In contrast to four-arm immobile DNA junctions, three-arm DNA junctions have unique structural and dynamic properties consistent with lack of a single dominant conformation. The effect of T-T base mismatches at the branch in a three-arm model junction has been investigated using a combination of electrophoretic mobility measurements, chemical footprinting experiments, and thermodynamic studies. The results indicate that three-arm junctions are only slightly destabilized by a mismatch flanking the branch, relative to four-arm junctions. The effect of a mismatch varies with the sequence and position of the site of the mismatch. Since a three-arm junction with two mismatches flanking the branch is as stable as two junctions each with a single mismatch, the stability of three-arm junctions is not determined by stacking interactions at the branch in a simple way. The properties of three-arm junctions with one or two mismatches are consistent with a picture in which the conformation is the result of several substrates in which base pairs flanking the branch are transiently open, allowing bases to interact with the remaining duplexes.
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