Maki S, Takiguchi S, Horiuchi T, Sekimizu K, Miki T
Faculty of Pharmaceutical Sciences, Kyushu Univeristy, Fukuoka, Japan.
J Mol Biol. 1996 Mar 1;256(3):473-82. doi: 10.1006/jmbi.1996.0102.
Escherichia coli DNA gyrase, as well as a free form of its A subunit (GyrA), exists in an inactivated form in cells that overproduce the F plasmid protein LetD (CcdB). We found that the inactivated DNA gyrase and GyrA protein can be rejuvenated in vitro by another F plasmid protein, LetA (CcdA). Using this rejuvenation as an assay, we purified the inactivated GyrA protein to near homogeneity and found it to be complexed with the LetD protein. The complex has a molecular mass of 230 kDa and was suggested to be a complex of two molecules each of GyrA and LetD proteins. The GyrA-LetD complex, in the presence of purified GyrB protein, does not cause DNA cleavage. Therefore, the LetD protein in the GyrA-LetD complex inhibits the gyrase action by a mechanism different from one that involves trapping a covalently linked gyrase-DNA complex. In as much as a free form of the LetD protein has been shown to induce DNA cleavage by gyrase, the LetD protein seems to have two distinct modes of action on DNA gyrase. Rejuvenation of the inactivated GyrA protein by the LetA protein was achieved in vitro, and mechanisms governing this process were examined using the purified proteins. The rejuvenated GyrA protein sediments through sucrose gradients as a single protein species of 190 kDa and is indistinguishable from a free form of GyrA protein. In the same sedimentation experiment, the LetD protein was seen to be complexed with the added LetA protein. Thus, the LetA protein apparently rejuvenates the GyrA protein by removing the bound LetD protein from the inactivated form, followed by formation of a LetA-LetD complex.
大肠杆菌DNA促旋酶及其A亚基（GyrA）的游离形式，在过量产生F质粒蛋白LetD（CcdB）的细胞中以失活形式存在。我们发现，失活的DNA促旋酶和GyrA蛋白可在体外被另一种F质粒蛋白LetA（CcdA）恢复活性。利用这种恢复活性作为检测方法，我们将失活的GyrA蛋白纯化至接近均一状态，发现它与LetD蛋白形成了复合物。该复合物的分子量为230 kDa，推测是由两个GyrA蛋白分子和两个LetD蛋白分子组成的复合物。在纯化的GyrB蛋白存在的情况下，GyrA-LetD复合物不会导致DNA切割。因此，GyrA-LetD复合物中的LetD蛋白通过一种不同于涉及捕获共价连接的促旋酶-DNA复合物的机制来抑制促旋酶的作用。由于已证明LetD蛋白的游离形式可诱导促旋酶切割DNA，所以LetD蛋白似乎对DNA促旋酶有两种不同的作用模式。在体外实现了LetA蛋白对失活GyrA蛋白的恢复活性，并使用纯化的蛋白研究了控制这一过程的机制。恢复活性的GyrA蛋白在蔗糖梯度中沉降为单一的190 kDa蛋白种类，与游离形式的GyrA蛋白无法区分。在相同的沉降实验中，发现LetD蛋白与添加的LetA蛋白形成了复合物。因此，LetA蛋白显然是通过从失活形式中去除结合的LetD蛋白，随后形成LetA-LetD复合物，从而使GyrA蛋白恢复活性。
