PAX-3 DNA INTERACTION - FLEXIBILITY IN THE DNA-BINDING AND INDUCTION OF DNA CONFORMATIONAL-CHANGES BY PAIRED DOMAINS

The mouse Pax-3 gene encodes a protein that is a member of the Pax fam ily of DNA binding proteins. Pax-3 contains two DNA binding domains: a paired domain (PD) and a paired type homeodomain (HD). Both domains a re separated by 53 amino acids and interact synergistically with a seq uence harboring an ATTA motif (binding to the HD) and a GTTCC site (bi nding to the PD) separated by 5 base pairs. Here we show that the inte raction of Pax-3 with these two binding sites is independent of their angular orientation. In addition, the protein spacer region between th e HD and the PD can be shortened without changing the spatial flexibil ity of the two DNA binding domains which interact with DNA. Furthermor e, by using circular permutation analysis we determined that binding o f Pax-3 to a DNA fragment containing a specific binding site causes co nformational changes in the DNA, as indicated by the different mobilit ies of the Pax-3-DNA complexes. The ability to change the conformation of the DNA was found to be an intrinsic property of the Pax-3 PD and of all Pax proteins that we tested so far. These in vitro studies sugg est that interaction of Pax proteins with their specific sequences in vivo may result in an altered DNA conformation.