Properties and secondary structure analysis of BanI endonuclease: Identification of putative active site

Biochemical properties of Type II restriction enzyme BanI were characterize d, Kinetic parameters were evaluated and an enhancement of rate was observe d when the recognition site was located in a more central position in the s ubstrate, suggesting that BanI locates its recognition site by a sliding me chanism, As BanI has three cysteine residues in its primary sequence, the e ffect of thiol inhibitors on BanI activity was also studied. Partial inhibi tion was observed only at a very high concentration of the inhibitor indica ting that cysteine residues are not directly involved in catalysis. The gel electrophoretic mobility shift assay demonstrated specific complex formati on between BanI and the DNA substrate in the presence of poly dI-dC and Mg2 +. A secondary structure analysis and comparison with EcoRI and BamHI cryst al structure revealed a putative active site similar to that seen in BamHI but different in the order in which the catalytic domain (central beta -she et) and recognition domain (adjacent alpha -helix) were arranged in the pro tein, (C) 2000 Academic Press.