The BisI family of restriction endonucleases is unique in requiring multiple methylations or hydroxymethylations within a short cytosine rich recognition sequence (GCNGC), and in cleaving directly within this sequence, rather than at a distance. Here, we report that the number of modified cytosines that are required for cleavage can be tuned by the salt concentration. We present crystal structures of the catalytic domain of Eco15I alone and at higher resolution of NhoI, with quadruple methylated DNA. The structures show that NhoI senses and recognizes modified cytosines in the context of double-stranded DNA without base flipping. Each protomer has two pockets for the 5’ (internal, G5mCNGC) and 3’ (external, GCNG5mC) methyl/hydroxymethyl groups. In our NhoI-DNA co-crystal structure, the internal methyl groups interact with the side chains of an (H/R)(V/I/T/M) diamino acid motif near the C-terminus of the domain. The external methyl groups make mostly main chain contacts, and in case of NhoI, interact also with a non-conserved arginine two residues downstream of the QXK motif (suggesting tight coupling of 5mC recognition and cleavage). Surface plasmon resonance analysis shows that for Eco15I, the internal and external methyl binding pockets contribute about equally to methylcytosine sensing.
Work in the Bochtler laboratory is supported by grants from the Polish National Science Center (NCN, grant 2018/30/Q/NZ2/00669), the Foundation for Polish Science (FNP, grant POIR.04.04.00-00-5D81/17-0), and the Polish Agency for Academic exchange (NAWA, grant PPI/APM/2018/1/00034).