The Type II restriction endonuclease MvaI has dual specificity

The MvaI restriction endonuclease cuts 5′-CC↓AGG-3′/5′-CC↑TGG-3′ sites as indicated by the arrows. N4-methylation of the inner cytosines (C^m4CAGG/C^m4CTGG) protects the site against MvaI cleavage. Here, we show that MvaI nicks the G-strand of the related sequence (CCGGG/CCCGG, BcnI site) if the inner cytosines are C5-methylated: C^m5C↓GGG/CC^m5CGG. At M.SssI-methylated SmaI sites, where two oppositely oriented methylated BcnI sites partially overlap, double-nicking leads to double-strand cleavage (CC^m5C↓GGG/CC^m5C↑GGG) generating fragments with blunt ends. The double-strand cleavage rate and the stringency of substrate site recognition is lower at the methylation-dependent site than at the canonical target site. MvaI is the first restriction endonuclease shown to possess, besides the ‘normal’ activity on its unmethylated recognition site, also a methylation-directed activity on a different sequence.