CHARACTERIZATION OF PURINE-MOTIF TRIPLEX DNA-BINDING PROTEINS IN HELAEXTRACTS

One piece of evidence indicating that triple-helical DNAs exist in viv o would be the demonstration of cellular proteins that recognize such structures. Using oligonucleotide probes containing a GT-rich purine-m otif tripler, proteins from either HeLa nuclear or cytoplasmic extract s and electrophoretic mobility shift assays, we identified four specif ic human protein-tripler complexes. Proteins in these complexes did no t recognize an analogous homopurine/homopyrimidine duplex DNA or a pyr imidine-motif tripler but did recognize purine-motif triplexes regardl ess of whether they possessed a phosphodiester or phosphorothioate bac kbone in the third strand or involved AAT instead of T*AT base triple ts. For each of these proteins, binding affinity increased with increa sing tripler length. For some tripler-binding proteins, a weak affinit y was noted for individual G-rich oligonucleotides, though this may ac tually reflect an affinity for quadruplex structures, which these olig onucleotides are prone to adopt. Ion exchange chromatographic fraction ation of HeLa nuclear extracts indicated that at least three different proteins were responsible for the observed electrophoretic mobility s hifts. Southwestern blotting methods identified three major polypeptid es, with apparent molecular masses of 100, 60, and 15 kDa, that prefer entially recognized purine-motif triplexes. These data demonstrate the existence of eukaryotic proteins that specifically recognize one trip ler motif and support the idea of a biological role for triple helical DNA.