DESIGN AND NMR-STUDY OF AN IMMOBILE DNA 4-WAY JUNCTION CONTAINING 38 NUCLEOTIDES

The DNA Holliday junction is a central intermediate in genetic recombi nation, We have designed and synthesized a DNA oligomer, J1a. as a mod el compound for the Holliday junction suitable to be studied by NMR sp ectroscopy and future molecular modelling. The design was based on a 4 6-base oligomer, J4, previously studied by Pikkemaat, J. A., van den F ist, H., van Boom, J. H. & Altona, C. [Biochemistry 33, 14896-14907 (1 994)], including the propensity to undergo a self-folding process to g ive a four-way junction in which three of the four arms are capped wit h a hairpin loop. J1a, however, is considerably shortened by eight bas es and thus contains only 35 residues which significantly facilitates the proton resonance assignments. The base sequence at the branch poin t is identical to that, in J4. H-1-NMR data clearly point to the prese nce of three hairpin loops in J1a and show that the double-helical arm s adopt the B-DNA form. Quasicontinuous pairwise stacking between heli cal arms to give a single preferred stacked X-conformation is evident. The extent of folding into this slacked conformation is strongly depe ndent upon the magnesium concentration. Full Watson-Crick base pairing at the branch point iii completely preserved. The A/D-stacking prefer ence of the small junction is the same as that exhibited by J4.