Site-specific inhibition of transcription factor binding to DNA by a metallointercalator.

Abstract

The metallointercalator Lambda-1-Rh(MGP)2phi5+ binds tightly and specifically to the site 5'-CATATG-3' in the major groove of double helical DNA by a combination of direct readout and shape selection. To examine competitive interactions between this small metal complex and a DNA-binding transcription factor, the preferred binding site for Lambda-1-Rh(MGP)2phi5+ was engineered into the AP-1 recognition element (ARE) of the major-groove binding bZIP transcription factor yAP-1, the yeast analogue of mammalian AP-1. Binding experiments confirmed that the modified ARE retained normal yAP-1 binding affinity. Photocleavage experiments demonstrated that the modified ARE contained a high-affinity binding site for Lambda-1-Rh(MGP)2phi5+, whereas the native ARE showed no interaction. Competition experiments using gel shift mobility assays demonstrated that Lambda-1-Rh(MGP)2phi5+ at 120 nM competes 50% of yAP-1 binding to the 5'-CATATG-3' containing oligonucleotide. In contrast, competitive disruption of protein binding to the native ARE requires 3 microM Lambda-1-Rh(MGP)2phi5+. Metallointercalator derivatives, including geometric isomers of Lambda-1-Rh(MGP)2phi5+, show no specific binding to the target site and show no inhibition of yAP-1/DNA complexes at concentrations as high as 20 microM. Thus, metallointercalators can be tuned to show selectivity for major groove sites on DNA comparable to transcription factors and indeed can inhibit transcription factor binding site selectively.