Characterization of a novel N4 -methylcytosine restriction-modification system in Deinococcus radiodurans

Deinococcus radiodurans is an extremophilic microorganism that possesses a unique DNA damage repair system, conferring strong resistance to radiation, desiccation, oxidative stress, and chemical damage. Recently, we discovered that D. radiodurans possesses a N4-methylation (m4C) methyltransferase called M.DraR1, which recognizes the 5'-CCGCGG-3' sequence and methylates the second cytosine. Here, we revealed its cognate restriction endonuclease R.DraR1 and recognized that it is the only endonuclease specially for non-4C-methylated 5'-CCGCGG-3' sequence so far. We designated the particular m4C R.DraR1-M.DraR1 as DraI R-M system. Bioinformatics searches displayed the rarity of the DraI R-M homologues system. Meanwhile, recombination and transformation efficiency experiments demonstrated the important role of the DraI R-M system in response to oxidative stress. Besides, in vitro activity experiments showed that R.DraR1 could exceptionally cleave DNA substrates with m5C-methlated 5'-CCGCGG-3' sequence besides its routine activity, suggesting that this particular R-M component possesses a broader substrate choice. Furthermore, an imbalance of the DraI R-M system led to cell death through regulating genes involved in the maintenance of cell survival such as genome stability, transporter, energy production. Thus, our research revealed a novel m4C R-M system that plays key roles in maintaining cell viability and defending foreign DNA in D. radiodurans. RNA-seq for KO_R.DraR1 and WT strains. All of the strains were cultured to OD600=1.0 without any stress treatment.