Abstract

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.