with small amounts of incorporation of dGMP and dAMP. In addition, two-base deletion was observed only when the exo- Klenow fragment was used. The thermodynamic stability of 8-MedG in the duplex was also studied. The duplex containing 8-MedG:dG was more thermally and thermodynamically stable than that of dG:dG. The duplex containing 8-MedG:dA was more thermodynamically stable than that of dG:dA. We conclude that 8-MedG is a miscoding lesion and capable of generating G --> C and G --> T transversions and deletion in cells.
8-甲基-2'-脱氧鸟苷（8-MedG）是通过在甲基自由基生成系统中使dG反应合成的，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-MedG的位点特异性修饰寡脱氧核苷酸用作由大肠杆菌DNA聚合酶I和哺乳动物DNA聚合酶α的3'→5'无外切核酸酶（exo-）Klenow片段催化的引物延伸反应的模板。由exo-Klenow片段催化的引物延伸很容易通过模板中的8-MedG损伤，而由polα催化的引物延伸在损伤对面则受到阻碍。分析DNA合成过程中形成的完全延伸产物，以量化8-MedG的错配特异性。两种DNA聚合酶主要掺入dCMP，即损伤对面的正确碱基，同时还少量掺入dGMP和dAMP。此外，仅当使用exo-Klenow片段时才观察到两碱基缺失。还研究了8-MedG在双链体中的热力学稳定性。含有8-MedG:dG的双链体比dG:dG的双链体在热稳定性和热力学稳定性上更高。含有8-MedG:dA的双链体比dG:dA的双链体在热力学上更稳定。我们得出结论，8-MedG是一种错配损伤，能够在细胞中产生G→C和G→T颠换以及缺失。
