Ca2+-mediated Site-specific DNA Cleavage and Suppression of Promiscuous Activity of KpnI Restriction Endonuclease*

The characteristic feature of type II restriction endonucleases (REases) is their exquisite sequence specificity and obligate Mg2+ requirement for catalysis. Efficient cleavage of DNA only in the presence of Ca2+ ions, comparable with that of Mg2+, is previously not described. Most intriguingly, KpnI REase exhibits Ca2+-dependent specific DNA cleavage. Moreover, the enzyme is highly promiscuous in its cleavage pattern on plasmid DNAs in the presence of Mn2+ or Mg2+, with the complete suppression of promiscuous activity in the presence of Ca2+. KpnI methyltransferase does not exhibit promiscuous activity unlike its cognate REase. The REase binds to oligonucleotides containing canonical and mapped noncanonical sites with comparable affinities. However, the extent of cleavage is varied depending on the metal ion and the sequence. The ability of the enzyme to be promiscuous or specific may reflect an evolutionary design. Based on the results, we suggest that the enzyme KpnI represents an REase evolving to attain higher sequence specificity from an ancient nonspecific nuclease.

Type II REases recognize specific DNA sequences, which vary from 4 to 8 bp and cleave both the strands in Mg 2ϩ -dependent reaction. They exhibit remarkable features in discriminating between specific and nonspecific DNA, not only in DNA binding but also during DNA cleavage (1). Based on the divalent metal ion requirement for DNA binding, REases have been classified into two groups (2). For example, EcoRI, BamHI, RsrI, and a few other enzymes bind to DNA preferentially at their recognition sites in the absence of divalent metal ions (3)(4)(5). In contrast, EcoRV, PvuII, TaqI, and a few other enzymes bind to cognate sequences in a divalent metal ion-dependent manner (6 -8). However, both the groups of enzymes need Mg 2ϩ for catalysis. The role of metal ions in DNA binding and the subsequent hydrolysis of the phosphodiester bond at the recognition site have been studied in detail for several REases. 1 Although the natural cofactor for all type II REases is Mg 2ϩ , Ca 2ϩ can replace Mg 2ϩ for specific DNA binding, with-out leading to the formation of productive complex (9). Some type II REases form stable protein-DNA complexes in the presence of Ca 2ϩ but do not support the DNA cleavage even though Ca 2ϩ is a nearly perfect analogue for Mg 2ϩ (10). Ca 2ϩ also increases the binding specificity of several REases such as MunI, TaqI, and Cfr10I toward their cognate sites (9,11).
Several type II REases show reduced sequence specificity at suboptimal reaction conditions such as low ionic strength, elevated pH, in the presence of water-miscible solvents, and when Mg 2ϩ is substituted with Mn 2ϩ . This relaxed specificity was first observed for EcoRI REase and termed as star activity (12). Subsequently, relaxed specificity has been characterized for several REases such as BamHI (13), DdeI (14), EcoRV (15), PvuII (16), and RsrI (17).
KpnI REase isolated from Klebsiella pneumoniae recognizes the palindromic double-stranded DNA sequence 5Ј-GGTAC2C-3Ј and cleaves DNA by generating a 3Ј, 4-base overhang (18). The enzyme binds to specific DNA in the absence of divalent metal ions (19). In this paper, we describe the highly promiscuous cleavage of DNA in the presence of Mg 2ϩ and Mn 2ϩ . The promiscuous activity is completely suppressed in the presence of Ca 2ϩ , which induces site-specific DNA cleavage.

EXPERIMENTAL PROCEDURES
Enzymes and DNA-KpnI REase was purified as described previously (18). The enzyme was diluted in binding buffer (20 mM Tris-HCl (pH 7.4), 25 mM NaCl, and 5 mM 2-mercaptoethanol) for all the studies. The concentration of the enzyme was estimated by the method of Bradford (20). One unit of KpnI REase is defined as the amount of enzyme required for complete digestion of 1 g of DNA at 37°C for 1 h by using assay buffer containing 5 mM Mg 2ϩ . T4 polynucleotide kinase and Klenow polymerase were purchased from New England Biolabs. Oligonucleotides were from Microsynth Inc., Switzerland, and purified on 18% urea-polyacrylamide gel (21). The purified oligonucleotides were end-labeled with T4 polynucleotide kinase and [␥-32 P]ATP (6000 Ci/ mmol). Pfu and Taq DNA polymerases, pUC18 and pBR322, were obtained from Bangalore Genei Pvt. Ltd., India. The plasmid clones used for primer extension studies were from the laboratory collections.
REase Digestion-Standard assay conditions involved lower enzyme units to DNA ratio (up to 10 units of enzyme), whereas in relaxed assay conditions more than 15 units of enzyme were used. Digestions were carried out by incubating different units of KpnI REase with plasmid DNA or 0.2 pmol of labeled oligonucleotides in assay buffer containing 10 mM Tris-HCl (pH 7.4), 5 mM ␤-mercaptoethanol, and appropriate concentrations of divalent metal ions at 37°C for 1 h. The reactions were terminated by adding stop dye containing 0.6% SDS and 25 mM EDTA. The cleavage products of plasmid DNA and oligonucleotides were analyzed on 1% agarose or 12% urea-polyacrylamide gel, respectively. The Ca 2ϩ chase reactions were carried out at a fixed concentration of Mg 2ϩ (2 mM) or Mn 2ϩ (0.5 mM) by using increasing concentrations of Ca 2ϩ .
Primer Extension Analysis-The different plasmids (2 g) were incubated with 20 units of KpnI REase in a buffer containing 20 mM Tris-HCl (pH 7.4), 5 mM 2-mercaptoethanol, and 5 mM Mn 2ϩ at 37°C for * This work was supported by the Department of Science and Technology, Government of India. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ʈ To whom correspondence should be addressed. Tel.: 91-80-23600668; Fax: 91-80-23602697; E-mail: vraj@mcbl.iisc.ernet.in. 1 The abbreviations used are: REase, restriction endonuclease; MTase, methyltransferase.
1 h. The digested plasmids were purified by phenol/chloroform extraction, and end-labeled primers were used for primer extension reaction as described by Balke et al. (22). The cleavage sites were mapped by standard dideoxy sequencing reactions using TaqDNA polymerase (23).
DNA Binding Assay-Different concentrations of KpnI REase (12-1200 nM for noncanonical sites; 3-500 nM KpnI for cognate sites) were incubated with 3.75 nM of end-labeled, double-stranded oligonucleotides containing cognate and noncognate sites in the buffer (20 mM Tris-HCl (pH 7.4), 25 mM NaCl, and 5 mM 2-mercaptoethanol) on ice for 15 min. The free DNA and enzyme-bound complexes were separated on 8% native polyacrylamide gel and autoradiographed. The amounts of DNA in free and bound form were quantitated using PhosphorImager, and the results were analyzed by Scatchard plot.

Metal Ion-dependent Cleavage Reactions-
The effect of divalent metal ions on the activity of KpnI REase was studied by using plasmid DNA as substrates. KpnI REase exhibits relaxed specificity at high enzyme to substrate ratios. A high degree of promiscuous activity was observed when more than 15 units of the enzyme was used in the presence of Mg 2ϩ (Fig. 1A). The promiscuous activity was further enhanced in the presence of Mn 2ϩ as compared with Mg 2ϩ under similar reaction conditions (Fig. 1B). This activity of the enzyme was observed at a range of Mg 2ϩ (1-5 mM) or Mn 2ϩ (0.5-5 mM) concentrations and was more pronounced than the conventional star activity exhibited by several type II REases. Most surprisingly, unlike other type II REases, KpnI REase exhibits specific DNA cleavage in presence of Ca 2ϩ (Fig. 1C) showing greater specificity even at high enzyme to substrate ratios in contrast to the cleavage pattern with Mg 2ϩ and Mn 2ϩ . The Ca 2ϩ -mediated high fidelity, efficient DNA cleavage is a unique property of KpnI REase. In the presence of Mg 2ϩ or Mn 2ϩ , the enzyme exhibited promiscuous cleavage with pBR322 that lacks the specific KpnI recognition sequence (supplemental Fig. 1).
KpnI MTase Does Not Exhibit Promiscuous Behavior-In order to assess the promiscuous activity of KpnI on methylated DNA and to evaluate whether the MTase exhibits promiscuous behavior, both pUC18 and pUC⌬K (pUC⌬K was generated by deleting the KpnI site from pUC18 by digesting with EcoRI and HindIII) were isolated from Escherichia coli DH10B harboring KpnI MTase expressing plasmid, pACMK (18). The methylated pUC18 DNA was not cleaved at a canonical site by the KpnI REase under standard assay conditions (1-10 units of enzyme). However, under relaxed conditions both pUC18 and pUC⌬K DNA isolated from KpnI MTase expressing cells exhibited promiscuous cleavage patterns by KpnI REase in the presence of Mg 2؉ (Fig. 1, D and E). From these results, we infer that the MTase does not exhibit promiscuous activity as only the cognate site was methylated. Furthermore, the modification of the cognate site has no influence on the promiscuous activity of the REase. Relaxed cleavage specificity was observed only in the presence of either Mg 2ϩ or Mn 2ϩ , and the modified DNA substrates were refractile to cleavage by KpnI REase in the presence of Ca 2ϩ .
Suppression of Promiscuous Activity-It has been shown by a large number of biochemical and structural studies with several type II REases that Mg 2ϩ and Ca 2ϩ occupy the same pocket in the active site (24 -26). High fidelity cleavage in the presence of Ca 2ϩ , irrespective of the enzyme concentrations and robust promiscuity at low concentrations of the enzyme in the presence of low amounts of Mg 2ϩ (2 mM) and Mn 2ϩ (0.5 mM), prompted us to carry out competition experiments between the different metal ions. In order to assess the effect of Ca 2ϩ on Mg 2ϩ -and Mn 2ϩ -dependent promiscuous activity of KpnI REase, Ca 2ϩ chase experiments were carried on Mg 2ϩ -or Mn 2ϩ -bound enzyme-DNA complex under relaxed conditions. The Mg 2ϩ -or Mn 2ϩ -mediated promiscuous activity was completely suppressed by an increase in Ca 2ϩ ion concentrations (Fig. 2, A and B). In a converse experiment with fixed amounts of Ca 2ϩ (2 mM) and increasing concentrations of Mg 2ϩ or Mn 2ϩ , the promiscuous activity of the enzyme was restored (data not shown). These observations indicate that Mg 2ϩ , Mn 2ϩ , and Ca 2ϩ compete with each other to occupy the active site of the enzyme.
Binding and Cleavage Analysis of KpnI REase with Oligonucleotide Containing Noncanonical Sites-Primer extension strategy was used to map the noncanonical sites, and the representative data are shown in the supplemental Fig. 2. Analysis of the data obtained from primer extension products of different plasmid substrates revealed that many hexameric sequences with one nucleotide change from the canonical sequence are efficient cleavage sites. The sequences that have been mapped include tGTACC, GtTACC, GaTACC, GGaACC, GGTcCC, GGTAtC, GGTACg, and GGTACt, where the lowercase letter indicates noncanonical nucleotide. The cleavage position within each of these noncanonical recognition sequences was identical to that observed with the canonical sequence. The relaxation in the specificity of KpnI occurred at every nucleotide position within the hexameric recognition sequence.
To study the DNA binding properties of KpnI at noncanonical sites, several oligonucleotides were designed based on the results from the primer extension data (Table I). Doublestranded oligonucleotides containing different mapped sites with one of the strands labeled at the 5Ј end were used as substrates for binding studies. Electrophoretic mobility shift assays were carried out in the absence of divalent metal ions, and the representative results are shown in Fig. 3. KpnI REase formed a single protein-DNA complex with oligonucleotide containing GGTACC (canonical site) and GGaACC and GGatCC (noncanonical sites) sequences (Fig. 3, A, C and D). All other noncanonical sequence containing oligonucleotides described above also formed similar complexes (data not shown). Scatchard analysis of the binding data revealed that the affinity of the enzyme to the various oligonucleotides varied from 8 to 34 nM (Table II). KpnI REase failed to show a stable complex with a completely nonspecific oligonucleotide (Fig. 3B) as observed earlier (19).
Cleavage reactions were carried out using double-stranded oligonucleotides in the presence of Mg 2ϩ or Mn 2ϩ as a cofactor. KpnI REase cleaves oligonucleotides containing various noncanonical sites having single nucleotide substitution, albeit less efficiently when compared with the cleavage with oligonucleotides containing canonical sequence (Fig. 4A). Noncanonical sites GtTACC, GaTACC, and GGTcCC were preferred over GGaACC, tGTACC, and GGTACg (Fig. 4A). No DNA cleavage was detected at the noncanonical sites in the presence of Ca 2ϩ even at a high enzyme to substrate ratio (Fig. 4B). The Ca 2ϩmediated DNA cleavage is confined to the canonical site irrespective of plasmid or oligonucleotide substrates. DISCUSSION DNA-binding enzymes involved in phosphoryl transfer reactions generally require divalent metal ions to promote their Nonspecific oligonucleotide 5Ј-GAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCT-3 5Ј-AGGAAGCGGAAGACGCCCAATACGCAAACCGCCTCTC-3Ј activity. In most cases Mg 2ϩ is used as a cofactor. The preference for Mg 2ϩ over other metal ions is probably due to its favorable physical and chemical properties, such as a small ionic radius with extensive hydration, high charge density, high transport numbers, and its abundance in the cell (27). In addition to DNA cleavage, Mg 2ϩ is also required for specific binding of several type II REases to their recognition sequences. Divalent metal ion Ca 2ϩ can substitute Mg 2ϩ for DNA binding (9). The comparative DNA binding studies revealed that EcoRV exhibits a greater degree of specificity in the presence of Ca 2ϩ for the canonical site (28). Binding of several other REases to their recognition sites is enhanced in the presence of Ca 2ϩ (8,9,11). Thus, substitution of Ca 2ϩ to Mg 2ϩ in DNA binding studies indicates that these two metal ions occupy the same DNA-binding pocket. Tertiary structures of several REases reveal that Ca 2ϩ indeed occupies the same position as that of Mg 2ϩ in the active site (10, 24 -26, 29, 30).
KpnI REase binds to cognate DNA sequence in the absence of metal ion cofactor (19). The exquisite cleavage specificity in the presence of Ca 2ϩ and suppression of Mg 2ϩ /Mn 2ϩ -mediated promiscuous activity by Ca 2ϩ are unique characteristics of KpnI REase. This suppression could probably be due to conformational changes at the active site of the protein leading to the formation of inactive or unstable complex at the noncanonical site ceasing DNA cleavage in the presence of Ca 2ϩ . The metal ion competition experiments of EcoRI and EcoRV showed that the Mg 2ϩ -dependent endonuclease activity was inhibited in the presence of Ca 2ϩ . However, in a Mn 2ϩ -dependent reaction, EcoRV showed enhanced activity by Ca 2ϩ (9). In these two well studied enzymes, Ca 2ϩ by itself does not induce DNA cleavage. The ionic radius of Ca 2ϩ is larger compared with Mg 2ϩ and Mn 2ϩ which favors different coordination geometry. The bulk effect and different coordination chemistry of Ca 2ϩ may cause steric interference and destabilize the bound oxygens that could perturb the transition state rather than the initial binding to the active site. The change in solvation at the interface of the enzyme-DNA complex is also known to play an important role in site-specific recognition. Influence of bound water molecules on site-specific recognition and cleavage of DNA was studied for several REases (31,32). In the case of Mg 2ϩ -mediated phosphodiester hydrolysis, an outer-sphere pathway that requires a solvated metal cofactor is favored as Mg 2ϩ maintains a moderate to high hydration state. According to this mecha-nism the metal cofactor is involved in stabilizing the transition state, either electrostatically and/or through hydrogen bonding from the metal-bound water molecules. The solvation state of the metal cofactor is of critical importance in site-specific DNA recognition by these enzymes. Ca 2ϩ -bound KpnI REase with fewer bound water molecules at the active site could be responsible for higher specificity in DNA recognition and cleavage than the Mg 2ϩ /Mn 2ϩ -bound enzyme.
The Ca 2ϩ -dependent cleavage specificity of KpnI REase could probably be due to the involvement of additional residues neighboring or distal to the active site influencing the stabilization of the KpnI-Ca 2ϩ -DNA transition state. Delineation of the KpnI active site architecture may open up the possibilities to engineer other type II REases for specific cleavage, resulting in high fidelity REases devoid of any promiscuous activity.
The ability of REases to recognize a wide range of sequences having one or two nucleotide changes in the recognition sequence is of evolutionary significance. The R-M systems appear to have evolved from two totally unrelated genes such as a nonspecific nuclease and a possible repair protein, which acquired an MTase activity (33,34). While evolving to recognize the specific sequence, some of the REases would have been evolutionarily stuck at an earlier step to retain broader cleavage specificity. KpnI REase seems to represent one such system. Under most physiological conditions, the enzyme is unlikely to express the promiscuous behavior as it exhibits remarkable specificity in the presence of Ca 2ϩ . However, the promiscuity of the enzyme could be advantageous to the bacteria if allowed to be expressed under certain circumstances especially when encountering the invasion by foreign genomes.