Discrimination between DNA sequences by the EcoRV restriction endonuclease

The EcoRV restriction endonuclease cleaves not only its recognition sequence on DNA, GATATC, but also, at vastly reduced rates, a number of alternative DNA sequences. The plasmid pAT153 contains 12 alternative sites, each of which differs from the recognition sequence by one base pair. The EcoRV nuclease showed a marked preference for one particular site from among these alternatives. This noncognate site was located at the sequence GTTATC, and the mechanism of action of EcoRV at this site was analyzed. The mechanism differed from that at the cognate site in three respects. First, the affinity of the enzyme for the noncognate site was lower than that for the cognate site, but, by itself, this cannot account for the specificity of EcoRV as measured from the values of kcat/Km. Second, the enzyme had a lower affinity for Mg2+ when it was bound to the noncognate site than when it was bound to its cognate site: this appears to be a key factor in limiting the rates of DNA cleavage at alternative sites. Third, the reaction pathway at the noncognate site differed from that at the cognate site. At the former, the EcoRV enzyme cleaved first one strand of the DNA and then the other while at the latter, both strands were cut in one concerted reaction. The difference in reaction pathway allows DNA ligase to proofread the activity of EcoRV by selective repair of single-strand breaks at noncognate sites, as opposed to double-strand breaks at the cognate site. The addition of DNA ligase to reactions with EcoRV made no difference to product formation at the cognate site, but products from reactions at noncognate sites were no longer detected.     

Fidelity of DNA recognition by the EcoRV restriction/modification system in vivo

The EcoRV restriction/modification system consists of two enzymes that recognize the DNA sequence GATATC. The EcoRV restriction endonuclease cleaves DNA at this site, but the DNA of Escherichia coli carrying the EcoRV system is protected from this reaction by the EcoRV methyltransferase. However, in vitro, the EcoRV nuclease also cleaves DNA at most sites that differ from the recognition sequence by one base pair. Though the reaction of the nuclease at these sites is much slower than that at the cognate site, it still appears to be fast enough to cleave the chromosome of the cell into many fragments. The possibility that the EcoRV methyltransferase also protects the noncognate sites on the chromosome was examined. The modification enzyme methylated alternate sites in vivo, but these were not the same as the alternate sites for the nuclease. The excess methylation was found at GATC sequences, which are also the targets for the dam methyltransferase of E. coli, a protein that is homologous to the EcoRV methyltransferase. Methylation at these sites gave virtually no protection against the EcoRV nuclease: even when the EcoRV methyltransferase had been overproduced, the cellular DNA remained sensitive to the EcoRV nuclease at its noncognate sites. The viability of E. coli carrying the EcoRV restriction/modification system was found instead to depend on the activity of DNA ligase. Ligase appears to proofread the EcoRV R/M system in vivo: DNA, cut initially in one strand at a noncognate site for the nuclease, is presumably repaired by ligase before the scission of the second strand.     

EcoRV restriction endonuclease: communication between catalytic metal ions and DNA recognition.

In the absence of magnesium ions, the EcoRV restriction endonuclease binds all DNA sequences with equal affinity but cannot cleave DNA. In the presence of Mg2+, the EcoRV endonuclease cleaves DNA at one particular sequence, GATATC, at least a million times more readily than any other sequence. To elucidate the role of the metal ion, the reactions of the EcoRV restriction enzyme were studied in the presence of MnCl2 instead of MgCl2. The reaction at the EcoRV recognition site was slower with Mn2+. This was caused partly by reduced rates for phosphodiester hydrolysis but also by the translocation of the enzyme along the DNA after cleaving it in one strand. In contrast, alternative sites that differ from the recognition site by one base pair were cleaved faster in the presence of Mn2+ relative to Mg2+. When located at an alternative site on the DNA, the EcoRV enzyme bound Mn2+ ions readily but had a very low affinity for Mg2+. The EcoRV nuclease is thus restrained from cleaving DNA at alternate sites in the presence of Mg2+, but the restraint fails to operate with Mn2+. A discrimination factor, which measures the ratio of the activity of the EcoRV nuclease at its recognition site over that at an alternative site, had values of 3 x 10(5) in MgCl2 and 6 in MnCl2.     

Molecular enzymology of the EcoRV DNA-(Adenine-N (6))-methyltransferase: kinetics of DNA binding and bending, kinetic mechanism and linear diffusion of the enzyme on DNA

The EcoRV DNA-(adenine-N(6))-methyltransferase recognizes GATATC sequences and modifies the first adenine residue within this site. We show here, that the enzyme binds to the DNA and the cofactor S-adenosylmethionine (AdoMet) in an ordered bi-bi fashion, with AdoMet being bound first. M.EcoRV binds DNA in a non-specific manner and the enzyme searches for its recognition site by linear diffusion with a range of approximately 1800 bp. During linear diffusion the enzyme continuously scans the DNA for the presence of recognition sites. Upon specific M.EcoRV-DNA complex formation a strong increase in the fluorescence of an oligonucleotide containing a 2-aminopurine base analogue at the GAT-2AP-TC position is observed which, most likely, is correlated with DNA bending. In contrast to the GAT-2AP-TC substrate, a G-2AP-TATC substrate in which the target base is replaced by 2-aminopurine does not show an increase in fluorescence upon M.EcoRV binding, demonstrating that 2-aminopurine is not a general tool to detect base flipping. Stopped-flow experiments show that DNA bending is a fast process with rate constants >10 s(-1). In the presence of cofactor, the specific complex adopts a second conformation, in which the target sequence is more tightly contacted by the enzyme. M.EcoRV exists in an open and in a closed state that are in slow equilibrium. Closing the open state is a slow process (rate constant approximately 0.7 min(-1)) that limits the rate of DNA methylation under single turnover conditions. Product release requires opening of the closed complex which is very slow (rate constant approximately 0.05-0.1 min(-1)) and limits the rate of DNA methylation under multiple turnover conditions. M.EcoRV methylates DNA sequences containing more than one recognition sites in a distributive manner. Since the dissociation rate from non-specific DNA does not depend on the length of the DNA fragment, DNA dissociation does not preferentially occur at the ends of the DNA.     

Relaxed specificity of the EcoRV restriction endonuclease

The EcoRV restriction endonuclease normally shows a high specificity for its recognition site on DNA, GATATC. In standard reactions, it cleaves DNA at this site several orders of magnitude more readily than at any alternative sequence. But in the presence of dimethyl sulphoxide and at high pH, the EcoRV enzyme cleaves DNA at several sites that differ from its recognition site by one nucleotide. Of the 18 (3 X 6) possible sequences that differ from GATATC by one base, all were cleaved readily except for the following 4 sites: TATATC, CATATC, GATATA and GATATG. However, two of the sites that could be cleaved by EcoRV in the presence of dimethyl sulphoxide, GAGATC and GATCTC, were only cleaved on DNA that lacked dam methylation: both contain the sequence GATC, the recognition site for the dam methylase of Escherichia coli.     

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.     

Reactions of BglI and other type II restriction endonucleases with discontinuous recognition sites

Type II restriction enzymes generally recognize continuous sequences of 4-8 consecutive base pairs on DNA, but some recognize discontinuous sites where the specified sequence is interrupted by a defined length of nonspecific DNA. To date, a mechanism has been established for only one type II endonuclease with a discontinuous site, SfiI at GGCCNNNNNGGCC (where N is any base). In contrast to orthodox enzymes such as EcoRV, dimeric proteins that act at a single site, SfiI is a tetramer that interacts with two sites before cleaving DNA. BglI has a similar recognition sequence (GCCNNNNNGGC) to SfiI but a crystal structure like EcoRV. BglI and several other endonucleases with discontinuous sites were examined to see if they need two sites for their DNA cleavage reactions. The enzymes included some with sites containing lengthy segments of nonspecific DNA, such as XcmI (CCANNNNNNNNNTGG). In all cases, they acted at individual sites. Elongated recognition sites do not necessitate unusual reaction mechanisms. Other experiments on BglI showed that it bound to and cleaved DNA in the same manner as EcoRV, thus further delineating a distinct group of restriction enzymes with similar structures and a common reaction mechanism.     

How does a DNA interacting enzyme change its specificity during molecular evolution? A site-directed mutagenesis study at the DNA binding site of the DNA-(adenine-N6)-methyltransferase EcoRV

The EcoRV DNA-(adenine-N6)-methyltransferase (MTase) recognizes GATATC sequences and modifies the first adenine residue within this site. Parts of its DNA interface show high sequence homology to DNA MTases of the dam family which recognize and modify GATC sequences. A phylogenetic analysis of M.EcoRV and dam-MTases suggests that EcoRV arose in evolution from a primordial dam-MTase in agreement to the finding that M.EcoRV also methylates GATC sites albeit at a strongly reduced rate. GATCTC sites that deviate in only one position from the EcoRV sequence are preferred over general dam sites. We have investigated by site-directed mutagenesis the function of 17 conserved and nonconserved residues within three loops flanking the DNA binding cleft of M.EcoRV. M.EcoRV contacts the GATATC sequence with two highly cooperative recognition modules. The contacts to the GAT-part of the recognition sequence are formed by residues conserved between dam MTases and M.EcoRV. Mutations at these positions lead to an increase in the discrimination between GATATC and GATC substrates. Our data show that the change in sequence specificity from dam (GATC) to EcoRV (GATATC) was accompanied by the generation of a second recognition module that contacts the second half of the target sequence. The new DNA contacts are formed by residues from all three loops that are not conserved between M.EcoRV and dam MTases. Mutagenesis at important residues within this module leads to variants that show a decreased ability to recognize the TC-part of the GATATC sequence.     

Investigation of the inhibitory role of phosphorothioate internucleotidic linkages on the catalytic activity of the restriction endonuclease EcoRV

The inhibitory effect of phosphorothioate residues, located within one strand of double-stranded DNA, on the hydrolytic activity of the restriction endonuclease EcoRV was investigated. Specific incorporation of a phosphorothioate group at the site of cleavage yielded the sequence 5'-GATsATC-3'. This modified sequence was cleaved at a relative rate of 0.1 compared to the unmodified substrate. Substrates 5'-GATsAsTC-3' and 5'-GsATsATC-3', both containing one additional phosphorothioate substitution, were linearized at a rate of 0.04 relative to unmodified DNA. However, under the same conditions, fully dAMPS-substituted DNA was found to be virtually resistant to the hydrolytic activity of EcoRV. Further experiments showed that double-stranded DNA fragments generated by PCR containing phosphorothioate groups within both strands are potent inhibitors of EcoRV catalysis. The inhibition was independent of whether the inhibitor fragment contained an EcoRV recognition site. We concluded that substitution of the phosphate group at the site of cleavage by a phosphorothioate residue decreases the rate of EcoRV-catalyzed hydrolysis most significantly. Substitution of other phosphate groups within the recognition sequence plays a limited role in enzyme inhibition. The presence of multiple dNMPS residues at regions of the DNA removed from the EcoRV recognition site may decrease the amount of enzyme available for catalysis by nonspecific binding to EcoRV.     

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.     

The M.EcoRV DNA-(adenine N6)-methyltransferase uses DNA bending for recognition of an expanded EcoDam recognition site

The M.EcoRV DNA methyltransferase recognizes GATATC sites. It is related to EcoDam, which methylates GATC sites. The DNA binding domain of M.EcoRV is similar to that of EcoDam suggesting a similar mechanism of DNA recognition. We show that amino acid residue Lys11 of M.EcoRV is involved in recognition of Gua1 and Arg128 contacts the Gua in base pair 6. These residues correspond to Lys9 and Arg124 in EcoDam, which recognize the Gua residues in both strands of the Dam recognition sequence, indicating that M.EcoRV and EcoDam make similar contacts to outermost base pairs of their recognition sequences and M.EcoRV recognizes its target site as an expanded GATC site. In contrast to EcoDam, M.EcoRV considerably bends the DNA (59+/-4 degrees) suggesting indirect readout of the AT-rich inner sequence. Recognition of an expanded target site by DNA bending is a new principle for changing DNA recognition specificity of proteins during molecular evolution. R128A is inefficient in DNA bending and binding, whereas K11A bends DNA with relaxed sequence specificity. These results suggest a temporal order of the formation of protein-DNA contacts in which the Gua6-Arg128 contact forms early followed by DNA bending and, finally, the formation of the Lys11-Gua1 contact.     

On the possibilities and limitations of rational protein design to expand the specificity of restriction enzymes: a case study employing EcoRV as the target

The restriction endonuclease EcoRV has been characterized in structural and functional terms in great detail. Based on this detailed information we employed a structure-guided approach to engineer variants of EcoRV that should be able to discriminate between differently flanked EcoRV recognition sites. In crystal structures of EcoRV complexed with d(CGGGATATCCC)(2) and d(AAAGATATCTT)(2), Lys104 and Ala181 closely approach the two base pairs flanking the GATATC recognition site and thus were proposed to be a reasonable starting point for the rational extension of site specificity in EcoRV [Horton,N.C. and Perona,J.J. (1998) J. Biol. Chem., 273, 21721-21729]. To test this proposal, several single (K104R, A181E, A181K) and double mutants of EcoRV (K104R/A181E, K104R/A181K) were generated. A detailed characterization of all variants examined shows that only the substitution of Ala181 by Glu leads to a considerably altered selectivity with both oligodeoxynucleotide and macromolecular DNA substrates, but not the predicted one, as these variants prefer cleavage of a TA flanked site over all other sites, under all conditions tested. The substitution of Lys104 by Arg, in contrast, which appeared to be very promising on the basis of the crystallographic analysis, does not lead to variants which differ very much from the EcoRV wild-type enzyme with respect to the flanking sequence preferences. The K104R/A181E and K104R/A181K double mutants show nearly the same preferences as the A181E and A181K single mutants. We conclude that even for the very well characterized restriction enzyme EcoRV, properties that determine specificity and selectivity are difficult to model on the basis of the available structural information.     

Mechanism of DNA Recognition by the Restriction Enzyme EcoRV

EcoRV, a restriction enzyme in Escherichia coli, destroys invading foreign DNA by cleaving it at the center step of a GATATC sequence. In the EcoRV-cognate DNA crystallographic complex, a sharp kink of 50° has been found at the center base-pair step (TA). Here, we examine the interplay between the intrinsic propensity of the cognate sequence to kink and the induction by the enzyme by performing all-atom molecular dynamics simulations of EcoRV unbound and interacting with three DNA sequences: the cognate sequence, GATATC (TA); the non-cognate sequence, GAATTC (AT); and with the cognate sequence methylated on the first adenine GA_CH3TATC (TA-CH₃). In the unbound EcoRV, the cleft between the two C-terminal subdomains is found to be open. Binding to AT narrows the cleft and forms a partially bound state. However, the intrinsic bending propensity of AT is insufficient to allow tight binding. In contrast, the cognate TA sequence is easier to bend, allowing specific, high-occupancy hydrogen bonds to form in the complex. The absence of cleavage for this methylated sequence is found to arise from the loss of specific hydrogen bonds between the first adenine of the recognition sequence and Asn185. On the basis of the results, we suggest a three-step recognition mechanism. In the first step, EcoRV, in an open conformation, binds to the DNA at a random sequence and slides along it. In the second step, when the two outer base pairs, GAxxTC, are recognized, the R loops of the protein become more ordered, forming strong hydrogen-bonding interactions, resulting in a partially bound EcoRV-DNA complex. In the third step, the flexibility of the center base pair is probed, and in the case of the full cognate sequence the DNA bends, the complex strengthens and the protein and DNA interact more closely, allowing cleavage.     

Structure–function correlation for the EcoRV restriction enzyme: from non-specific binding to specific DNA cleavage

The EcoRV restriction endonuclease cleaves DNA at its recognition sequence at least a million times faster than at any other DNA sequence. The only cofactor it requires for activity is Mg²⁺: but in binding to DNA in the absence of Mg²⁺, the EcoRV enzyme shows no specificity for its recognition site. Instead, the reason why EcoRV cuts one DNA sequence faster than any other is that the rate of cleavage is controlled by the binding of Mg²⁺ to EcoRV-DNA complexes: the complex at the recognition site has a high affinity for Mg²⁺, while the complexes at other DNA sequences have low affinities for Mg²⁺. The structures of the EcoRV endonuclease, and of its complexes with either 8pecific or non-specific DNA, have been solved by X-ray crystallography. In the specific complex, the protein interacts with the bases in the recognition sequence and the DNA takes up a highly distorted structure. In the non-specific complex with an unrelated DNA sequence, there are virtually no interactions with the bases and the DNA retains a B-like structure. Since the free energy changes for the formation of specific and non-specific complexes are the same, the energy from the specific interactions balances that required for the distortion of the DNA. The distortion inserts the phosphate at the scissile bond into the active site of the enzyme, where it forms part of the binding site for Mg²⁺. Without this distortion, the EcoRV–DNA complex would be unable to bind Mg²⁺ and thus unable to cleave DNA. The specificity of the EcoRV restriction enzyme is therefore governed, not by DNA binding as such, but by its ability to organize the structure of the DNA to which it is bound.     

Probing the DNA interface of the EcoRV DNA-(adenine-N6)-methyltransferase by site-directed mutagenesis,fluorescence spectroscopy,and UV cross-linking

The EcoRV DNA-(adenine-N6)-methyltransferase recognizes GATATC sites and methylates the DNA as indicated. It is related to the large family of dam methyltransferases which modify GATC sites. We have studied the interaction of DNA with M.EcoRV and 12 M.EcoRV variants using oligonucleotides containing 2-aminopurine as a fluorescence probe in equilibrium and stopped-flow DNA binding studies and 5-iododeoxyuracil for UV cross-linking. M.EcoRV binds to DNA in a multistep binding reaction, including two different conformations of the specific enzyme-DNA complex, and induces a strong conformational change of the DNA at the fourth position of the recognition site. Mutations at residues forming contacts to the GAT part of the recognition site reduce the stability of both specific enzyme-DNA complexes. Two enzyme variants which fail to recognize the ATC part do not induce the deformation of the DNA which explains why they cannot interact properly with the recognition site. Other mutations at residues which interact with the ATC part selectively reduce the stability of the second enzyme-DNA complex. These results show that when approaching the DNA M.EcoRV first contacts the GAT part of the target site. Since the residues mediating these contacts are conserved among M.EcoRV and dam MTases, the kinetics of formation of the enzyme-DNA complex correspond to the evolutionary history of the protein. Whether the observation that evolutionarily conserved contacts are formed early during complex formation is a general rule for DNA interacting enzymes or proteins that change their specificity during evolution remains to be seen.     

Nonidentical DNA-binding sites of endonuclease NaeI recognize different families of sequences flanking the recognition site.

NaeI endonuclease uses a two-site binding mechanism to cleave substrate DNA: reaction-rate studies imply that occupancy of the second DNA site causes an allosteric change in the protein that enables DNA cleavage at the first site [Conrad, M., & Topal, M. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9707-9711]. Measurements of relative binding affinities for 14-base-pair DNA fragments containing the NaeI recognition sequence GCCGGC and various flanking sequences showed that the two DNA-binding sites are not identical. G.C-rich flanking sequences were preferred by the activator binding site, whereas A.T-rich flanking sequences were preferred by the substrate binding site: GGGTGCCGGCAGGG was preferred 8-fold more by the activator site but 14-fold less by the substrate site than TTTCGCCGGCGTTT. Substitution of pyrimidine or 7-deazapurine for purine immediately 3' to GCCGGC reduced DNA affinity for only the activator site by up to 26-fold, implying that the activator DNA-binding site requires N-7 base contacts immediately flanking GCCGGC. The implications of nonidentical DNA-binding sites, one of which binds a specific DNA site to allosterically activate the other, are discussed.     

EcoRV restriction endonuclease: communication between DNA recognition and catalysis.

A genetic system was constructed for the mutagenesis of the EcoRV restriction endonuclease and for the overproduction of mutant proteins. The system was used to make two mutants of EcoRV, with Ala in place of either Asn185 or Asn188. In the crystal structure of the EcoRV-DNA complex, both Asn185 and Asn188 contact the DNA within the EcoRV recognition sequence. But neither mutation affected the ability of the protein to bind to DNA. In the absence of metal ion cofactors, the mutants bound DNA with almost the same affinity as that of the wild-type enzyme. In the presence of Mg2+, both mutants retained the ability to cleave DNA specifically at the EcoRV recognition sequence, but their activities were severely depressed relative to that of the wild-type. In contrast, with Mn2+ as the cofactor, the mutant enzymes cleaved the EcoRV recognition site with activities that were close to that of the wild-type. When bound to DNA at the EcoRV recognition site, the mutant proteins bound Mn2+ ions readily, but they had much lower affinities for Mg2+ ions than the wild-type enzyme. This was the reason for their low activities with Mg2+ as the cofactor. The arrangement of the DNA recognition functions, at one location in the EcoRV restriction enzyme, are therefore responsible for organizing the catalytic functions at a separate location in the protein.     

Divalent metal dependence of site-specific DNA binding by EcoRV endonuclease.

Measurements of binding equilibria of EcoRV endonuclease to DNA, for a series of base-analogue substrates, demonstrate that expression of sequence selectivity is strongly enhanced by the presence of Ca2+ ions. Binding constants were determined for short duplex oligodeoxynucleotides containing the cognate DNA site, three cleavable noncognate sites, and a fully nonspecific site. At pH 7.5 and 100 mM NaCl, the full range of specificity from the specific (tightest binding) to nonspecific (weakest binding) sites is 0.9 kcal/mol in the absence of metal ions and 5.8 kcal/mol in the presence of Ca2+. Precise determination of binding affinities in the presence of the active Mg2+ cofactor was found to be possible for substrates retaining up to 1.6% of wild-type activity, as determined by the rate of phosphoryl transfer. These measurements show that Ca2+ is a near-perfect analogue for Mg2+ in binding reactions of the wild-type enzyme with DNA base-analogue substrates, as it provides identical DeltaDeltaG degrees bind values among the cleavable noncognate sites. Equilibrium dissociation constants of wild-type and base-analogue sites were also measured for the weakly active EcoRV mutant K38A, in the presence of either Mg2+ or Ca2+. In this case, Ca2+ allows expression of a greater degree of specificity than does Mg2+. DeltaDeltaG degrees bind values of K38A toward specific versus nonspecific sites are 6.1 kcal/mol with Ca2+ and 3.9 kcal/mol with Mg2+, perhaps reflecting metal-specific conformational changes in the ground-state ternary complexes. The enhancement of binding specificity provided by divalent metal ions is likely to be general to many restriction endonucleases and other metal-dependent nucleic acid-modifying enzymes. These results strongly suggest that measurements of DNA binding affinities for EcoRV, and likely for many other restriction endonucleases, should be performed in the presence of divalent metal ions.     

DNA strand arrangement within the SfiI-DNA complex: atomic force microscopy analysis

The SfiI restriction enzyme binds to DNA as a tetramer holding two usually distant DNA recognition sites together before cleavage of the four DNA strands. To elucidate structural properties of the SfiI-DNA complex, atomic force microscopy (AFM) imaging of the complexes under noncleaving conditions (Ca2+ instead of Mg2+ in the reaction buffer) was performed. Intramolecular complexes formed by protein interaction between two binding sites in one DNA molecule (cis interaction) as well as complexes formed by the interaction of two sites in different molecules (trans interaction) were analyzed. Complexes were identified unambiguously by the presence of a tall spherical blob at the DNA intersections. To characterize the path of DNA within the complex, the angles between the DNA helices in the proximity of the complex were systematically analyzed. All the data show clear-cut bimodal distributions centered around peak values corresponding to 60 degrees and 120 degrees. To unambiguously distinguish between the crossed and bent models for the DNA orientation within the complex, DNA molecules with different arm lengths flanking the SfiI binding site were designed. The analysis of the AFM images for complexes of this type led to the conclusion that the DNA recognition sites within the complex are crossed. The angles of 60 degrees or 120 degrees between the DNA helices correspond to a complex in which one of the helices is flipped with respect to the orientation of the other. Complexes formed by five different recognition sequences (5'-GGCCNNNNNGGCC-3'), with different central base pairs, were also analyzed. Our results showed that complexes containing the two possible orientations of the helices were formed almost equally. This suggests no preferential orientation of the DNA cognate site within the complex, suggesting that the central part of the DNA binding site does not form strong sequence specific contacts with the protein.