Chao L, Tillman D M
J Bacteriol. 1982 Aug;151(2):764-70. doi: 10.1128/jb.151.2.764-770.1982.
The role of DNA gyrase in handling DNA damages induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined with two Escherichia coli strains, KL161 and KL166. The two strains are isogenic except that KL166 harbors a mutation at the nalA (gyrA) locus which specifies one of the two subunits of DNA gyrase. We treated the two strains with several different types of mutagenic agents and found the nalA strain to be highly resistant to MNNG-induced killing and mutagenic effects as compared with the parental strain. The MNNG resistance was specific, since the two strains were about equally sensitive to methyl methane sulfonate, ethyl methane sulfonate, and UV and gamma radiations. We pulse-labeled the two strains with [(3)H]uridine and (14)C-amino acids after MNNG treatment to analyze RNA and protein synthetic rates. The pulse-labeled proteins were also separated on polyacrylamide gels. The results show that pulse-labeled RNA and proteins persisted in the nalA strain but declined rapidly in the parental strain after MNNG treatment. We compared membrane-free nucleoid preparations from the two strains by sucrose density gradient centrifugation and found a difference in nucleoid organization between the two strains. The nucleoid of the nalA strain, unlike that of the parental strain, may have a highly ordered structure, as indicated by its resistance to ethidium bromide-induced relaxation. The ability of the two strains to express an adaptive response to MNNG was determined. We found that the resistance to MNNG killing and mutagenesis by the nalA strain cannot be further increased by adaptive treatment. These results suggest that an alteration in DNA gyrase may have profound effects on E. coli chromosome organization and base methylation by MNNG.
利用两株大肠杆菌KL161和KL166研究了DNA促旋酶在处理由N-甲基-N'-硝基-N-亚硝基胍（MNNG）诱导的DNA损伤中的作用。这两株菌是同基因的，只是KL166在nalA（gyrA）位点存在一个突变，该位点决定DNA促旋酶两个亚基之一。我们用几种不同类型的诱变剂处理这两株菌，发现与亲本菌株相比，nalA菌株对MNNG诱导的杀伤和诱变作用具有高度抗性。MNNG抗性是特异性的，因为这两株菌对甲磺酸甲酯、乙磺酸甲酯以及紫外线和γ射线的敏感性大致相同。在MNNG处理后，我们用[³H]尿苷和¹⁴C氨基酸对这两株菌进行脉冲标记，以分析RNA和蛋白质的合成速率。脉冲标记的蛋白质也在聚丙烯酰胺凝胶上进行分离。结果表明，MNNG处理后，脉冲标记的RNA和蛋白质在nalA菌株中持续存在，但在亲本菌株中迅速下降。我们通过蔗糖密度梯度离心比较了这两株菌的无膜类核制剂，发现两株菌之间类核组织存在差异。nalA菌株的类核与亲本菌株不同，可能具有高度有序的结构，这可由其对溴化乙锭诱导的松弛的抗性表明。测定了这两株菌对MNNG表达适应性反应的能力。我们发现，nalA菌株对MNNG杀伤和诱变的抗性不能通过适应性处理进一步提高。这些结果表明，DNA促旋酶的改变可能对大肠杆菌染色体组织和MNNG引起的碱基甲基化有深远影响。
