Abstract
SummarySpecific 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 the mutational paths that led to changes in specificity are unknown. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB [Partitioning Protein B] and Noc [Nucleoid Occlusion Factor], which both play 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 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 for understanding the evolution of other classes of DNA-binding proteins.
Highlights
In living organisms, hundreds of DNA-binding proteins carry out a plethora of roles in homeostasis, in transcriptional regulation in response to stress, and in the maintenance and transmission of genetic information
We show that specificity to phosphate groups of thymine À6 (parS) or NBS is encoded by a small set of four residues at the protein-DNA interface and that mutations in these residues are enough to reprogram DNA-binding specificity
DNA-Binding Specificity for parS and NBS Is Conserved within ParB and Noc Family To test whether ParB and Noc family members retained their DNAbinding specificity, we selected a group of 17 ParB and 4 Noc from various bacterial clades for characterization (Figures 1B and S1A)
Summary
Hundreds of DNA-binding proteins carry out a plethora of roles in homeostasis, in transcriptional regulation in response to stress, and in the maintenance and transmission of genetic information These DNA-binding proteins do so faithfully due to their distinct DNA-binding specificity toward their cognate DNA sites. Among the many ways to evolve new biological innovations, gene duplication and neo-functionalization have been widely implicated as major forces in evolution (Conrad and Antonarakis, 2007; Kaessmann, 2010; Lynch and Conery, 2000; Qian and Zhang, 2014; Teichmann and Babu, 2004) In this process, after a gene was duplicated, one copy retained the original function, whereas the other accumulated beneficial and diverging mutations that produced a different protein with a new function. We used a pair of related DNA-binding proteins (ParB [Partitioning Protein B] and Noc [Nucleoid Occlusion Factor]) that are crucial for bacterial chromosome segregation and maintenance to better understand factors that might have influenced the evolution of a new DNA-binding specificity
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