CHARACTERIZATION AND COMPARISON OF SYNTHETIC IMMOBILE AND MOBILE HOLLIDAY JUNCTIONS

Eight synthetic Holliday junction (HJ) oligonucleotides containing an immobile or a mobile junction were characterized by gel electrophoresi s, ultraviolet absorption and circular dichroism (CD) spectroscopy. Fo ur 24-mer deoxyribonucleotides formed stable immobile and mobile HJs i n 0.1 M NaCl at 5 mu M strand concentration at room temperature. Howev er, the immobile HJ constructed from four 18-mers was less stable, and four 12-mers did not form the HJ structure under the conditions used. A comparison of the melting profiles of the HJs with those of the dup lexes corresponding to the arms of four-way junctions indicated that t he thermal stability of the HJ was similar to that of the individual a rm and the cooperativity of the melting behavior of the HJ was relativ ely higher than that of the individual arm duplex. The T(m)s of the mo bile HJs containing 4, 6, 8, and 10 base-pair homologous cores at junc tions were essentially identical with that of the immobile HJ of the s ame size. There is a tendency that the HJ containing a larger homologo us core region becomes more resistant to thermal denaturation. The add ition of divalent metal cations, Mg2+ and Ca2+, to the solutions of th e HJs raised their melting temperatures. The difference found for the CD spectra of the HJs which differ only in the arrangement of the HJ d epended primarily upon the DNA sequence flanking the junction. The Ruv C protein binds to the immobile and mobile HJs, regardless of the pres ence and the size of the homologous core at the junction.