Characterization of purine-motif triplex DNA-binding proteins in HeLa extracts.
One piece of evidence indicating that triple-helical DNAs exist in vivo would be the demonstration of cellular proteins that recognize such structures. Using oligonucleotide probes containing a GT-rich purine-motif triplex, proteins from either HeLa nuclear or cytoplasmic extracts and electrophoretic mobility shift assays, we identified four specific human protein-triplex complexes. Proteins in these complexes did not recognize an analogous homopurine/homopyrimidine duplex DNA or a pyrimidine-motif triplex but did recognize purine-motif triplexes regardless of whether they possessed a phosphodiester or phosphorothioate backbone in the third strand or involved A*AT instead of T*AT base triplets. For each of these proteins, binding affinity increased with increasing triplex length. For some triplex-binding proteins, a weak affinity was noted for individual G-rich oligonucleotides, though this may actually reflect an affinity for quadruplex structures, which these oligonucleotides are prone to adopt. Ion exchange chromatographic fractionation of HeLa nuclear extracts indicated that at least three different proteins were responsible for the observed electrophoretic mobility shifts. Southwestern blotting methods identified three major polypeptides, with apparent molecular masses of 100, 60, and 15 kDa, that preferentially recognized purine-motif triplexes. These data demonstrate the existence of eukaryotic proteins that specifically recognize one triplex motif and support the idea of a biological role for triple helical DNA.
Musso
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Department of Tumor Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Nelson
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LD
Van Dyke
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MW
D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D017433:Protein Structure, Secondary; D011687:Purines / Q000378:metabolism*; D018911:Trinucleotide Repeats
D004268:DNA-Binding Proteins; D011687:Purines; C051802:triplex DNA; D004247:DNA