A bacterial defense system targeting modified cytosine of phage genomic DNA

Abstract The evolutionary arms race between bacteria and phages drives the development of bacterial antiviral defense systems and phage counter-defense strategies. Restriction–modification (RM) systems protect bacteria by methylating ‘self’ DNA and cleaving unmodified phage DNA. Phages like T-even coliphages evade RM systems by substituting cytosine with 5-hydroxymethyl cytosine (5hmC) or 5-glucosylated hmC (5ghmC). Here, we characterize ‌a single-component antiviral defense system featuring a GIY-YIG endonuclease domain. Biochemical and structural analyses demonstrate that this defense system is a type IV modification-dependent restriction endonuclease that specifically degrades 5hmC- or 5ghmC-modified DNA, and we accordingly name it CMoRE ( C ytosine Mo dification ‌R ‌estriction E ndonuclease). The crystal structures reveal an N-terminal GIY-YIG nuclease domain and a C-terminal modification-sensing domain. Unique features, including a ‘GIYxY-YIG’ motif and an inhibitory negatively charged loop, distinguish CMoRE as an additional member of the GIY-YIG family. This system not only highlights the evolutionary interplay between phages and bacteria but also presents CMoRE as a potential tool for precise genomic detection of 5hmC in mammals, with implications for epigenetics research and disease diagnostics.