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Volume 272, Number 22, Issue of May 30, 1997 pp. 14175-14182
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

The Paired-domain Regulates DNA Binding by the Homeodomain within the Intact Pax-3 Protein

(Received for publication, January 27, 1997, and in revised form, March 24, 1997)

D. Alan Underhill and Philippe Gros

From the  Department of Biochemistry, McGill University, Montreal, Quebec, H3G 1Y6 Canada

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 (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 -turn portion of a -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 Sp^d arginine mutation, suggesting that the -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 -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 Sp^d 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 -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.

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