Reciprocal effect of Waardenburg syndrome mutations on DNA binding by the Pax-3 paired domain and homeodomain |
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Authors: | Fortin, AS Underhill, DA Gros, P |
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Affiliation: | Department of Biochemistry, McGill University, Montreal, Quebec, Canada. |
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Abstract: | The Pax-3 protein contains two DNA-binding domains, a paired domain and ahomeodomain. Mutations in Pax-3 cause Waardenburg syndrome (WS) in humansand the mouse Splotch (Sp) phenotype. In the Sp-delayed mouse, a mutationin the Pax-3 paired domain (G9R) abrogates the DNA-binding activity of boththe paired domain and the homeodomain, suggesting that they mayfunctionally interact. To investigate this possibility further, we haveanalyzed the DNA-binding properties of additional point mutants in thePax-3 paired domain and homeodomain that occur in WS patients (F12L, N14H,G15S, P17L, R23L, G48A, S51F and G66D in the paired domain, V47F and R53Gin the homeodomain), the Pax-1 un mutation (G15A) and a substitutionassociated with Peters' anomaly in the PAX-6 gene (R23G). Within the paireddomain, seven of 10 mutations were found to abrogate DNA-binding by thepaired domain. Remarkably, these seven mutations also affected DNA bindingby the homeodomain, causing either a complete loss (P17L and G66D), areduction (R23G, R23L, G15S and G15A) or an increase in DNA-bindingactivity (N14H). In addition, the effect of paired domain mutationsoccurred at the level of monomer formation by the homeodomain, while thedimerization potential of this domain seemed unaffected in mutants where itcould be analyzed. Furthermore, while both homeodomain mutations were foundto abolish DNA binding by this domain, the R53G mutation also abrogated DNAbinding by the paired domain. The important observation that independentmutations in either domain can affect DNA binding by the other in theintact Pax- 3 protein strongly suggests that the two domains are notfunctionally independent but bind DNA through cooperative interactions.Modeling the deleterlous mutations on the three-dimensional structure ofthe paired domain of Drosophila Prd shows that these mutations cluster atthe DNA interface, thus suggesting that a series of DNA contacts areessential for DNA binding by both the paired domain and the homeodomain ofPax-3. |
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