Characterization of a triplex DNA-binding protein encoded by an alternative reading frame of loricrin.
In an attempt to identify genes encoding triple-helical DNA-binding proteins, we performed South-Western screening of a human keratinocyte cDNA expression library using a purine (Pu)-rich triplex DNA probe. We isolated two independent clones containing part of the loricrin gene. Both were translated with a different reading frame than that of the loricrin protein, the major component of the cell envelope during normal keratinocyte cornification. The affinity of the encoded polypeptide for Pu-triplex DNA was confirmed by electrophoretic mobility shift assays using a bacterially expressed N-terminal loricrin deletion fused with the maltose-binding protein (MBP-LOR3ARF). Interactions between Pu-triplex DNA and MBP-LOR3ARF are characterized by a distribution of four increasingly slower mobility complexes, suggesting that multiple MBP-LOR3ARF molecules can recognize a single triplex. Binding was also observed between MBP-LOR3ARF and a pyrimidine-motif triplex DNA, although at lower affinity than Pu-triplex DNA. No apparent binding was observed between MBP-LOR3ARF and double-stranded DNA, suggesting that MBP-LOR3ARF is a bona fide Pu-triplex binding protein. Finally, purified specific rabbit antibodies against LORARF detected four human proteins with apparent molecular masses of 210, 110, 68, and 66 kDa on Western blot analysis. The 210-, 110-, and 68-kDa proteins also showed specific Pu-triplex DNA binding in a South-Western experiment, suggesting that LORARF shares common domains with other human Pu-triplex DNA-binding proteins.
Ciotti
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Dipartimento di Oncologia, Biologia e Genetica, Sezione di Biologia e Genetica, Università degli Studi di Genova, viale Benedetto XV, 6-16132 Genova, Italy.
Van Dyke
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Bianchi-Scarrà
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Musso
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D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D011485:Protein Binding; D016364:Reading Frames / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology
D004268:DNA-Binding Proteins; C422449:LORARF protein, human; C520206:MBP-LOR3(ARF) protein, human; D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C064554:loricrin; C051802:triplex DNA; D004247:DNA