The PD-(D/E)XK nuclease superfamily, initially identified in type II restriction endonucleases and later in many enzymes involved in DNA recombination and repair, is one of the most challenging targets for protein sequence analysis and structure prediction. Typically, the sequence similarity between these proteins is so low, that most of the relationships between known members of the PD-(D/E)XK superfamily were identified only after the corresponding structures were determined experimentally. Thus, it is tempting to speculate that among the uncharacterized protein families, there are potential nucleases that remain to be discovered, but their identification requires more sensitive tools than traditional PSI-BLAST searches.
The low degree of amino acid conservation hampers the possibility of identification of new members of the PD-(D/E)XK superfamily based solely on sequence comparisons to known members. Therefore, we used a recently developed method HHsearch for sensitive detection of remote similarities between protein families represented as profile Hidden Markov Models enhanced by secondary structure. We carried out a comparison of known families of PD-(D/E)XK nucleases to the database comprising the COG and PFAM profiles corresponding to both functionally characterized as well as uncharacterized protein families to detect significant similarities. The initial candidates for new nucleases were subsequently verified by sequence-structure threading, comparative modeling, and identification of potential active site residues.
In this article, we report identification of the PD-(D/E)XK nuclease domain in numerous proteins implicated in interactions with DNA but with unknown structure and mechanism of action (such as putative recombinase RmuC, DNA competence factor CoiA, a DNA-binding protein SfsA, a large human protein predicted to be a DNA repair enzyme, predicted archaeal transcription regulators, and the head completion protein of phage T4) and in proteins for which no function was assigned to date (such as YhcG, various phage proteins, novel candidates for restriction enzymes). Our results contributes to the reduction of "white spaces" on the sequence-structure-function map of the protein universe and will help to jump-start the experimental characterization of new nucleases, of which many may be of importance for the complete understanding of mechanisms that govern the evolution and stability of the genome.