SITE-DIRECTED MUTAGENESIS STUDY ON DNA-BINDING REGIONS OF THE MOUSE HOMOLOG OF SUPPRESSOR OF HAIRLESS, RBP-J-KAPPA

To map regions important for DNA binding of the mouse homologue of Sup pressor of Hairless or RBP-J kappa protein, mutated mouse RBP-J kappa cDNAs were made by insertion of oligonucleotide linkers or base replac ement. DNA binding assays using the mutated proteins expressed in COS cells showed that various mutations between 218 Arg and 227 Arg decrea sed the DNA binding activity drastically. The DNA binding activity was not affected by amino acid replacements within the integrase motif of the RBP-J kappa protein (230His-269His). Replacements between 291Arg and 323Tyr affected the DNA binding activity slightly but reproducibly . These results indicate that the region encompassing 218Arg-227Arg is critical for the DNA binding activity of RBP-J kappa. This region did not show any significant homology to motifs or domains of the previou sly described DNA binding proteins. Using a truncation mutant protein RBP-J kappa was shown to associate with DNA as a monomer.