Differences between EcoRI nonspecific and "star" sequence complexes revealed by osmotic stress

The binding of the restriction endonuclease EcoRI to DNA is exceptionally specific. Even a single basepair change ("star" sequence) from the recognition sequence, GAATTC, decreases the binding free energy of EcoRI to values nearly indistinguishable from nonspecific binding. The difference in the number of waters sequestered by the protein-DNA complexes of the "star" sequences TAATTC and CAATTC and by the specific sequence complex determined from the dependence of binding free energy on water activity is also practically indistinguishable at low osmotic pressures from the 110 water molecules sequestered by nonspecific sequence complexes. Novel measurements of the dissociation rates of noncognate sequence complexes and competition equilibrium show that sequestered water can be removed from "star" sequence complexes by high osmotic pressure, but not from a nonspecific complex. By 5 Osm, the TAATTC "star" sequence complex has lost almost 90 of the approximately 110 waters initially present. It is more difficult to remove water from the CAATTC "star" sequence complex. The sequence dependence of water loss correlates with the known sequence dependence of "star" cleavage activity.     

Novel subtype of type IIs restriction enzymes. BfiI endonuclease exhibits similarities to the EDTA-resistant nuclease Nuc of Salmonella typhimurium

The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleotide sequence 5'-ACTGGG-3' and cleaves complementary DNA strands 5/4 nucleotides downstream of the recognition sequence. The genes coding for the BfiI restriction-modification (R-M) system were cloned/sequenced and biochemical characterization of BfiI restriction enzyme was performed. The BfiI R-M system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme. Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzyme amino acid sequence revealed intriguing similarities to an EDTA-resistant nuclease of Salmonella typhimurium. Biochemical analyses demonstrated that BfiI, like the nuclease of S. typhimurium, cleaves DNA in the absence of Mg(2+) ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate. However, unlike the nonspecific S. typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically. We propose that the DNA-binding specificity of BfiI stems from the C-terminal part of the protein. The catalytic N-terminal subdomain of BfiI radically differs from that of type II restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S. typhimurium; therefore, BfiI did not require metal ions for catalysis. We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism.     

Trapping DNA-protein binding reactions with neutral osmolytes for the analysis by gel mobility shift and self-cleavage assays

We take advantage of our previous observation that neutral osmolytes can strongly slow down the rate of DNA–protein complex dissociation to develop a method that uses osmotic stress to ‘freeze’ mixtures of DNA–protein complexes and prevent further reaction enabling analysis of the products. We apply this approach to the gel mobility shift assay and use it to modify a self-cleavage assay that uses the nuclease activity of the restriction endonucleases to measure sensitively their specific binding to DNA. At sufficiently high concentrations of neutral osmolytes the cleavage reaction can be triggered at only those DNA fragments with initially bound enzyme. The self-cleavage assay allows measurement of binding equilibrium and kinetics directly in solution avoiding the intrinsic problems of gel mobility shift and filter binding assays while providing the same sensitivity level. Here we compare the self-cleavage and gel mobility shift assays applied to the DNA binding of EcoRI and BamHI restriction endonucleases. Initial results indicate that BamHI dissociation from its specific DNA sequence is strongly linked to water activity with the half-life time of the specific complex increasing ~20-fold from 0 to 1 osmolal betaine.     

Macromolecular hydration changes associated with BamHI binding and catalysis

In this report, the effects of osmotic pressure on BamHI cognate binding and catalysis were investigated and compared with a previous study on EcoRI (Robinson, C. R. and Sligar, S. G. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2186-2191). Our observation of the dependence of binding and catalytic parameters on osmotic pressure has allowed for the comparison of hydration changes associated with site-specific DNA recognition for both endonucleases. Over a large range of osmotic pressures (pi), the dependence of BamHI on osmotic stress during cognate binding and catalysis was very different from that of the related endonuclease EcoRI. The binding of EcoRI to cognate DNA was dominated by a dehydration of the endonuclease-DNA complex, whereas binding by BamHI to its cognate sequence was accompanied by a solvent release corresponding to some 125 fewer waters. Catalytic analysis at elevated osmotic pressures indicated that both endonucleases had undergone a net hydration of the complex with BamHI displaying a much greater dependence on osmotic stress than EcoRI. Although the enzymes shared core structural motifs, comparisons of high resolution x-ray structures revealed many different secondary structural features of the complexed endonucleases. The large difference in hydration changes by both BamHI and EcoRI could be attributed to these dissimilar secondary structural features, as well as the functional differences of the two endonucleases during site-specific DNA recognition.     

A comparative structure-function analysis and molecular mechanism of action of endonucleases from Serratia marcescens and Physarum polycephalum

Structural and functional characteristics were compared for wild-type nuclease from Serratia marcescens, which belongs to the family of DNA/RNA nonspecific endonucleases, its mutational forms, and the nuclease I-PpoI from Physarum polycephalum, which is a representative of the Cys-His box-containing subgroup of the superfamily of extremely specific intron-encoded homing DNases. Despite the lack of sequence homology and the overall different topology of the Serratia marcescens and I-PpoI nucleases, their active sites have a remarkable structural similarity. Both of them have a unique magnesium atom in the active site, which is a part of the coordinatively bonded water-magnesium complex involved in their catalytic acts. In the enzyme-substrate complexes, the Mg2+ ion is chelated by an Asp residue, coordinates two oxygen atoms of DNA, and stabilizes the transition state of the phosphate anion and 3'-OH group of the leaving nucleotide. A new mechanism of the phosphodiester bond cleavage, which is common for the Serratia marcescens and I-PpoI nucleases and differs from the known functioning mechanism of the restriction and homing endonucleases, was proposed. It presumes a His residue as a general base for the activation of a non-cluster water molecule at the nucleophilic in line displacement of the 3'-leaving group. A strained metalloenzyme-substrate complex is formed during hydrolysis and relaxes to the initial state after the reaction. The English version of the paper.     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies.

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.     

Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

The DNA binding stringency of restriction endonucleases is crucial for their proper function. The X-ray structures of the specific and non-cognate complexes of the restriction nuclease EcoRV are considerably different suggesting significant differences in the hydration and binding free energies. Nonetheless, the majority of studies performed at pH 7.5, optimal for enzymatic activity, have found a < 10-fold difference between EcoRV binding constants to the specific and nonspecific sequences in the absence of divalent ions. We used a recently developed self-cleavage assay to measure EcoRV-DNA competitive binding and to evaluate the influence of water activity, pH and salt concentration on the binding stringency of the enzyme in the absence of divalent ions. We find the enzyme can readily distinguish specific and nonspecific sequences. The relative specific-nonspecific binding constant increases strongly with increasing neutral solute concentration and with decreasing pH. The difference in number of associated waters between specific and nonspecific DNA-EcoRV complexes is consistent with the differences in the crystal structures. Despite the large pH dependence of the sequence specificity, the osmotic pressure dependence indicates little change in structure with pH. The large osmotic pressure dependence means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowded environment of the cell. In addition to divalent ions, water activity and pH are key parameters that strongly modulate binding specificity of EcoRV.     

Crystal structures of type II restriction endonuclease EcoO109I and its complex with cognate DNA

EcoO109I is a type II restriction endonuclease that recognizes the DNA sequence of RGGNCCY. Here we describe the crystal structures of EcoO109I and its complex with DNA. A comparison of the two structures shows that the catalytic domain moves drastically to capture the DNA. One metal ion and two water molecules are observed near the active site of the DNA complex. The metal ion is a Lewis acid that stabilizes the pentavalent phosphorus atom in the transition state. One water molecule, activated by Lys-126, attacks the phosphorus atom in an S(N)2 mechanism, whereas the other water interacts with the 3'-leaving oxygen to donate a proton to the oxygen. EcoO109I is similar to EcoRI family enzymes in terms of its DNA cleavage pattern and folding topology of the common motif in the catalytic domain, but it differs in the manner of DNA recognition. Our findings propose a novel classification of the type II restriction endonucleases and lead to the suggestion that EcoO109I represents a new subclass of the EcoRI family.     

Differences in hydration coupled to specific and nonspecific competitive binding and to specific DNA Binding of the restriction endonuclease BamHI

Using the osmotic stress technique together with a self-cleavage assay we measure directly differences in sequestered water between specific and nonspecific DNA-BamHI complexes as well as the numbers of water molecules released coupled to specific complex formation. The difference between specific and nonspecific binding free energy of the BamHI scales linearly with solute osmolal concentration for seven neutral solutes used to set water activity. The observed osmotic dependence indicates that the nonspecific DNA-BamHI complex sequesters some 120-150 more water molecules than the specific complex. The weak sensitivity of the difference in number of waters to the solute identity suggests that these waters are sterically inaccessible to solutes. This result is in close agreement with differences in the structures determined by x-ray crystallography. We demonstrate additionally that when the same solutes that were used in competition experiments are used to probe changes accompanying the binding of free BamHI to its specific DNA sequence, the measured number of water molecules released in the binding process is strikingly solute-dependent (with up to 10-fold difference between solutes). This result is expected for reactions resulting in a large change in a surface exposed area.     

Diffusion of the Restriction Nuclease EcoRI along DNA

Many specific sequence DNA binding proteins locate their target sequence by first binding to DNA nonspecifically, then by linearly diffusing or hopping along DNA until either the protein dissociates from the DNA or it finds the recognition sequence. We have devised a method for measuring one-dimensional diffusion along DNA based on the ratio of the dissociation rate of protein from DNA fragments containing one specific binding site to the dissociation rate from DNA fragments containing two specific binding sites. Our extensive measurements of dissociation rates and specific-nonspecific relative binding constants of the restriction nuclease EcoRI enable us to determine the diffusion rate of nonspecifically bound protein along the DNA. By varying the distance between the two binding sites, we confirm a linear diffusion mechanism. The sliding rate is relatively insensitive to salt concentration and osmotic pressure, indicating that the protein moves smoothly along the DNA probably following the helical phosphate-sugar backbone of DNA. We calculate a diffusion coefficient for EcoRI of 3 × 10⁴ bp² s^− 1 EcoRI is able to diffuse ∼ 150 bp, on average, along the DNA in 1 s. This diffusion rate is about 2000-fold slower than the diffusion of free protein in solution. A factor of 40-50 can be accounted for by rotational friction resulting from following the helical path of the DNA backbone. Two possibilities could account for the remaining activation energy: salt bridges between the DNA and the protein are transiently broken, or the water structure at the protein-DNA interface is disrupted as the two surfaces move past each other.     

Dynamic and Structural Changes in the Minimally Restructuring EcoRI Bound to a Minimally Mutated DNA Chain

The dynamics of a protein plays an important role in protein functionality. Here, we examine the differences in the dynamics of a minimally restructuring protein, EcoRI, when it is bound to its cognate DNA and to a noncognate sequence which differs by just a single basepair. Molecular dynamics simulations of the complexes and essential dynamics analyses reveal that the overall dynamics of the protein subunits change from a coordinated motion in the cognate complex to a scrambled motion in the noncognate complex. This dynamical difference extends to the protein-DNA interface where EcoRI tries to constrict the DNA in the cognate complex. In the noncognate complex, absence of the constricting motion of interfacial residues, overall change in backbone dynamics and structural relaxation of the arms enfolding the DNA leave the DNA less-kinked relative to the situation in the cognate complex, thus indicating that the protein is poised for linear diffusion along the DNA rather than for catalytic action. In a larger context, the results imply that the DNA sequences dictate protein dynamics and that when a protein chances upon the recognition sequence some of the key domains of the protein undergo dynamical changes that prepare the protein for eventual catalytic action.     

Dynamic and Structural Changes in the Minimally Restructuring EcoRI Bound to a Minimally Mutated DNA Chain

Abstract

The dynamics of a protein plays an important role in protein functionality. Here, we examine the differences in the dynamics of a minimally restructuring protein, EcoRI, when it is bound to its cognate DNA and to a noncognate sequence which differs by just a single basepair. Molecular dynamics simulations of the complexes and essential dynamics analyses reveal that the overall dynamics of the protein subunits change from a coordinated motion in the cognate complex to a scrambled motion in the noncognate complex. This dynamical difference extends to the protein-DNA interface where EcoRI tries to constrict the DNA in the cognate complex. In the noncognate complex, absence of the constricting motion of interfacial residues, overall change in backbone dynamics and structural relaxation of the arms enfolding the DNA leave the DNA less-kinked relative to the situation in the cognate complex, thus indicating that the protein is poised for linear diffusion along the DNA rather than for catalytic action. In a larger context, the results imply that the DNA sequences dictate protein dynamics and that when a protein chances upon the recognition sequence some of the key domains of the protein undergo dynamical changes that prepare the protein for eventual catalytic action.     

The specific binding, bending, and unwinding of DNA by RsrI endonuclease, an isoschizomer of EcoRI endonuclease

To determine whether RsrI endonuclease recognizes and cleaves the sequence GAATTC in duplex DNA similarly to its isoschizomer EcoRI we initiated a functional comparison of the two enzymes. Equilibrium binding experiments showed that at 20 degrees C RsrI endonuclease binds to specific and nonspecific sequences in DNA with affinities similar to those of EcoRI. At 0 degrees C the affinity of RsrI for its specific recognition sequence is reduced 7-fold whereas the affinity for noncanonical sequences remains relatively unchanged. Unlike EcoRI, incubation of RsrI endonuclease with N-ethylmaleimide inactivates the enzyme; however, preincubation with DNA prevents the inactivation. The N-ethylmaleimide-treated enzyme fails to bind DNA as assayed by gel mobility shift assays. Comparison of the deduced amino acid sequences of RsrI and EcoRI endonucleases suggests that modification of Cys245 is responsible for the inactivation. Fe(II). EDTA and methidiumpropyl-EDTA.Fe(II) footprinting results indicate that RsrI, like EcoRI, protects 12 base pairs from cleavage when bound to its specific recognition sequence in the absence of Mg2+. RsrI bends DNA by approximately 50 degrees, as determined by measuring the relative electrophoretic mobilities of specific RsrI-DNA complexes with the binding site in the center or near the end of the DNA fragment. This value is similar to that reported for EcoRI. RsrI also unwinds the DNA helix by 25 degrees +/- 5 degrees, a value close to that reported for EcoRI endonuclease. Collectively, these results indicate that the overall structural changes induced in the DNA by the binding of RsrI and EcoRI endonucleases to DNA in the absence of Mg2+ are similar. In the accompanying paper (Aiken, C. R., McLaughlin, L. W., and Gumport, R. I. (1991) J. Biol. Chem. 266, 19070-19078) we present results of studies of RsrI endonuclease using oligonucleotide substrates containing base analogues which suggest differences in the ways the two enzymes cleave DNA.     

EcoRV restriction endonuclease binds all DNA sequences with equal affinity

In the presence of MgCl2, the EcoRV restriction endonuclease cleaves its recognition sequence on DNA at least a million times more readily than any other sequence. In this study, the binding of the EcoRV restriction enzyme to DNA was examined in the absence of Mg2+. With each DNA fragment tested, several DNA-protein complexes were detected by electrophoresis through polyacrylamide. No differences were observed between isogenic DNA molecules that either contained or lacked the EcoRV recognition site. The number of complexes with each fragment varied with the length of the DNA. Three complexes were formed with a DNA molecule of 55 base pairs, corresponding to the DNA bound to 1, 2, or 3 molecules of the protein, while greater than 15 complexes were formed with a DNA of 381 base pairs. A new method was developed to analyze the binding of a protein to multiple sites on DNA. The method showed that the EcoRV enzyme binds to all DNA sequences, including the EcoRV recognition site, with the same equilibrium constant, though two molecules of the protein bind preferentially to adjacent sites on the DNA in a cooperative fashion. All of the complexes with a substrate that contained the EcoRV site dissociated upon addition of competitor DNA, but when the competitor was mixed with MgCl2, a fraction of the substrate was cleaved at the EcoRV site. The fraction cleaved was due mainly to the translocation of the enzyme from nonspecific sites on the DNA to the specific site.     

Structural and functional insight into sugar-nonspecific nucleases in host defense

In prokaryotes, sugar-nonspecific nucleases that cleave DNA and RNA in a sequence-independent manner take part in host defense, as well as site-specific restriction enzymes. Examples include the periplasmic nuclease Vvn and the secreted nuclease ColE7, which degrade foreign nucleic acid molecules in the host periplasm and in the cytoplasm of foreign cells, respectively. Recently determined crystal structures of Vvn and ColE7 in complex with double-stranded DNA provide structural insight into nonspecific DNA interactions and cleavage by sugar-nonspecific nucleases. Both nucleases bind DNA at the minor groove through a common 'betabetaalpha-metal' endonuclease motif and primarily contact the DNA phosphate backbone, probably to avoid sequence-dependent base recognition. In eukaryotes, several apoptotic endonucleases that are responsible for DNA degradation in programmed cell death also contain a betabetaalpha-metal fold at the active site, suggesting that they may recognize and cleave DNA in a comparable way.     

Facilitated diffusion during catalysis by EcoRI endonuclease. Nonspecific interactions in EcoRI catalysis

The potential for processive EcoRI endonuclease hydrolysis has been examined on several DNA substrates containing two EcoRI sites which were embedded in identical sequence environments. With a 388-base pair circular DNA, in which the two recognition sites are separated by 51 base pairs (shorter distance) or 337 base pairs (longer distance), 77 and 34% of all events involved processive hydrolysis at ionic strengths of 0.059 and 0.13, respectively. However, the frequency of processive action on linear substrates, in which the two sites were separated by 51 base pairs, was only 42 and 17% at these ionic strengths, values half those observed with the circular DNA. Processive action was not detectable on circular or linear substrates at an ionic strength of 0.23. These findings indicate that DNA search by the endonuclease occurs by facilitated diffusion, a mechanism in which the protein locates and leaves its recognition sequence by interacting with nonspecific DNA sites. We suggest that processivity on linear substrates is limited to values half that for small circles due to partitioning of the enzyme between the two products generated by cleavage of a linear molecule. Given such topological effects, measured processivity values imply that the endonuclease can diffuse within a DNA domain to locate and recognize an EcoRI site 50 to 300 base pairs distant from an initial binding site, with minimum search efficiencies being 80 and 30% at ionic strengths of 0.059 and 0.13, respectively. The high efficiency of processive action indicates that a positionally correlated mode of search plays a major role in facilitated diffusion in this system under such conditions. Also consistent with this view was the identification of a striking position effect when two closely spaced EcoRI sites were asymmetrically positioned near the end of a linear DNA. The endonuclease displays a substantial preference for the more centrally located recognition sequence. This preference does not reflect differential sensitivity of the two sites to cleavage per se, but can be simply explained by preferential entry of the enzyme via the larger nonspecific target available to the more centrally positioned recognition sequence. These conclusions differ from those of a previous qualitative analysis of endonuclease processivity over short distances (Langowski, J., Alves, J., Pingoud, A., and Maass, G. (1983) Nucleic Acids Res. 11, 501-513).     

Interfacial water as a "hydration fingerprint" in the noncognate complex of BamHI

The molecular code of specific DNA recognition by proteins as a paradigm in molecular biology remains an unsolved puzzle primarily because of the subtle interplay between direct protein-DNA interaction and the indirect contribution from water and ions. Transformation of the nonspecific, low affinity complex to a specific, high affinity complex is accompanied by the release of interfacial water molecules. To provide insight into the conversion from the loose to the tight form, we characterized the structure and energetics of water at the protein-DNA interface of the BamHI complex with a noncognate sequence and in the specific complex. The fully hydrated models were produced with Grand Canonical Monte Carlo simulations. Proximity analysis shows that water distributions exhibit sequence dependent variations in both complexes and, in particular, in the noncognate complex they discriminate between the correct and the star site. Variations in water distributions control the number of water molecules released from a given sequence upon transformation from the loose to the tight complex as well as the local entropy contribution to the binding free energy. We propose that interfacial waters can serve as a "hydration fingerprint" of a given DNA sequence.     

A Comparative Structure–Function Analysis and Molecular Mechanism of Action of Endonucleases from Serratia marcescens and Physarum polycephalum

Structural and functional characteristics were compared for wild-type nuclease from Serratia marcescens, which belongs to the family of DNA/RNA nonspecific endonucleases, its mutational forms, and the nuclease I-PpoI from Physarum polycephalum, which is a representative of the Cys-His box-containing subgroup of the superfamily of extremely specific intron-encoded homing DNases. Despite the lack of sequence homology and the overall different topology of the Serratia marcescens and I-PpoI nucleases, their active sites have a remarkable structural similarity. Both of them have a unique magnesium atom in the active site, which is a part of the coordinatively bonded water–magnesium complex involved in their catalytic acts. In the enzyme–substrate complexes, the Mg²⁺ ion is chelated by an Asp residue, coordinates two oxygen atoms of DNA, and stabilizes the transition state of the phosphate anion and 3"-OH group of the leaving nucleotide. A new mechanism of the phosphodiester bond cleavage, which is common for the Serratia marcescens and I-PpoI nucleases and differs from the known functioning mechanism of the restriction and homing endonucleases, was proposed. It presumes a His residue as a general base for the activation of a non-cluster water molecule at the nucleophilic in line displacement of the 3"-leaving group. A strained metalloenzyme–substrate complex is formed during hydrolysis and relaxes to the initial state after the reaction.     

Crystallization of restriction endonuclease BamHI with nonspecific DNA

Restriction endonucleases show extraordinary specificity in distinguishing specific from nonspecific DNA sequences. A single basepair change within the recognition sequence results in over a million-fold loss in activity. To understand the basis of this sequence discrimination, it is just as important to study the nonspecific complex as the specific complex. We describe here the crystallization of restriction endonuclease BamHI with several nonspecific oligonucleotides. The 11-mer, 5'-ATGAATCCATA-3', yielded cocrystals with BamHI, in the presence of low salt, that diffracted to 1.9 A with synchrotron radiation. The cocrystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 114.8 A, b = 91.1 A, c = 66.4 A, alpha = 90 degrees, beta = 90 degrees, gamma = 90 degrees. This success in the cocrystallization of BamHI with a nonspecific DNA provides insights for future attempts at crystallization of other nonspecific DNA-protein complexes.     

Mva1269I: a monomeric type IIS restriction endonuclease from Micrococcus varians with two EcoRI- and FokI-like catalytic domains

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN / -3'/5'-NG / CATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "/" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two "PD-(D/E)XK" domains. The N-terminal domain is related to the 5'-GAATTC-3'-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.