Katiliene Zivile, Katilius Evaldas, Woodbury Neal W
Department of Chemistry and Biochemistry and the Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287-1604, USA.
Biophys J. 2003 Jun;84(6):4053-61. doi: 10.1016/S0006-3495(03)75131-5.
Single molecule fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy were used to investigate DNA looping by NgoMIV restriction endonuclease. Using a linear double-stranded DNA (dsDNA) molecule labeled with a fluorescence donor molecule, Cy3, and fluorescence acceptor molecule, Cy5, and by varying the concentration of NgoMIV endonuclease from 0 to 3 x 10(-6) M, it was possible to detect and determine diffusion properties of looped DNA/protein complexes. FRET efficiency distributions revealed a subpopulation of complexes with an energy transfer efficiency of 30%, which appeared upon addition of enzyme in the picomolar to nanomolar concentration range (using 10(-11) M dsDNA). The concentration dependence, fluorescence burst size analysis, and fluorescence correlation analysis were all consistent with this subpopulation arising from a sequence specific interaction between an individual enzyme and a DNA molecule. A 30% FRET efficiency corresponds to a distance of approximately 65 A, which correlates well with the distance between the ends of the dsDNA molecule when bound to NgoMIV according to the crystal structure of this complex. Formation of the looped complexes was also evident in measurements of the diffusion times of freely diffusing DNA molecules with and without NgoMIV. At very high protein concentrations compared to the DNA concentration, FRET and fluorescence correlation spectroscopy results revealed the formation of larger DNA/protein complexes.
单分子荧光共振能量转移（FRET）和荧光相关光谱法被用于研究NgoMIV限制性内切酶介导的DNA环化。使用一个标记有荧光供体分子Cy3和荧光受体分子Cy5的线性双链DNA（dsDNA）分子，通过将NgoMIV内切酶的浓度从0变化到3×10⁻⁶ M，得以检测并确定环状DNA/蛋白质复合物的扩散特性。FRET效率分布显示存在一个能量转移效率为30%的复合物亚群，当加入皮摩尔到纳摩尔浓度范围的酶时（使用10⁻¹¹ M的dsDNA）该亚群出现。浓度依赖性、荧光猝发大小分析和荧光相关分析均与该亚群由单个酶与DNA分子之间的序列特异性相互作用产生这一情况一致。30%的FRET效率对应于大约65埃的距离，根据该复合物的晶体结构，这与结合NgoMIV时dsDNA分子两端之间的距离高度相关。在有和没有NgoMIV的情况下对自由扩散的DNA分子的扩散时间进行测量时，环状复合物的形成也很明显。在蛋白质浓度相对于DNA浓度非常高时，FRET和荧光相关光谱法的结果显示形成了更大的DNA/蛋白质复合物。
