THE PAIRED-DOMAIN REGULATES DNA-BINDING BY THE HOMEODOMAIN WITHIN THEINTACT PAX-3 PROTEIN

Pax-3 contains two structurally independent DNA-binding domains, a pai red-domain and a homeodomain, Their functional interdependence has bee n suggested by the analysis of the Sp-delayed (Sp(d)) mouse mutant, in which a glycine to arginine substitution at position 9 of the paired- domain abrogates DNA binding by both domains, This glycine is located in the beta-turn portion of a beta-hairpin motif, and the requirement for this structure was investigated by mutagenesis at this and neighbo ring positions, At position 9, only substitution with proline increase d DNA binding by the paired-domain and homeodomain above the level obs erved with the Sp(d) arginine mutation, suggesting that the beta-turn is necessary for the function of both DNA-binding domains, Alanine sca nning mutagenesis also identified a number of flanking residues import ant for DNA binding by both domains, emphasizing the requirement of th e beta-hairpin for the interaction of Pax-3 with DNA. Furthermore, we show that these mutations reduce binding by the homeodomain at the mon omeric level and do not impair dimerization on a TAAT(N)(2)ATTA consen sus motif, In contrast, the wild-type paired-domain was found to preve nt dimerization on consensus motifs with 3-base pair spacing of the ty pe TAAT(N)(3)ATTA. Importantly, both the deleterious effect of the Sp( d) mutation on homeodomain DNA binding and the loss of dimerization on TAAT(N)(3)ATTA motifs can be transferred to a heterologous homeodomai n from the human phox protein. Moreover, the presence of the paired-do main affects sequence discrimination within the 3-base pair spacer in this context, These analyses establish that the beta-hairpin motif is essential for paired domain and homeodomain DNA binding, and suggest a novel mechanism by which the paired-domain can influence sequence spe cificity of the homeodomain within the Pax-3 polypeptide.