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Abstract:

The most prominent of the prokaryotic defense systems are restriction-modification (RM) and CRISPR-Cas. Type IV RM systems are modification-dependent restriction enzymes that target DNA containing modified bases using dedicated specificity domains. The Type IV RM class includes the two-component McrBC system, consisting of McrB, a GTPase which can be fused to a wide range of specificity domains, and McrC, a PD-DxK nuclease. We identified abundant varieties of McrBC which we termed CoCoNuT (coiled-coil nuclease tandem) systems. CoCoNuTs are often encoded in complex operonic contexts implying a role in a novel form of viral RNA restriction regulated by CBASS (cyclic oligonucleotide-based antiphage signaling system), which also serves an activating function for Type III CRISPR. Furthermore, we detected many McrB homologs with several fused specificity domains, up to 8 in a single protein, a majority of which are uncharacterized or only distantly homologous to known domains. This vast pool of novel domains and cognate DNA modifications represents a significant area of viral DNA chemistry and corresponding prokaryotic immunity that remains poorly understood. We addressed this with a comprehensive search for McrBC systems, followed by an unprecedented census and AlphaFold structural predictions of all fused domains. Surprisingly, this analysis led to the identification of a wide-spread, highly divergent type of CoCoNuT, as well as an intriguing hypothesis concerning the origins and mechanisms of all the CoCoNuT systems.