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The TypeⅡ restriction endonuclease NgoMIV recognizes sequence 5-GCCGGC and cleaves it after the first G to yield 4 nt 5-overhangs.To achieve its specificity NgoMIV builds an intricate network of hydrogen bonds to the bases at target site.Restriction endonuclease Ecl 18kI is specific for the sequence/CCNGG and cleaves it before the outer C to generate 5 nt 5-overhangs.Crystal structure unexpectedly revealed that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotide,flipping it out from the helix.It tumed out that nucleotide flipping allows Ecl18kI to adapt the conserved sequence readout machinery for the interrupted target site.EcoRII/PspGI enzymes specific for the CCWGG sequence (where W =A or T) are evolutionarily related to Ecl18kI and recognize target sequences that differ only in the central base pair.Using 2-aminopurine fluorescence assay we demonstrate that EcoRII and PspGI,like Ecl18kI un-stack bases at the center of their recognition sequences and flip them into pockets that are formed by the enzymes.