Structure of three-way DNA junctions. 1. Non-planar DNA geometry

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.