Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains

Abstract

The mouse Pax-3 gene encodes a protein that is a member of the Pax family of DNA binding proteins. Pax-3 contains two DNA binding domains: a paired domain (PD) and a paired type homeodomain (HD). Both domains are separated by 53 amino acids and interact synergistically with a sequence harboring an ATTA motif (binding to the HD) and a GTTCC site (binding to the PD) separated by 5 base pairs. Here we show that the interaction of Pax-3 with these two binding sites is independent of their angular orientation. In addition, the protein spacer region between the HD and the PD can be shortened without changing the spatial flexibility of the two DNA binding domains which interact with DNA. Furthermore, by using circular permutation analysis we determined that binding of Pax-3 to a DNA fragment containing a specific binding site causes conformational changes in the DNA, as indicated by the different mobilities 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 suggest that interaction of Pax proteins with their specific sequences in vivo may result in an altered DNA conformation.

Cell-type-specific regulation of distinct sets of gene targets by Pax3 and Pax3/FKHR

“…EPHA4 is induced by both Pax3 and Pax3/FKHR in SaOS-2 cells. Inspection of the region 5 0 of the EphA4 gene reveals at least one potential Pax3 PD binding site (Chalepakis et al, 1994b;Chalepakis and Gruss, 1995;Underhill et al, 1995;Underhill and Gros, 1997) at À83 (5 0 CACGTCACCGGC). Likewise, EFNB2, another factor involved in somite development (Durbin et al, 1998;Johnson et al, 2001;Barrios et al, 2003) and polarized trafficking of axons and neural crest cells through the somite (Wang and Anderson, 1997;Koblar et al, 2000;De Bellard et al, 2002), was induced by Pax3 and Pax3/FKHR in SaOS-2 cells.…”

Interaction of the pRB-family proteins with factors containing paired-like homeodomains

“…These two structurally distinct domains clearly in¯uence each other's functional activities (Chalepakis et al, 1994;Underhill et al, 1995). We tested whether deletion of speci®c helices from the paired-like homeodomain in Pax-3 would abrogate its binding to p107 (Figure 6).…”

Pax-3 contains domains for transcription activation and transcription inhibition.

“…Efficient binding of Pax-3 to the PRS sequences requires the presence of both DNA recognition motifs (ATTA and either GTTCC or GT-TAC), suggesting a synergistic interaction of the two DNAbinding domains with the PRS sequences. The length of the spacer sequence between the ATTA and GTTCC motifs can vary considerably without affecting the binding of Pax-3 (16). In addition, the space between the PD and the HD (53 amino acids) can be shortened without changing the flexibility of Pax-3 to interact with the PRS sequences (16).…”

“…The length of the spacer sequence between the ATTA and GTTCC motifs can vary considerably without affecting the binding of Pax-3 (16). In addition, the space between the PD and the HD (53 amino acids) can be shortened without changing the flexibility of Pax-3 to interact with the PRS sequences (16). Finally, the protein region between the PD and the HD was found to participate in the homodimerization of truncated Pax-3 protein products when bound to the DNA (15).…”