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 domain and a homeodomain, and alterations in the Pax-3 gene are responsible for the neural tube defects observed in the Splotch (Sp) mouse mutant. Of five Sp alleles, Splotch-delayed (Spd) is the only one that encodes a full-length Pax-3 protein, containing a single glycine-to-arginine 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 Spd Pax-3, using oligonucleotides that bind primarily to the paired domain (e5) or exclusively to the homeodomain (P2). Wild-type Pax-3 was found to bind e5 in a specific manner. In contrast, the Spd mutation reduced binding of Pax-3 to e5 17-fold, revealing a defect in DNA binding by the paired domain. Surprisingly, the Spd mutation also drastically reduced the homeodomain-specific binding to P2 by 21-fold when compared with the wild-type protein. Interestingly, a deletion which removes the Spd mutation was found to restore P2-binding activity, suggesting that within the full-length Pax-3 protein, the paired domain and homeodomain may interact. We conclude, therefore, that the Spd mutation is phenotyically expressed in vitro by a defect in the DNA-binding properties of Pax-3. Furthermore, it is apparent that the paired domain and homeodomain of Pax-3 do not function as independent domains, since a mutation in the former impairs the DNA-binding activity of the latter.
Underhill
D A
DA
Department of Biochemistry, McGill University, Montreal, PQ, Canada.
Vogan
K J
KJ
Gros
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D005998:Glycine; D018398:Homeodomain Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D007106:Immune Sera; D007668:Kidney; D007700:Kinetics; D051379:Mice; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D009436:Neural Tube Defects / Q000235:genetics; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D016364:Reading Frames; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D014157:Transcription Factors; D014162:Transfection
D017931:DNA Primers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007106:Immune Sera; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D011994:Recombinant Proteins; D014157:Transcription Factors; C068686:Pax3 protein, mouse; D004247:DNA; D001120:Arginine; D005998:Glycine