CONFORMATIONAL DIFFERENCES BETWEEN BULGED PYRIMIDINES (C-C) AND PURINES (A-A, I-I) AT THE BRANCH POINT OF 3-STRANDED DNA JUNCTIONS

We have synthesized DNA oligomers that can combine to form three-way j unctions containing six base pairs in each stem and two unpaired bases at the branch point. Gel electrophoresis experiments indicate that th e oligomers form stable complexes with equimolar stoichiometry. Using two- and three-dimensional proton nuclear magnetic resonance spectrosc opy, we have completed nonexchangeable proton chemical shift assignmen ts for three junctions which differ only in the identity of the unpair ed bases (C-C, A-A, or I-I) at the branch point. Our results indicate that unpaired pyrimidines at the branch point of junctions behave diff erently than do unpaired purines. In a junction with two unpaired cyti dines, the 5' base loops out from the molecule to lie along the minor groove of the preceding duplex stem of the junction. The 3. unpaired c ytidine also demonstrates an unusual pattern of NOE connectivities wit h detected cross peaks to the subsequent base in the 3' direction. Jun ctions with unpaired purines at the branch point exhibit different beh avior. Our data suggests that in these molecules the unpaired bases pa rticipate in stacking interactions among themselves and with the neigh boring bases in the molecule. Despite these differences, the NOE patte rns from each junction suggest the presence of a preferred, pair-wise stacking between two of the helices within the molecule. The structura l differences between bulge-pyrimidine and bulge-purine junctions are discussed in light of the functional significance unpaired bases might have in the structure and dynamics of multistranded DNA junctions and , by extension, to junctions within cellular RNAs.