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 protein s, we performed South-Western screening of a human keratinocyte cDNA expres sion library using a purine (Pu)-rich triplex DNA probe. We isolated two in dependent clones containing part of the loricrin gene. Both were translated with a different reading frame than that of the loricrin protein, the majo r 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-term inal loricrin deletion fused with the maltose-binding protein (MBP-LOR3(ARF )). Interactions between Pu-triplex DNA and MBP-LOR3(ARF) are characterized by a distribution of four increasingly slower mobility complexes, suggesti ng that multiple MBP-LOR3(ARF) molecules can recognize a single triplex. Bi nding was also observed between MBP-LOR3(ARF) and a pyrimidine-motif triple x DNA, although at lower affinity than Pu-triplex DNA. No apparent binding was observed between MBP-LOR3(ARF) and double-stranded DNA, suggesting that MBP-LOR3(ARF) is a bona fide Pu-triplex binding protein. Finally, purified specific rabbit antibodies against LORARF detected four human proteins wit h apparent molecular masses of 210, 110, 68, and 66 kDa on Western blot ana lysis. The 210-, 110-, and 68-kDa proteins also showed specific Pu-triplex DNA binding in a South-Western experiment, suggesting that LORARF shares co mmon domains with other human Pu-triplex DNA-binding proteins.