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Data from: Ancestral proteins trace the emergence of substrate specificity and oligomerization within bacterial DEDDy dinucleases

Mortensen, Sofia1

Published Jul 29, 2026 on Dryad. https://doi.org/10.5061/dryad.bnzs7h4sc

Data files

Jul 29, 2026 version files 67.10 MB

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Abstract

Nucleases play essential roles in bacterial genome maintenance and host defense. This dataset accompanies a study investigating the evolutionary divergence of Gram-positive diDNases and Gram-negative nanoRNase C (NrnC), two homologous nuclease families that differ in oligomeric state, substrate specificity, and catalytic efficiency. Using ancestral sequence reconstruction, biochemical characterization, structural analyses, and functional assays, the study identifies a common ancestor of diDNases and NrnC that forms a dimer and displays intermediate preference for DNA dinucleotides. Comparative analyses of ancestral and extant enzymes reveal how gradual structural changes drove the evolution of substrate specificity, oligomerization, and catalytic activity while preserving a conserved protein fold, providing insight into the evolution and biological function of DNA dinucleotide-processing nucleases.

The deposited dataset includes raw fluorescence time-course measurements from enzyme kinetics assays used to determine nuclease activity on fluorescent substrate analogs, raw data from multi-angle light scattering (MALS) and mass photometry experiments used to assess protein oligomeric states, colony images and quantification data from Pseudomonas aeruginosa small-colony phenotype assays used to evaluate in vivo protein function, and protein primary sequences needed to reproduce the AlphaFold structural models analyzed in the study. These data provide the experimental basis for the biochemical, biophysical, structural, and functional analyses presented in the associated publication.