Sugino A, Peebles C L, Kreuzer K N, Cozzarelli N R
Proc Natl Acad Sci U S A. 1977 Nov;74(11):4767-71. doi: 10.1073/pnas.74.11.4767.
A target protein for nalidixic and oxolinic acids in Escherichia coli, the nalA gene product (Pnal), was purified to homogeneity as judged by gel electrophoresis, using an in vitro complementation assay. It is a dimer of identical 110,000-dalton subunits. A polypeptide of this molecular weight is uniquely induced by a lambda nalA transducing phage, thereby showing that the purified Pnal is a product of the nalA gene. Nalidixic and oxolinic acids inhibit DNA gyrase activity and induce formation of a relaxation complex analogue. Treatment of the complex with sodium dodecyl sulfate causes a doublestrand break in the DNA substrate and the resulting linear molecule seems covalently bound to protein. Complex formation, unlike the introduction of supertwists, does not require ATP or relaxed circular DNA and is insensitive to novobiocin. DNA gyrase from a strain with a nalA mutation conferring drug resistance (nalA(r)) is 1/100 as sensitive to oxolinic and nalidixic acids with respect to inhibition of supertwisting and induction of the pre-linearization complex. Addition of Pnal restores drug sensitivity and stimulates DNA gyrase activity. DNA gyrase preparations and Pnal catalyze a third reaction sensitive to nalidixic and oxolinic acids, the ATP-independent relaxation of supertwister DNA. Relaxation by gyrase from nalA(r) cells is drug resistant. The nicking-closing activity is distinct from E. coli omega protein in several properties, including the ability to relax positively supertwisted DNA. We postulate that the nalA gene product occurs in two molecular forms, as Pnal and as a gyrase component. Both forms catalyze nicking-closing, and inhibition of this activity by nalidixic and oxolinic acids may account for the inhibition of DNA synthesis by these drugs.
利用体外互补分析，将大肠杆菌中萘啶酸和恶喹酸的靶蛋白——nalA基因产物（Pnal）纯化至凝胶电泳判断的均一状态。它是由相同的110,000道尔顿亚基组成的二聚体。这种分子量的多肽由λ nalA转导噬菌体特异性诱导产生，从而表明纯化的Pnal是nalA基因的产物。萘啶酸和恶喹酸抑制DNA回旋酶活性并诱导形成松弛复合物类似物。用十二烷基硫酸钠处理该复合物会导致DNA底物出现双链断裂，产生的线性分子似乎与蛋白质共价结合。与引入超螺旋不同，复合物的形成不需要ATP或松弛的环状DNA，并且对新生霉素不敏感。来自具有赋予耐药性的nalA突变（nalA(r)）菌株的DNA回旋酶，在抑制超螺旋形成和诱导线性化前复合物方面，对恶喹酸和萘啶酸的敏感性仅为野生型的1/100。添加Pnal可恢复药物敏感性并刺激DNA回旋酶活性。DNA回旋酶制剂和Pnal催化第三种对萘啶酸和恶喹酸敏感的反应，即超螺旋DNA的不依赖ATP的松弛。来自nalA(r)细胞的回旋酶介导的松弛具有耐药性。切口封闭活性在几个特性上与大肠杆菌ω蛋白不同，包括松弛正超螺旋DNA的能力。我们推测nalA基因产物以两种分子形式存在，即Pnal和作为回旋酶的一个组分。两种形式都催化切口封闭，萘啶酸和恶喹酸对这种活性的抑制可能解释了这些药物对DNA合成的抑制作用。
