Abbott P J, Mehta J R, Ludlum D B
Biochemistry. 1980 Feb 19;19(4):643-7. doi: 10.1021/bi00545a006.
To study the nature and repair of the promutagenic DNA lesion O6-methylguanine, we have synthesized 8-14C-labeled O6-methyldeoxyguanosine triphosphate and investigated the kinetics of its incorporation into the synthetic copolymers poly(dC'm6dG) and poly(dT,m6dG). Deoxy[8-14C]guanosine was methylated with ethereal diazomethane and the products were separated by high-pressure liquid chromatography. O6-Methyldeoxy[14C]guanosine was converted to the 5'-monophosphate with carrot phosphotransferase and then to the 5'-triphosphate via the phosphorimidazolidate formed by the action of N,N'-carbonyldiimidazole. Although m6dGTP was a poor substrate for Escherichia coli DNA polymerase I, copolymers could be synthesized from dCTP or dTTP and m6dGTP with terminal deoxynucleotidyl transferase. The percent of m6dG in the polymer increased linearly as the percentage of m6dGTP in the polymerization mixture was increased to 20% of the total. The percent incorporation of m6dGTP into poly(dT,m6dG) was, however, higher than into poly(dC,m6dG). Good yields of both polymers were readily obtained. The stability of O6-methyldeoxyguanosine in poly(dT,m6dG) was found to be pH dependent, and the half-life has been measured at four different pH values.
为了研究前诱变DNA损伤O6-甲基鸟嘌呤的性质和修复，我们合成了8-14C标记的O6-甲基脱氧鸟苷三磷酸，并研究了其掺入合成共聚物聚（dC'm6dG）和聚（dT，m6dG）的动力学。脱氧[8-14C]鸟苷用乙醚重氮甲烷甲基化，产物通过高压液相色谱分离。O6-甲基脱氧[14C]鸟苷用胡萝卜磷酸转移酶转化为5'-单磷酸，然后通过N，N'-羰基二咪唑作用形成的磷酰咪唑啉酸盐转化为5'-三磷酸。尽管m6dGTP是大肠杆菌DNA聚合酶I的不良底物，但可以用末端脱氧核苷酸转移酶从dCTP或dTTP和m6dGTP合成共聚物。随着聚合混合物中m6dGTP的百分比增加到总量的20%，聚合物中m6dG的百分比呈线性增加。然而，m6dGTP掺入聚（dT，m6dG）的百分比高于掺入聚（dC，m6dG）的百分比。两种聚合物都很容易获得高产率。发现O6-甲基脱氧鸟苷在聚（dT，m6dG）中的稳定性取决于pH值，并在四个不同的pH值下测量了半衰期。
