Nose K, Okamoto H
Biochem Biophys Res Commun. 1983 Mar 16;111(2):383-9. doi: 10.1016/0006-291x(83)90317-0.
A nick-translation reaction with E. coli DNA polymerase I (pol. I) was used to detect in situ DNA breaks produced by chemical carcinogens. Normal human fibroblasts treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in various doses were permeabilized with lysolecithin, and were nick translated in the presence of [3H]dCTP and pol. I. The radioactivity incorporated increased with MNNG concentration, and was directly proportional to the poly(ADP-ribose) synthetase activity. Other DNA-damaging agents such as bleomycin or 4-nitroquinoline 1-oxide also caused the nick translation rate to increase. When MNNG-treated cells were cultured in fresh medium containing no MNNG, the increase in the rate of nick translation in permeable cells became less and this decrease was abolished by addition of aphidicolin or cytosine arabinoside. The nick translation method described here may be a useful means for estimating intrinsic DNA breaks in cells treated with carcinogens.
利用大肠杆菌DNA聚合酶I（pol. I）进行缺口平移反应，以检测化学致癌物产生的原位DNA断裂。用不同剂量的N-甲基-N'-硝基-N-亚硝基胍（MNNG）处理正常人类成纤维细胞，并用溶血卵磷脂使其通透化，然后在[3H]dCTP和pol. I存在的情况下进行缺口平移。掺入的放射性随MNNG浓度增加而增加，且与聚（ADP-核糖）合成酶活性成正比。其他DNA损伤剂，如博来霉素或4-硝基喹啉-1-氧化物，也会导致缺口平移率增加。当用MNNG处理的细胞在不含MNNG的新鲜培养基中培养时，可通透细胞中缺口平移率的增加变小，并且通过添加阿非科林或阿糖胞苷可消除这种下降。这里描述的缺口平移方法可能是估计用致癌物处理的细胞中固有DNA断裂的有用手段。
