Diversification of DNA-binding specificity via permissive and specificity-switching mutations in the ParB/Noc protein family

Specific interactions between proteins and DNA are essential to many biological processes. Yet it remains unclear how the diversification in DNA-binding specificity was brought about, and what were the mutational paths that led to changes in specificity. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB and Noc: both having roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity, and that mutational paths to a new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc, and in a broader context, might be useful in understanding the evolution of other classes of DNA-binding proteins. Overall design: Chromatin-immunoprecipitation with deep sequencing experiments (ChIP-seq) were performed on exponential-growing Escherichia coli (see the supplementary Materials and Methods for the exact treatment that were applied to each strain). Bacterial one-hybrid (B1H) with deep seqeuncing i.e. deep mutational scanning experiments were performed using plasmid DNA extracted from cells that were either selected on M9 minimal medium lacking (or supplemented) with histidine.