Yang M, Millar D P
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037, USA.
Biochemistry. 1996 Jun 18;35(24):7959-67. doi: 10.1021/bi952892z.
Time-resolved fluorescence resonance energy transfer has been used to examine the global structure and conformational flexibility of three-way DNA junctions containing unpaired bases at the branch point. Three-way junctions were prepared with donor (fluorescein) and acceptor (tetramethyl-rhodamine) dyes attached to the ends of different helical arms in various pairwise combinations. The time-resolved fluorescence decay of the donor in each labeled junction was measured by time-correlated single photon counting. The distributions of donor-acceptor (D-A) distances present between each pair of labeled helices were recovered from analysis of the donor decay profiles using a Gaussian distribution model. The recovered D-A distance distributions reveal the mean distance between each pair of helices, as well as the range of distances that exists between each pair. For the junction lacking unpaired bases, the three mean interarm distances are similar, indicating an extended structure. In addition, a relatively broad range of distances is present between each pair of helices, showing that the structure is flexible. The addition of unpaired bases causes the junction to fold into a different structure, with one interarm distance being shorter than the other two. The change in overall geometry of the junction appears to be primarily due to the repositioning of one of the helices flanking the bulge. In bulged junctions containing unpaired thymine, cytosine, or adenine bases, the helix containing the 3' portion of the bulged strand appears to undergo the greatest change in its mean position relative to the other helices. In contrast, in the bulged junction containing unpaired guanine bases, the helix containing the 5' portion of the bulged strand is displaced. In all bulged junctions, there is a wide range of distances between the perturbed helix and the other two helices, indicating high mobility for the perturbed arm. These results indicate that the overall structure and conformational flexibility of three-way DNA junctions are sensitive to the presence of unpaired bases at the branch point of the junction and that the precise effect of a bulge depends on the nature of the unpaired bases.
时间分辨荧光共振能量转移已被用于研究在分支点含有未配对碱基的三链DNA连接体的整体结构和构象灵活性。通过将供体（荧光素）和受体（四甲基罗丹明）染料以各种两两组合的方式连接到不同螺旋臂的末端来制备三链连接体。通过时间相关单光子计数测量每个标记连接体中供体的时间分辨荧光衰减。使用高斯分布模型对供体衰减曲线进行分析，从而得到每对标记螺旋之间存在的供体-受体（D-A）距离分布。恢复的D-A距离分布揭示了每对螺旋之间的平均距离以及每对螺旋之间存在的距离范围。对于缺乏未配对碱基的连接体，三个平均臂间距离相似，表明其结构呈伸展状。此外，每对螺旋之间存在相对较宽的距离范围，表明该结构具有灵活性。添加未配对碱基会使连接体折叠成不同的结构，其中一个臂间距离比其他两个短。连接体整体几何形状的变化似乎主要是由于凸起侧翼的一个螺旋重新定位所致。在含有未配对胸腺嘧啶、胞嘧啶或腺嘌呤碱基的凸起连接体中，包含凸起链3'部分的螺旋相对于其他螺旋的平均位置似乎发生了最大变化。相比之下，在含有未配对鸟嘌呤碱基的凸起连接体中，包含凸起链5'部分的螺旋发生了位移。在所有凸起连接体中，受干扰的螺旋与其他两个螺旋之间存在广泛的距离，表明受干扰的臂具有高流动性。这些结果表明，三链DNA连接体的整体结构和构象灵活性对连接体分支点处未配对碱基的存在敏感，并且凸起的精确影响取决于未配对碱基的性质。
