Insights into the molecular recognition of the 5′-GNN-3′ family of DNA sequences by zinc finger domains

In order to construct zinc finger domains that recognize all of the possible 64 DNA triplets, it is necessary to understand the mechanisms of protein/DNA interactions on the molecular level. Previously we reported 16 zinc finger domains which had been characterized in detail to bind specifically to the 5′-GNN-3′ family of DNA sequences. Artificial transcription factors constructed from these domains can regulate the expression of endogenous genes. These domains were created by phage-display selection followed by site-directed mutagenesis. A total of 84 mutants of a three-domain zinc finger protein have been analyzed for their DNA-binding specificity. Here, we report the results of this systematic and extensive mutagenesis study. New insights into zinc finger/DNA interactions were obtained by combining specificity data with computer modeling and comparison with known structural data from NMR and crystallographic studies. This analysis suggests that unusual cross-strand and inter-helical contacts are made by some of these proteins, and the general orientation of the recognition helix to the DNA is flexible, even when constrained by flanking zinc finger domains. These findings disfavor the utility of existing simple recognition codes and suggest that highly specific domains cannot be obtained from phage display alone in most cases, but only in combination with rational design. The molecular basis of zinc finger/DNA interaction is complex and its understanding is dependent on the analysis of a large number of proteins. This understanding should enable us to refine rapidly the specificity of other zinc finger domains, as well as polydactyl proteins constructed with these domains to recognize extended DNA sequences.