A bacterial defense system targeting modified cytosine of phage genomic DNA

Abstract

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.