DNA RECOGNITION SITE ANALYSIS OF XENOPUS WINGED HELIX PROTEINS

DNA binding proteins of the winged helix family contain a conserved 11 0 amino acid region, the fork head/HNF-3 domain. Three members of the recently described XFD (Xenopus fork head domain related) multigene fa mily in the frog Xenopus laevis that contain this DNA-binding domain h ave been studied. We determined the in vitro DNA recognition sequences by means of two independent methods: PCR supported site selection wit h degenerated deoxyoligonucleotides and affinity chromatography of gen omic Xenopus DNA fragments. In contrast to a remarkable sequence diver gence within their protein sequence of the fork head domains, all thre e proteins share a similar 7 bp DNA target motif. The protein-DNA inte raction has been studied by means of DMS interference and hydroxyl rad ical footprinting. A region of 18 bp encompassing the 7 bp target moti f is sufficient to confer binding and specificity. The specificity of binding could be attributed on the DNA level to residues located 5' to the 7 bp core region, and on the protein level most likely to a regio n within the first half of the fork head domain. The possible role of specific nucleotides within the target site in binding the protein is discussed in the context of the current crystal structure of the compl ex of this domain with DNA.