Characterization of the specific DNA nicking activity of restriction endonuclease N.BstNBI

N.BstNBI is a unique restriction endonuclease isolated from Bacillus stearothermophilus, We have characterized the recognition sequence and the cleavage site of N.BstNBI. Mapping of cleavage sites of N.BstNBI showed that it recognizes an asymmetric sequence, 5' GAGTC 3', and cleaves only on the top strand 4 base pairs away from its recognition sequence. To verify the nicking activity of N.BstNBI, we have constructed two plasmids containing a single recognition sequence (pNB1) or no recognition site (pNB0). When pNB1 and pNB0 were incubated with the enzyme, N.BstNBI nicked only the plasmid pNB1, suggesting that N.BstNBI is a specific nicking endonuclease.