Brown P M, Madden C A, Fox K R
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, United Kingdom.
Biochemistry. 1998 Nov 17;37(46):16139-51. doi: 10.1021/bi981768n.
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.
我们基于160个碱基对的tyrT序列制备了一系列七个DNA片段，这些片段在不同位置包含12 - 14个碱基对的寡聚嘌呤序列，并在将它们重组到核小体核心颗粒上后，研究了它们形成三链螺旋的可能性。通过使用DNase I足迹法，我们发现一般来说，三链体只能在核小体DNA片段末端附近的位点形成。对于天然片段，碱基1 - 145与蛋白质表面接触。在这些与核小体结合的片段上，位于位置33之前和94之后的位点可以形成稳定的三链体。这些三链体可以由含CT的寡核苷酸形成，在pH 5.5时产生平行三链体，也可以由含GT的寡核苷酸在pH 7.5时形成反平行三链体。在这些位置之间的位点没有形成反平行三链体。当寡核苷酸浓度高达30 microM时，在位置39 - 50和43 - 54之间的位点也没有形成平行三链体。然而，在位置48和59之间的一个位点明显形成了平行三链体，尽管与游离DNA相比亲和力有所降低，这表明这个寡聚嘌呤序列与核小体表面的结合不太紧密，或者其平移位置发生了改变。在核小体二分体附近引入一个寡聚嘌呤序列会使片段采用不同的核小体位置，该位置可以被平行三链体靶向，但不能被反平行三链体靶向。
