Type II restriction endonuclease R.KpnI is a member of the HNH nuclease superfamily

Abstract

The restriction endonuclease (REase) R.KpnI is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves the DNA sequence 5'-GGTAC^C-3' in the presence of Mg2+ as showngenerating 3' four base overhangs. Bioinformatics analysis reveals thatR.KpnI contains a \beta\beta\alpha-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonucleaseI-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequencenon-specific Serratia nuclease and sequence-specific homing endonuclease I-PpoI. According to our homology model of R.KpnI, D148,H149 and Q175 correspond to the critical D, H and N or H residues ofthe HNH nucleases. Substitutions of these three conserved residues lead to the loss of the DNA cleavage activity by R.KpnI, confirming their importance. The mutant Q175E fails to bind DNA at the standard conditions, although the DNA binding and cleavage can be rescued at pH6.0, indicating a role for Q175 in DNA binding and cleavage. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD...D/EXK superfamily of nucleases, instead is a member of the HNH superfamily