ANALYSIS OF THE MOUSE SPLOTCH-DELAYED MUTATION INDICATES THAT THE PAX-3 PAIRED DOMAIN CAN INFLUENCE HOMEODOMAIN DNA-BINDING ACTIVITY

The murine Pax-3 protein contains two DNA-binding domains, a paired do main and a homeodomain, and alterations in the Pax-3 gene are responsi ble for the neural tube defects observed in the Splotch (Sp) mouse mut ant, Of five Sp alleles, Splotch-delayed (Sp(d)) is the only one that encodes a full-length Pax-3 protein, containing a single glycine-to-ar ginine substitution within the paired domain, To better understand the consequence of this mutation on Pax-3 function, we have analyzed the DNA-binding properties of wild-type and Sp(d) Pax-3, using oligonucleo tides that bind primarily to the paired domain (e(5)) or exclusively t o the homeodomain (P2), Wild-type Pax-3 was found to bind e(5) in a sp ecific manner, In contrast, the Sp(d) mutation reduced binding of Pax- 3 to e(5) 17-fold, revealing a defect in DNA binding by the paired dom ain. Surprisingly, the Sp(d) mutation also drastically reduced the hom eodomain-specific binding to P2 by 21-fold when compared with the wild -type protein, Interestingly, a deletion which removes the Sp(d) mutat ion was found to restore PZ-binding activity, suggesting that within t he full-length Pax-3 protein, the paired domain and homeodomain may in teract. We conclude, therefore, that the Sp(d) mutation is phenotypica lly expressed in vitro by a defect in the DNA-binding properties of Pa x-3. Furthermore, it is apparent that the paired domain and homeodomai n of Pax-3 do not function as independent domains, since a mutation in the former impairs the DNA-binding activity of the latter.