The paired-domain regulates DNA binding by the homeodomain within the intact Pax-3 protein.
Pax-3 contains two structurally independent DNA-binding domains, a paired-domain and a homeodomain. Their functional interdependence has been suggested by the analysis of the Sp-delayed (Spd) 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 neighboring positions. At position 9, only substitution with proline increased DNA binding by the paired-domain and homeodomain above the level observed with the Spd arginine mutation, suggesting that the beta-turn is necessary for the function of both DNA-binding domains. Alanine scanning mutagenesis also identified a number of flanking residues important for DNA binding by both domains, emphasizing the requirement of the beta-hairpin for the interaction of Pax-3 with DNA. Furthermore, we show that these mutations reduce binding by the homeodomain at the monomeric level and do not impair dimerization on a TAAT(N)2ATTA consensus motif. In contrast, the wild-type paired-domain was found to prevent dimerization on consensus motifs with 3-base pair spacing of the type TAAT(N)3ATTA. Importantly, both the deleterious effect of the Spd mutation on homeodomain DNA binding and the loss of dimerization on TAAT(N)3ATTA motifs can be transferred to a heterologous homeodomain from the human phox protein. Moreover, the presence of the paired-domain 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 specificity of the homeodomain within the Pax-3 polypeptide.
Underhill
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Department of Biochemistry, McGill University, Montreal, Quebec, H3G 1Y6 Canada.
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D010957:Plasmids; D011485:Protein Binding; D017421:Sequence Analysis; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D000072041:PAX3 Transcription Factor; C489409:PAX3 protein, human; D051761:Paired Box Transcription Factors; D014157:Transcription Factors; C068686:Pax3 protein, mouse; D004247:DNA