Shlyakhtenko L S, Rekesh D, Lindsay S M, Kutyavin I, Appella E, Harrington R E, Lyubchenko Y L
Department of Microbiology, Arizona State University, Tempe 85287-2701.
J Biomol Struct Dyn. 1994 Jun;11(6):1175-89. doi: 10.1080/07391102.1994.10508062.
Three-way junctions were obtained by annealing two synthetic DNA-oligomers. One of the strands contains a short palindrome sequence, leading to the formation of a hairpin with four base pairs in the stem and four bases in the loop. Another strand is complementary to the linear arms of the first hairpin-containing strand. Both strands were annealed to form a three-way branched structure with sticky ends on the linear arms. The branched molecules were ligated, and the ligation mixture was analysed on a two-dimensional gel in conditions which separated linear and circular molecules. Analysis of 2D-electrophoresis data shows that circular molecules with high mobility are formed. Formation of circular molecules is indicative of bends between linear arms. We estimate the magnitude of the angle between linear arms from the predominant size of the circular molecules formed. When the junction-to-junction distance is 20-21 bp, trimers and tetramers are formed predominately, giving an angle between linear arms as small as 60-90 degrees. Rotation of the hairpin position in the three-way junction allowed us to measure angles between other arms, yielding similar values. These results led us to conclude that the three-way DNA junction possesses a non-planar pyramidal geometry with 60-90 degrees between the arms. Computer modeling of the three-way junction with 60 degrees pyramidal geometry showed a predominantly B-form structure with local distortions at the junction points that diminish towards the ends of the helices. The size distributions of circular molecules are rather broad indicating a dynamic flexibility of three-way DNA junctions.
通过退火两条合成DNA寡聚物获得三向连接体。其中一条链包含一个短回文序列，导致形成一个茎部有四个碱基对且环部有四个碱基的发夹结构。另一条链与第一条含发夹结构链的线性臂互补。两条链退火形成一个三向分支结构，线性臂上带有粘性末端。将分支分子连接起来，并在二维凝胶上分析连接混合物，该条件可分离线性和环状分子。二维电泳数据分析表明形成了高迁移率的环状分子。环状分子的形成表明线性臂之间存在弯曲。我们根据形成的环状分子的主要大小估计线性臂之间的角度大小。当连接体到连接体的距离为20 - 21个碱基对时，主要形成三聚体和四聚体，线性臂之间的角度小至60 - 90度。通过旋转三向连接体中发夹的位置，我们能够测量其他臂之间的角度，得到类似的值。这些结果使我们得出结论，三向DNA连接体具有非平面金字塔几何结构，臂之间的角度为60 - 90度。对具有60度金字塔几何结构的三向连接体进行计算机建模显示，其主要为B型结构，连接点处有局部扭曲，且向螺旋末端逐渐减小。环状分子的大小分布相当宽，表明三向DNA连接体具有动态灵活性。
