STRUCTURAL FEATURES OF A 3-STRANDED DNA JUNCTION CONTAINING A C-C JUNCTIONAL BULGE

We have examined the stability of junctional base pairs in a three-way DNA junction with two unpaired cytidine residues at the branch point using two-dimensional nuclear Overhauser effect spectroscopy in H2O so lution. Our data directly support the presence of two of the three jun ctional Watson-Crick base pairs, with indirect support for the third a s well. These results complement the data presented in the preceding p aper, where we examined the nonexchangeable proton resonance assignmen ts of three-way DNA junctions from NOESY data in D2O solution. We have incorporated the NOE data from both sets of experiments, using this i nformation as input for a combined distance geometry (DG) and simulate d annealing (SA) protocol designed to derive three-dimensional structu res of the junction molecule consistent with the NMR data. Although th e data does not allow us to derive a unique solution for the structure of the molecule, certain conformational features are invariably prese nt in our models. We demonstrate the existence of a preferred, pair-wi se stacking arrangement between two of the three helices in the juncti on. Furthermore, the remaining duplex stem is situated so that it alwa ys forms an acute angle with just one of the arms from the quasi-conti nuous helix. The unpaired residues provide an extended backbone segmen t linking two of the helices together. The first unpaired base on the 5' end loops out from the interior of the molecule to reside along the minor groove of one helix. The second is located within the interior of the molecule, stacking below one of the junctional base pairs. Our findings suggest that junctional base pair stacking is an important de terminant in the conformation of multistranded nucleic acid junctions. In three-way junctions, the presence of unpaired bases at the branch point provides a relief from covalent constraints that would otherwise prevent the simultaneous realization of both base pairing and base pa ir stacking within the branch point of the molecule.