Moore C L, Klevan L, Wang J C, Griffith J D
J Biol Chem. 1983 Apr 10;258(7):4612-7.
Gyrase bound to duplex DNA in the absence of ATP is seen by electron microscopy as a nearly spherical particle frequently located at the intersection of two duplex DNA strands. Such looped structures with gyrase situated at the base of the loops are observed with both linear and circular DNA substrates, and two or three individual DNA molecules bound to the same protein are also seen at high DNA concentrations. Addition of the nonhydrolyzable beta,gamma-imido analog of ATP to the gyrase . DNA reaction mixture prior to sample fixation for microscopy reduces the frequency of gyrase molecules found at DNA intersections. Looped structures similar to those of the gyrase . DNA complex are also seen with the complex of DNA and the A subunit of gyrase. When negatively supercoiled DNA which has been partially relaxed by gyrase in the absence of ATP is fixed for electron microscopic examination, intermediate forms are observed that contain both supercoiled and relaxed loops in a single DNA molecule, with the enzyme located at the common base of the loops. These results suggest that gyrase possesses multiple DNA-binding sites, a feature which allows the enzyme to hold DNA in constrained loops. The relation of these observations to the mechanism of gyrase action is discussed.
在没有ATP的情况下，与双链DNA结合的促旋酶通过电子显微镜观察为一个近乎球形的颗粒，经常位于两条双链DNA链的交叉点处。无论是线性还是环状DNA底物，都能观察到促旋酶位于环底部的这种环状结构，并且在高DNA浓度下也能看到两个或三个与同一蛋白质结合的单个DNA分子。在对样品进行显微镜固定之前，向促旋酶-DNA反应混合物中添加不可水解的ATPβ，γ-亚氨基类似物，会降低在DNA交叉点处发现的促旋酶分子的频率。与促旋酶-DNA复合物类似的环状结构在DNA与促旋酶A亚基的复合物中也能看到。当在没有ATP的情况下被促旋酶部分松弛的负超螺旋DNA被固定用于电子显微镜检查时，会观察到中间形式，即在单个DNA分子中同时包含超螺旋环和松弛环，酶位于环的共同底部。这些结果表明促旋酶具有多个DNA结合位点，这一特征使该酶能够将DNA保持在受限的环中。讨论了这些观察结果与促旋酶作用机制的关系。
