Parallel and antiparallel holliday junctions differ in structure and stability

Two Holliday junction analogs, JA and JP, containing identical base-paired arms have been constructed from oligonucleotides. The former is constrained to adopt an antiparallel Sigal-Alberts structure, and the latter a parallel structure, by means of single strand d(T)₉ tethers. We evaluate here the free energy difference between JA and JP using two different methods. One is a direct measurement of the ratio of the equilibrium constants for formation of branched structures from intact duplexes using one labeled strand and a competition assay. The second method estimates the difference in stability from the difference in thermal denaturation temperatures of JA and JP, using urea to shift the t_m of the complexes. Both methods reveal a small free energy difference between the two complexes: JA is more stable than JP by -1.1(± 0.4) kcal (mol junction)^-1, at 25 °C, 5 mm-Mg²⁺, from the first method, and by -1.6(± 0.3) kcal (mol junction)^-1, according to the second. DNase I and the resolvase, endonuclease I from phage T7, cleave JA differently from JP in the vicinity of the branch, indicating that the structures of these two models differ at this site. Diethyl pyrocarbonate also reveals a difference in the major grooves. Comparison of the scission patterns of JA and JP by the reactive chemical probes methidium-propyl-EDTA · · Fe(II), [MPE · Fe(II)] and Cu(I)-[o-phenanthroline]₂, [(OP)₂Cu(I)], indicates that in both cases the branch point is a site of enhanced binding for drugs, as it is in the untethered four-arm junction containing the same core sequence at the branch.