The paired box encodes a second DNA-binding domain in the Paired homeo domain protein

The homeo box, which encodes the DNA-binding homeo domain, is a DNA sequence motif present in several Drosophila developmental genes; it has been used to identify many homologous genes involved in mammalian development. The paired box is another conserved sequence motif, first identified in the paired (prd) and gooseberry (gsb) Drosophila homeo domain genes. It encodes a 128-amino-acid domain, the paired domain, which has since been found in other fly and mouse gene products, in association with the homeo domain or in its absence. We show that the paired box of the prd gene encodes a DNA-binding activity, independent of the DNA-binding activity of the Paired (Prd) homeo domain and with a different sequence specificity. The amino-terminal region of the paired domain, including one of the three predicted α-helices, is necessary and sufficient for binding. We investigate the binding of the Prd protein to two sites in the even-skipped promoter, which are composed of overlapping sequences bound by the homeo domain and by the paired domain. We also show that a mutation in the paired box of Prd, corresponding to the mutation in the paired box of the mouse Pax-1 gene thought to cause the undulated skeletal phenotype, destroys the ability of the Prd protein to bind to the paired domain-specific site. This supports the view that the undulated phenotype results from the inactivation of the DNA-binding activity of the paired domain of Pax-1.