Crystallization of complexes of EcoRV endonuclease with cognate and non-cognate DNA fragments

Complexes of the type II restriction endonuclease EcoRV with a variety of short, selfcomplementary deoxyoligonucleotides have been crystallized. The best crystals diffract to about 2.7 A resolution and consist of 1:1 complexes between endonuclease dimers and duplexes of the cognate decamer GGGATATCCC containing the hexameric RV recognition sequence GATATC. Crystals with the non-cognate DNA octamer duplexes CGAGCTCG and CGAATTCG diffract to 3.0 and 3.5 A resolution, respectively, and contain two DNA duplexes per enzyme dimer.     

Cross-linking of SsoII restriction endonuclease to cognate and non-cognate DNAs

Specific and non-specific interactions SsoII restriction endonuclease (R·SsoII) were probed by the method of covalent attachment to modified DNA containing an active monosubstituted pyrophosphate internucleotide bond instead of a phosphodiester one. R·SsoII with six N-terminal His residues was shown to be cross-linked to duplexes with this type of modification, either containing or not the recognition sequence. Competition experiments with covalent attachment of R·SsoII to activated DNAs demonstrated the similar affinity of the enzyme to cognate and non-cognate DNAs in the absence of cofactor, Mg²⁺ ions.     

Structure of PvuII endonuclease with cognate DNA.

We have determined the structure of PvuII endonuclease complexed with cognate DNA by X-ray crystallography. The DNA substrate is bound with a single homodimeric protein, each subunit of which reveals three structural regions. The catalytic region strongly resembles structures of other restriction endonucleases, even though these regions have dissimilar primary sequences. Comparison of the active site with those of EcoRV and EcoRI endonucleases reveals a conserved triplet sequence close to the reactive phosphodiester group and a conserved acidic pair that may represent the ligands for the catalytic cofactor Mg2+. The DNA duplex is not significantly bent and maintains a B-DNA-like conformation. The subunit interface region of the homodimeric protein consists of a pseudo-three-helix bundle. Direct contacts between the protein and the base pairs of the PvuII recognition site occur exclusively in the major groove through two antiparallel beta strands from the sequence recognition region of the protein. Water-mediated contacts are made in the minor grooves to central bases of the site. If restriction enzymes do share a common ancestor, as has been proposed, their catalytic regions have been very strongly conserved, while their subunit interfaces and DNA sequence recognition regions have undergone remarkable structural variation.     

Sliding and jumping of single EcoRV restriction enzymes on non-cognate DNA

The restriction endonuclease EcoRV can rapidly locate a short recognition site within long non-cognate DNA using 'facilitated diffusion'. This process has long been attributed to a sliding mechanism, in which the enzyme first binds to the DNA via nonspecific interaction and then moves along the DNA by 1D diffusion. Recent studies, however, provided evidence that 3D translocations (hopping/jumping) also help EcoRV to locate its target site. Here we report the first direct observation of sliding and jumping of individual EcoRV molecules along nonspecific DNA. Using fluorescence microscopy, we could distinguish between a slow 1D diffusion of the enzyme and a fast translocation mechanism that was demonstrated to stem from 3D jumps. Salt effects on both sliding and jumping were investigated, and we developed numerical simulations to account for both the jump frequency and the jump length distribution. We deduced from our study the 1D diffusion coefficient of EcoRV, and we estimated the number of jumps occurring during an interaction event with nonspecific DNA. Our results substantiate that sliding alternates with hopping/jumping during the facilitated diffusion of EcoRV and, furthermore, set up a framework for the investigation of target site location by other DNA-binding proteins.     

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.     

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.     

Crystal structure of MunI restriction endonuclease in complex with cognate DNA at 1.7 A resolution.

The MunI restriction enzyme recognizes the palindromic hexanucleotide sequence C/AATTG (the '/' indicates the cleavage site). The crystal structure of its active site mutant D83A bound to cognate DNA has been determined at 1.7 A resolution. Base-specific contacts between MunI and DNA occur exclusively in the major groove. While DNA-binding sites of most other restriction enzymes are comprised of discontinuous sequence segments, MunI combines all residues involved in the base-specific contacts within one short stretch (residues R115-R121) located at the N-terminal region of the 3(10)4 helix. The outer CG base pair of the recognition sequence is recognized solely by R115 through hydrogen bonds made by backbone and side chain atoms to both bases. The mechanism of recognition of the central AATT nucleotides by MunI is similar to that of EcoRI, which recognizes the G/AATTC sequence. The local conformation of AATT deviates from the typical B-DNA form and is remarkably similar to EcoRI-DNA. It appears to be essential for specific hydrogen bonding and recognition by MunI and EcoRI.     

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.     

Modes of DNA cleavage by the EcoRV restriction endonuclease

The mechanism of action of the EcoRV restriction endonuclease at its single recognition site on the plasmid pAT153 was analyzed by kinetic methods. In reactions at pH 7.5, close to the optimum for this enzyme, both strands of the DNA were cut in a single concerted reaction: DNA cut in only one strand of the duplex was neither liberated from the enzyme during the catalytic turnover nor accumulated as a steady-state intermediate. In contrast, reactions at pH 6.0 involved the sequential cutting of the two strands of the DNA. Under these conditions, DNA cut in a single strand was an obligatory intermediate in the reaction pathway and a fraction of the nicked DNA dissociated from the enzyme during the turnover. The different reaction profiles are shown to be consistent with a single mechanism in which the kinetic activity of each subunit of the dimeric protein is governed by its affinity for Mg2+ ions. At pH 7.5, Mg2+ is bound to both subunits of the dimer for virtually the complete period of the catalytic turnover, while at pH 6.0 Mg2+ is bound transiently to one subunit at a time. The kinetics of the EcoRV nuclease were unaffected by DNA supercoiling.     

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.     

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.     

Evidence for two types of complexes formed by yeast tyrosyl-tRNA synthetase with cognate and non-cognate tRNA. Effect of ribonucleoside triphosphates

Polyacrylamide gel electrophoresis at pH 8.3 was used to detect and quantitate the formation of the yeast tyrosyl-tRNA synthetase (an alpha 2-type enzyme) complex with its cognate tRNA. Electrophoretic mobility of the complex is intermediate between the free enzyme and free tRNA; picomolar quantities can be readily detected by silver staining and quantitated by densitometry of autoradiograms when [32P]tRNA is used. Two kinds of complexes of Tyr-tRNA synthetase with yeast tRNA(Tyr) were detected. A slower-moving complex is formed at ratios of tRNA(Tyr)/enzyme less than or equal to 0.5; it is assigned the composition tRNA.(alpha 2)2. At higher ratios, a faster-moving complex is formed, approaching saturation at tRNA(Tyr)/enzyme = 1; any excess of tRNA(Tyr) remains unbound. This complex is assigned the composition tRNA.alpha 2. The slower, i.e. tRNA.(alpha 2)2 complex, but not the faster complex, can be formed even with non-cognate tRNAs. Competition experiments show that the affinity of the enzyme towards tRNA(Tyr) is at least 10-fold higher than that for the non-cognate tRNAs. ATP and GTP affect the electrophoretic mobility of the enzyme and prevent the formation of tRNA.(alpha 2)2 complexes both with cognate and non-cognate tRNAs, while neither tyrosine, as the third substrate of Tyr tRNA synthetase, nor AMP, AMP/PPi, or spermidine, have such effects. Hence, the ATP-mediated formation of the alpha 2 structure parallels the increase in specificity of the enzyme towards its cognate tRNA.     

Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure.

The 1.85 A crystal structure of endonuclease III, combined with mutational analysis, suggests the structural basis for the DNA binding and catalytic activity of the enzyme. Helix-hairpin-helix (HhH) and [4Fe-4S] cluster loop (FCL) motifs, which we have named for their secondary structure, bracket the cleft separating the two alpha-helical domains of the enzyme. These two novel DNA binding motifs and the solvent-filled pocket in the cleft between them all lie within a positively charged and sequence-conserved surface region. Lys120 and Asp138, both shown by mutagenesis to be catalytically important, lie at the mouth of this pocket, suggesting that this pocket is part of the active site. The positions of the HhH motif and protruding FCL motif, which contains the DNA binding residue Lys191, can accommodate B-form DNA, with a flipped-out base bound within the active site pocket. The identification of HhH and FCL sequence patterns in other DNA binding proteins suggests that these motifs may be a recurrent structural theme for DNA binding proteins.     

Mg2+ confers DNA binding specificity to the EcoRV restriction endonuclease.

The EcoRV mutant D90A which carries an amino acid substitution in its active center does not cleave DNA. Therefore, it is possible to perform DNA binding experiments with the EcoRV-D90A mutant both in the absence and in the presence of Mg2+. Like wild-type EcoRV [Taylor et al. (1991) Biochemistry 30, 8743-8753], it does not show a pronounced specificity for binding to its recognition site in the absence of Mg2+ as judged by the appearance of multiple shifted bands in an electrophoretic mobility shift assay with a 377-bp DNA fragment carrying a single EcoRV recognition sequence. In the presence of Mg2+, however, only one band corresponding to a 1:1 complex appears even with a high excess of protein over DNA. This complex most likely is the specific one, because its formation is suppressed much more effectively by a 13-bp oligodeoxynucleotide with an EcoRV site than by a corresponding oligodeoxynucleotide without an EcoRV site. The preferential interaction of the EcoRV-D90A mutant with specific DNA in the presence of Mg2+ was also demonstrated directly: a 20-bp oligodeoxynucleotide with an EcoRV site is bound with KAss = 4 x 10(8) M-1, while a corresponding oligodeoxynucleotide without an EcoRV site is bound with KAss less than or equal to 1 x 10(5) M-1. From these data it appears that Mg2+ confers DNA binding specificity to this mutant by lowering the affinity to nonspecific sites and raising the affinity to specific sites as compared to binding in the absence of Mg2+. It is concluded that this is also true for wild-type EcoRV.     

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.     

Simultaneous DNA binding and bending by EcoRV endonuclease observed by real-time fluorescence

The complete catalytic cycle of EcoRV endonuclease has been observed by combining fluorescence anisotropy with fluorescence resonance energy transfer (FRET) measurements. Binding, bending, and cleavage of substrate oligonucleotides were monitored in real time by rhodamine-x anisotropy and by FRET between rhodamine and fluorescein dyes attached to opposite ends of a 14-mer DNA duplex. For the cognate GATATC site binding and bending are found to be nearly simultaneous, with association and bending rate constants of (1.45-1.6) x 10(8) M(-1) s(-1). On the basis of the measurement of k(off) by a substrate-trapping approach, the equilibrium dissociation constant of the enzyme-DNA complex in the presence of inhibitory calcium ions was calculated as 3.7 x 10(-12) M from the kinetic constants. Further, the entire DNA cleavage reaction can be observed in the presence of catalytic Mg(2+) ions. These measurements reveal that the binding and bending steps occur at equivalent rates in the presence of either Mg(2+) or Ca(2+), while a slow decrease in fluorescence intensity following bending corresponds to k(cat), which is limited by the cleavage and product dissociation steps. Measurement of k(on) and k(off) in the absence of divalent metals shows that the DNA binding affinity is decreased by 5000-fold to 1.4 x 10(-8) M, and no bending could be detected in this case. Together with crystallographic studies, these data suggest a model for the induced-fit conformational change in which the role of divalent metal ions is to stabilize the sharply bent DNA in an orientation suitable for accessing the catalytic transition state.     

Cloning DNA restriction endonuclease fragments with protruding single-stranded ends

A new method of in vitro recombination was employed to construct plasmids containing lac promoter fragments 64 bp and 144 bp long. The 64 bp HpaII-HhaI fragment contains the binding site for the catabolite activator protein (CAP). The HpaII-HaeIII 144 bp fragment includes the binding sites for RNA polymerase, the lac repressor and CAP. The method utilizes the ability of T4 DNA polymerase to make flush-ended DNA either by filling in a recessed 3'-end or by exonucleolytic removal of a protruding 3'-end. The treated fragments were then blunt-end ligated to the filled-in EcoRI cloning sites of the plasmids pVH51 and pBR322 using T4 ligase. In this process, the EcoRI sites were regenerated on the fragment ends thus facilitating the subsequent isolation of the fragments from their cloning vectors.     

Role of magnesium ions in DNA recognition by the EcoRV restriction endonuclease

The restriction endonuclease EcoRV binds two magnesium ions. One of these ions, Mg(A)(2+), binds to the phosphate group where the cleavage occurs and is required for catalysis, but the role of the other ion, Mg(B)(2+) is debated. Here, multiple independent molecular dynamics simulations suggest that Mg(B)(2+) is crucial for achieving a tightly bound protein-DNA complex and stabilizing a conformation that allows cleavage. In the absence of Mg(B)(2+) in all simulations the protein-DNA hydrogen bond network is significantly disrupted and the sharp kink at the central base pair step of the DNA, which is observed in the two-metal complex, is not present. Also, the active site residues rearrange in such a way that the formation of a nucleophile, required for DNA hydrolysis, is unlikely.     

The structure of drug-deoxydinucleoside phosphate complex; generalized conformational behavior of intercalation complexes with RNA and DNA fragments.

A 2:2 complex of proflavine and deoxycytidylyl-3', 5'-guanosine has been crystallized and its structure determined by x-ray crystallography. The two dinucleoside phosphate strands form self complementary duplexes with Watson Crick hydrogen bonds. One proflavin is asymmetrically intercalated between the base pairs and the other is stacked above them. The conformations of the nucleotides are unusual in that one strand has C3',C2'endomixed sugar puckering and the other has C3',C3' endo deoxyribose sugars. These results show that the conformation of the 3'sugar is of secondary importance to the intercalated geometry.     

Mechanism of discrimination between cognate and non-cognate tRNAs by phenylalanyl-tRNA synthetase from yeast.

The interaction between phenylalanyl-tRNA synthetase from yeast and Escherichia coli and tRNAPhe (yeast), tRNASer (yeast), tRNA1Val (E. coli) has been investigated by ultracentrifugation analysis, fluorescence titrations and fast kinetic techniques. The fluorescence of the Y-base of tRNAPhe and the intrinsic fluorescence of the synthetases have been used as optical indicators. 1. Specific complexes between phenylalanyl-tRNA synthetase and tRNAPhe from yeast are formed in a two-step mechanism: a nearly diffusion-controlled recombination is followed by a fast conformational transition. Binding constants, rate constants and changes in the quantum yield of the Y-base fluorescence upon binding are given under a variety of conditions with respect to pH, added salt, concentration of Mg2+ ions and temperature. 2. Heterologous complexes between phenylalanyl-tRNA synthetase (E. coli) and tRNAPhe (yeast) are formed in a similar two-step mechanism as the specific complexes; the conformational transition, however, is slower by a factor 4-5. 3. Formation of non-specific complexes between phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) proceeds in a one-step mechanism. Phenylalanyl-tRNA synthetase (yeast) binds either two molecules of tRNAPhe (yeast) or only one molecule of tRNATyr (E. coli); tRNA1Val (E. coli) or tRNASer (yeast) are also bound in a 1:1 stoichiometry. Binding constants for complexes of phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) are determined under a variety of conditions. In contrast to specific complex formation, non-specific binding is disfavoured by the presence of Mg2+ ions, and is not affected by pH and the presence of pyrophosphate. The difference in the stabilities of specific and non-specific complexes can be varied by a factor of 2--100 depending on the ionic conditions. Discrimination of cognate and non-cognate tRNA by phenylalanyl-tRNA synthetase (yeast) is discussed in terms of the binding mechanism, the topology of the binding sites, the nature of interacting forces and the relation between specificity and ionic conditions.