Kreuzer K N, Cozzarelli N R
J Bacteriol. 1979 Nov;140(2):424-35. doi: 10.1128/jb.140.2.424-435.1979.
Temperature-sensitive nalA mutants of Escherichia coli have been used to investigate the structure and functions of deoxyribonucleic acid (DNA) gyrase. Extracts of one such mutant (nalA43) had thermosensitive DNA gyrase subunit A activity but normal gyrase subunit B activity, proving definitively that nalA is the structural gene for subunit A. Extracts of a second nalA (Ts) mutant (nalA45) had a 50-fold deficiency of gyrase subunit A activity. The residual DNA supertwisting was catalyzed by the mutant DNA gyrase rather than by a novel supertwisting enzyme. The nalA45(Ts) extract was also deficient in the nalidixic acid target, which is defined as the protein necessary to confer drug sensitivity to in vitro DNA replication directed by a nalidixic acid-resistant mutant extract. Thus, gyrase subunit A and the nalidixic acid target are one and the same protein, the nalA gene product. Shift of the nalA43(Ts) mutant to a nonpermissive temperature resulted in a precipitous decline in the rate of [(3)H]thymidine incorporation, demonstrating an obligatory role of the nalA gene product in DNA replication. The rates of incorporation of [(3)H]uridine pulses and continuously administered [(3)H]uracil were quickly reduced approximately twofold upon temperature shift of the nalA43(Ts) mutant, and therefore some but not all transcription requires the nalA gene product. The thermosensitive growth of bacteriophages phiX174 and T4 in the nalA43(Ts) host shows that these phages depend on the host nalA gene product. In contrast, the growth of phage T7 was strongly inhibited by nalidixic acid but essentially unaffected by the nalA43(Ts) mutation. The inhibition of T7 growth by nalidixic acid was, however, eliminated by temperature inactivation of the nal43 gene product. Therefore, nalidixic acid may block T7 growth by a corruption rather than a simple elimination of the nalidixic acid target. Possible mechanisms for such a corruption are considered, and their relevance to the puzzling dominance of drug sensitivity is discussed.
大肠杆菌的温度敏感型nalA突变体已被用于研究脱氧核糖核酸（DNA）促旋酶的结构和功能。一种这样的突变体（nalA43）的提取物具有温度敏感型DNA促旋酶亚基A活性，但促旋酶亚基B活性正常，这明确证明nalA是亚基A的结构基因。第二个nalA（Ts）突变体（nalA45）的提取物促旋酶亚基A活性有50倍的缺陷。残余的DNA超螺旋是由突变的DNA促旋酶催化的，而不是由一种新的超螺旋酶催化。nalA45（Ts）提取物在萘啶酸靶点上也有缺陷，该靶点被定义为赋予对由萘啶酸抗性突变体提取物指导的体外DNA复制药物敏感性所必需的蛋白质。因此，促旋酶亚基A和萘啶酸靶点是同一种蛋白质，即nalA基因产物。nalA43（Ts）突变体转移到非允许温度导致[（3）H]胸苷掺入率急剧下降，表明nalA基因产物在DNA复制中起必不可少的作用。nalA43（Ts）突变体温度转移后，[（3）H]尿苷脉冲掺入率和持续给予[（3）H]尿嘧啶的掺入率迅速降低约两倍，因此部分但不是所有转录需要nalA基因产物。噬菌体phiX174和T4在nalA43（Ts）宿主中的温度敏感生长表明这些噬菌体依赖宿主nalA基因产物。相比之下，噬菌体T7的生长受到萘啶酸的强烈抑制，但基本上不受nalA43（Ts）突变的影响。然而，nal43基因产物的温度失活消除了萘啶酸对T7生长的抑制。因此，萘啶酸可能通过破坏而不是简单消除萘啶酸靶点来阻断T7生长。考虑了这种破坏的可能机制，并讨论了它们与令人困惑的药物敏感性优势的相关性。
