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 shown generating 3' four base overhangs. Bioinformatics analysis reveals that R.KpnI contains a betabetaalpha-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonuclease I-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequence non-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 of the 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 pH 6.0, 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.

Figure 1

(A) Multiple sequence alignment of R.KpnI and the experimentally solved structures of HNH-superfamily nucleases used as modeling templates. The secondary structure of T4 Endonuclease VII is shown above the alignment as tubes (helices) and arrows (strands). Amino acid residues of the active site are highlighted in black, residues involved in formation of a Zn-finger in Endonuclease VII and R.KpnI are highlighted in grey. The numbers in parentheses indicate the size of terminal extensions of the conserved ‘ββα-finger’, which do not share either the sequence or the structure similarity. The arrow and the cylinder represent the β-strand and the α-helix, respectively. (B) Comparison of the modeled structure of the catalytic domain of KpnI (a) and the experimentally solved structures of HNH-superfamily nucleases used as modeling templates (b) T4 Endonuclease VII (1en7), (c) colicin E9 (1bxi), (d) intron-encoded homing endonuclease I-PpoI (1ipp) and (e) Serratia marcenscens endonuclease (1g8t). Amino acid residues of the active site and those involved in formation of a Zn-finger in Endonuclease VII and KpnI are indicated and labeled.

Figure 2

Restriction digestion of pUC18 DNA by R.KpnI and its mutants. Purified enzymes (A) R.KpnI (B) D148G (C) H149L and (D) Q175E mutants were incubated with 500 ng of pUC18 DNA and the products were electrophoresed on 1% agarose gel. The concentration of the enzyme used is depicted in each panel. SC, L and OC indicate the respective position of the supercoiled, linear and open circular forms of plasmid.

Figure 3

DNA binding properties of mutant proteins. (A) EMSA analysis: 50 nM of wt and mutant enzymes were incubated with 3.75 nM of ³²P-labeled 20mer duplex oligonucleotides containing recognition sequence (Table 1) under standard assay conditions as described in Materials and Methods. (B) Determination of DNA binding affinities of R.KpnI, D148G and H149L. The assays were carried out as above with the increasing amounts of proteins (0, 0.1, 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 nM) to carry out Scatchard analysis. Lane F in each panel refers to oligonucleotides incubated only with EMSA buffer in the absence of protein.

Figure 4

Effect of pH on DNA cleavage activities of ββα-Me motif mutants of KpnI REase. Increasing amounts of wt, Q175E, H149L and D148G were incubated with pUC18 in buffer with different pH (A) pH 6.0 (B) pH 7.0 (C) pH 8.0 and pH 9.0 (D) at 37°C for 1 h. The samples were electrophoresed through 1% agarose. SC, L and OC indicate the respective positions of the supercoiled, linear and open circular forms of pUC18.

Figure 5

Proposed catalytic mechanism of R.KpnI. Residues D148 and Q175 are shown in this scheme to be involved in metal coordination and H149 acting as a general base. H149 activates the nucleophilic water molecule to facilitate the phosphodiester bond cleavage.

Figure 6

Role of Q175E in DNA binding activity. Different concentrations of Q175E (1, 2.5, 5, 7.5, 10, 15, 20, 25, 50 and 100 nM) were incubated with 3.75 nM of ³²P-labeled 20 bp duplex DNA containing KpnI recognition sequence on ice for 15 min at pH 6.0. The products were analyzed on 8% PAGE as described in Materials and Methods.