Lovett M A, Helinski D R
J Biol Chem. 1975 Nov 25;250(22):8790-5.
The proteins of the DNA-protein relaxation complex of plasmid ColE1 were labeled with [3H]leucine by growth of ColE1 containing Escherichia coli cells in the presence of this radioactive labeled amino acid. Three major [3H]leucine-labeled proteins are found associated with the supercoiled DNA in the ColE1 relaxation complex. The molecular weights of these proteins, determined by sodium dodecyl sulfate-acrylamide gel electrophoresis, are 60,000, 16,000, and 11,000, respectively. Induction of relaxation of the supercoiled DNA by treatment of the complex with sodium dodecyl sulfate results in a dissociation of the two smaller proteins from the DNA. The 60,000 protein, however, remains associated specifically with the nicked strand of the open circular DNA. The strand-specific association of this protein with the relaxed DNA resists heat denaturation of the DNA, sedimentation through an alkaline (pH 12.5) sucrose gradient, and centrifugation to equilibrium in an alkaline (pH 12.5) CsCl gradient.
通过在含有放射性标记氨基酸的条件下培养携带质粒ColE1的大肠杆菌细胞，用[³H]亮氨酸对质粒ColE1的DNA - 蛋白质松弛复合物中的蛋白质进行标记。在ColE1松弛复合物中，发现三种主要的[³H]亮氨酸标记蛋白与超螺旋DNA相关联。通过十二烷基硫酸钠 - 丙烯酰胺凝胶电泳测定，这些蛋白质的分子量分别为60,000、16,000和11,000。用十二烷基硫酸钠处理该复合物诱导超螺旋DNA松弛，导致两种较小的蛋白质从DNA上解离。然而，60,000的蛋白质仍然特异性地与开环DNA的切口链相关联。这种蛋白质与松弛DNA的链特异性关联能够抵抗DNA的热变性、通过碱性（pH 12.5）蔗糖梯度的沉降以及在碱性（pH 12.5）CsCl梯度中离心至平衡。
