Identification of a triplex DNA-binding protein from human cells

Abstract

Intramolecular or intermolecular triple helices could be recognized by specific proteins that stabilize triplex structures and might play a role in gene regulation. In order to identify such proteins, we designed a 55 nucleotide-long DNA oligomer that could fold on itself to form an intramolecular triple helix of the Py·Pu × Py motif. The stability of this triplex under physiological conditions was demonstrated by gel retardation and thermal denaturation experiments. We have identified a protein from HeLa cell nuclear extracts that binds to this synthetic oligonucleotide. The protein has an apparent molecular mass of 55 kDa. Electrophoretic mobility shift assays revealed that the protein did not have any affinity for the single-stranded and double-stranded oligonucleotides corresponding respectively to the third strand and the Watson-Crick duplex of the triple helix. This protein also binds to an intramolecular Py·Pu × Pu triplex but with a lower affinity than to a Py·Pu × Py triple helix.