Triple-helix formation at different positions on nucleosomal DNA.

Abstract

We have prepared a series of seven DNA fragments, based on the 160 base-pair tyrT sequence, which contain 12-14 base-pair oligopurine tracts at different positions, and have examined their availability for triple-helix formation after reconstituting onto nucleosome core particles. By using DNase I footprinting we find that in general, triplexes can only be formed at sites located toward the ends of nucleosomal DNA fragments. For the native fragment, bases 1-145 are in contact with the protein surface. Stable triplexes can be formed on these nucleosome-bound fragments for sites located before position 33 and beyond position 94. These are formed with both CT-containing oligonucleotides, generating parallel triplexes at pH 5.5, and GT-containing oligonucleotides forming antiparallel triplexes at pH 7.5. No antiparallel triplexes were formed at sites located between these positions. Parallel triplexes were also not formed at sites between positions 39-50 and 43-54 with oligonucleotide concentrations as high as 30 microM. However parallel triplex formation was evident at a site between positions 48 and 59, albeit with a reduced affinity compared to free DNA, suggesting that this oligopurine tract is less tightly associated with the nucleosome surface or that it has an altered translational position. The introduction of an oligopurine tract in the vicinity of the nucleosome dyad caused the fragment to adopt a different nucleosomal position, which could be targeted with parallel, but not antiparallel triplexes.