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1.
19 F NMR spectroscopy have been applied to evaluate metal ion binding by the representative PvuII endonuclease in the absence of substrate. In separate experiments, ITC data demonstrate that PvuII endonuclease binds 2.16 Mn(II) ions and 2.05 Ca(II) metal ions in each monomer active site with K d values of  ≈ 1 mM. While neither calorimetry nor protein NMR spectroscopy is directly sensitive to Mg(II) binding to the enzyme, Mn(II) competes with Mg(II) for common sites(s) on PvuII endonuclease. Substitution of the conserved active site carboxylate Glu68 with Ala resulted in a loss of affinity for both equivalents of both Ca(II) and Mn(II). Interestingly, the active site mutant D58A retained an affinity for Mn(II) with K d  ≈ 2 mM. Mn(II) paramagnetic broadening in 19F spectra of wild-type and mutant 3-fluorotyrosine PvuII endonucleases are consistent with ITC results. Chemical shift analysis of 3-fluorotyrosine mutant enzymes is consistent with a perturbed conformation for D58A. Therefore, free PvuII endonuclease binds metal ions, and metal ion binding can precede DNA binding. Further, while Glu68 is critical to metal ion binding, Asp58 does not appear to be critical to the binding of at least one metal ion and appears to also have a role in structure. These findings provide impetus for exploring the roles of multiple metal ions in the structure and function of this representative endonuclease. Received: 30 March 1999 / Accepted: 28 September 1999  相似文献   

2.
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.  相似文献   

3.
Conlan LH  Dupureur CM 《Biochemistry》2002,41(50):14848-14855
Restriction enzymes serve as important model systems for understanding the role of metal ions in phosphodiester hydrolysis. To this end, a number of laboratories have reported dramatic differences between the metal ion-dependent and metal ion-independent DNA binding behaviors of these systems. In an effort to illuminate the underlying mechanistic details which give rise to these differences, we have quantitatively dissected these equilibrium behaviors into component association and dissociation rates for the representative PvuII endonuclease and use these data to assess the stoichiometry of metal ion involvement in the binding process. The dependence of PvuII cognate DNA on Ca(II) concentration binding appears to be cooperative, exhibiting half-saturation at 0.6 mM metal ion and yielding an n(H) of 3.5 +/- 0.2 per enzyme homodimer. Using both nitrocellulose filter binding and fluorescence assays, we observe that the cognate DNA dissociation rate (k(-)(1) or k(off)) is very slow (10(-)(3) s(-)(1)) and exhibits a shallow dependence on metal ion concentration. DNA trap cleavage experiments with Mg(II) confirm the general irreversibility of DNA binding relative to cleavage, even at low metal ion concentrations. More dramatically, the association rate (k(1) or k(on)) also appears to be cooperative, increasing more than 100-fold between 0.2 and 10 mM Ca(II), with an optimum value of 2.7 x 10(7) M(-)(1) s (-)(1). Hill analysis of the metal ion dependence of k(on) indicates an n(H) of 3.6 +/- 0.2 per enzyme dimer. This value is consistent with the involvement in DNA association of two metal ions per subunit active site, a result which lends new strength to arguments for two-metal ion mechanisms in restriction enzymes.  相似文献   

4.
Horton NC  Perona JJ 《Biochemistry》2004,43(22):6841-6857
Four crystal structures of EcoRV endonuclease mutants K92A and K38A provide new insight into the mechanism of DNA bending and the structural basis for metal-dependent phosphodiester bond cleavage. The removal of a key active site positive charge in the uncleaved K92A-DNA-M(2+) substrate complex results in binding of a sodium ion in the position of the amine nitrogen, suggesting a key role for a positive charge at this position in stabilizing the sharp DNA bend prior to cleavage. By contrast, two structures of K38A cocrystallized with DNA and Mn(2+) ions in different lattice environments reveal cleaved product complexes featuring a common, novel conformation of the scissile phosphate group as compared to all previous EcoRV structures. In these structures, the released 5'-phosphate and 3'-OH groups remain in close juxtaposition with each other and with two Mn(2+) ions that bridge the conserved active site carboxylates. The scissile phosphates are found midway between their positions in the prereactive substrate and postreactive product complexes of the wild-type enzyme. Mn(2+) ions occupy two of the three sites previously described in the prereactive complexes and are plausibly positioned to generate the nucleophilic hydroxide ion, to compensate for the incipient additional negative charge in the transition state, and to ionize a second water for protonation of the 3'-oxyanion. Reconciliation of these findings with earlier X-ray and fluorescence studies suggests a novel mechanism in which a single initially bound metal ion in a third distinct site undergoes a shift in position together with movement of the scissile phosphate deeper into the active site cleft. This reconfigures the local environment to permit binding of the second metal ion followed by movement toward the pentacovalent transition state. The new mechanism suggested here embodies key features of previously proposed two- and three-metal catalytic models, and offers a view of the stereochemical pathway that integrates much of the copious structural and functional data that are available from exhaustive studies in many laboratories.  相似文献   

5.
Structure of PvuII endonuclease with cognate DNA.   总被引:9,自引:2,他引:9       下载免费PDF全文
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.  相似文献   

6.
Recognizing the methylation status of specific DNA sequences is central to the function of many systems in eukaryotes and prokaryotes. Restriction–modification systems have to distinguish between ‘self’ and ‘non-self’ DNA and depend on the inability of restriction endonucleases to cleave their DNA substrates when the DNA is appropriately methylated. These endonucleases thus provide a model system for studying the recognition of DNA methylation by proteins. We have characterized the interaction of R·PvuII with DNA containing the physiologically relevant N4-methylcytosine modification. R·PvuII binds N4mC-modified DNA and cleaves it very slowly. Methylated strands in hemimethylated duplexes were cleaved at a higher rate than in fully methylated duplexes, in parallel with a higher binding affinity for hemimethylated DNA. The co-crystal structures of R·PvuII–DNA, together with a mutagenesis study, have implicated specific amino acids in recognition of the methylatable base; one of these is His84. We report that replacing His84 with Ala reduced the rate of cleavage of unmodified DNA but, in contrast, slightly increased the cleavage of N4mC-modified DNA.  相似文献   

7.
Alteration of the specificity of PvuII restriction endonuclease.   总被引:2,自引:1,他引:2       下载免费PDF全文
The restriction endonuclease PvuII which cleaves the sequence CAGCTG, at the position indicated by the arrow, was found to decrease its substrate specificity in the presence of organic solvents. Thirty-three sites, that we have named PvuII sites, were identified on the nucleotide sequence of pBR322 DNA. The new recognition sequences cleaved in pBR322 DNA, at the positions indicated by the arrows, were shown to be AAGCTG, GAGCTG, CNGCTG, CANCTG, CAGNTG, CAGCNG, CAGCTC and CAGCTT. (TAGCTG and the complementary sequence CAGCTA are not present in pBR322 DNA). From these recognition sequences, we deduced that PvuII activity recognizes and cleaves degenerate sequences which differ from the standard PvuII sequence CAGCTG at only one of the recognition site. Any substitution can occur at any one of the six positions in the hexanucleotide sequence. The optimum incubation medium for PvuII activity was found to be: 10-50 mM Tris-HCl, pH 8.5, 12-15 mM MgCl2, 50 mM NaCl, 10% ethanol + 10% dimethylsulfoxide (DMSO).  相似文献   

8.
PI-SceI, a homing endonuclease of the LAGLIDADG family, consists of two domains involved in DNA cleavage and protein splicing, respectively. Both domains cooperate in binding the recognition sequence. Comparison of the structures of PI-SceI in the absence and presence of substrate reveals major conformational changes in both the protein and DNA. Notably, in the protein-splicing domain the loop comprising residues 53-70 and adopts a "closed" conformation, thus enabling it to interact with the DNA. We have studied the dynamics of DNA binding and subsequent loop movement by fluorescence techniques. Six amino acids in loop53-70 were individually replaced by cysteine and modified by fluorescein. The interaction of the modified PI-SceI variants with the substrate, unlabeled or labeled with tetramethylrhodamine, was analyzed in equilibrium and stopped-flow experiments. A kinetic scheme was established describing the interaction between PI-SceI and DNA. It is noteworthy that the apparent hinge-flap motion of loop53-70 is only observed in the presence of a divalent metal ion cofactor. Substitution of the major Mg2+-binding ligands in PI-SceI, Asp-218 and Asp-326, by Asn or "nicking" PI-SceI with trypsin at Arg-277, which interferes with formation of an active enzyme.substrate complex, both prevent the conformational change of loop53-70. Deletion of the loop inactivates the enzyme. We conclude that loop53-70 is an important structural element that couples DNA recognition by the splicing domain with DNA cleavage by the catalytic domain and as such "communicates" with the Mg2+ binding sites at the catalytic centers.  相似文献   

9.
The ability to construct molecular motifs with predictable properties in aqueous solution requires an extensive knowledge of the relationships between structure and energetics. The design of metal binding motifs is currently an area of intense interest in the bioorganic community. To date synthetic motifs designed to bind metal ions lack the remarkable affinities observed in biological systems. To better understand the structural basis of metal ion affinity, we report here the thermodynamics of binding of divalent zinc ions to wild-type and mutant carbonic anhydrases and the interpretation of these parameters in terms of structure. Mutations were made both to the direct His ligand at position 94 and to indirect, or second-shell, ligands Gln-92, Glu-117, and Thr-199. The thermodynamics of ligand binding by several mutant proteins is complicated by the development of a second zinc binding site on mutation; such effects must be considered carefully in the interpretation of thermodynamic data. In all instances modification of the protein produces a complex series of changes in both the enthalpy and entropy of ligand binding. In most cases these effects are most readily rationalized in terms of ligand and protein desolvation, rather than in terms of changes in the direct interactions of ligand and protein. Alteration of second-shell ligands, thought to function primarily by orienting the direct ligands, produces profoundly different effects on the enthalpy of binding, depending on the nature of the residue. These results suggest a range of activities for these ligands, contributing both enthalpic and entropic effects to the overall thermodynamics of binding. Together, our results demonstrate the importance of understanding relationships between structure and hydration in the construction of novel ligands and biological polymers.  相似文献   

10.
Understanding the energetic consequences of molecular structure in aqueous solution is a prerequisite to the rational design of synthetic motifs with predictable properties. Such properties include ligand binding and the collapse of polymer chains into discrete three-dimensional structures. Despite advances in macromolecular structure determination, correlations of structure with high-resolution thermodynamic data remain limited. Here we compare thermodynamic parameters for the binding of Zn(II), Cu(II), and Co(II) to human carbonic anhydrase II. These calorimetrically determined values are interpreted in terms of high-resolution X-ray crystallographic data. While both zinc and cobalt are bound with a 1:1 stoichiometry, CAII binds two copper ions. Considering only the high-affinity site, there is a diminution in the enthalpy of binding through the series Co(II) --> Zn(II) --> Cu(II) that mirrors the enthalpy of hydration; this observation reinforces the notion that the thermodynamics of solute association with water is at least as important as the thermodynamics of solute-solute interaction and that these effects must be considered when interpreting association in aqueous solution. Additionally, DeltaC(p) data suggest that zinc binding to CAII proceeds with a greater contribution from desolvation than does binding of either copper or cobalt, suggesting Nature optimizes binding by optimizing desolvation.  相似文献   

11.
Homing endonucleases (HE) have emerged as precise tools for achieving gene targeting events. Redesigned HEs with tailored specificities can be used to cleave new sequences, thereby considerably expanding the number of targetable genes and loci. With HEs, as well as with other protein scaffolds, context dependence of DNA/protein interaction patterns remains one of the major limitations for rational engineering of new DNA binders. Previous studies have shown strong crosstalk between different residues and regions of the DNA binding interface. To investigate this phenomenon, we systematically combined mutations from three groups of amino acids in the DNA binding regions of the I-CreI HE. Our results confirm that important crosstalk occurs throughout this interface in I-CreI. Detailed analysis of success rates identified a nearest-neighbour effect, with a more pronounced level of dependence between adjacent regions. Taken together, these data suggest that combinatorial engineering does not necessarily require the identification of separable functional or structural regions, and that groups of amino acids provide acceptable building blocks that can be assembled, overcoming the context dependency of the DNA binding interface. Furthermore, the present work describes a sequential method to engineer tailored HEs, wherein three contiguous regions are individually mutated and assembled to create HEs with engineered specificity.  相似文献   

12.
Xie F  Qureshi SH  Papadakos GA  Dupureur CM 《Biochemistry》2008,47(47):12540-12550
Ester hydrolysis is one of the most ubiquitous reactions in biochemistry. Many of these reactions rely on metal ions for various mechanistic steps. A large number of metal-dependent nucleases have been crystallized with two metal ions in their active sites. In spite of an ongoing discussion about the roles of these metal ions in nucleic acid hydrolysis, there are very few studies which examine this issue using the native cofactor Mg(II) and global fitting of reaction progress curves. As part of a comprehensive study of the representative homodimeric PvuII endonuclease, we have collected single-turnover DNA cleavage data as a function of Mg(II) concentration and globally fit these data to a number of models which test various aspects of the metallonuclease mechanism. DNA association rate constants are approximately 100-fold higher in the presence of the catalytically nonsupportive Ca(II) versus the native cofactor Mg(II), highlighting an interesting cofactor difference. A pathway in which metal ions bind prior to DNA is kinetically favored. The data fit well to a model in which both one and two metal ions per active site (EM(2)S and EM(4)S, respectively) support cleavage. Interestingly, the cleavage rate for EM(2)S is approximately 100-fold slower than that displayed by EM(4)S. Collectively, these data indicate that for the PvuII system, catalysis involving one metal ion per active site can indeed occur, but that a more efficient two-metal ion mechanism can be operative under saturating metal ion (in vitro) conditions.  相似文献   

13.
Schierling B  Wende W  Pingoud A 《FEBS letters》2012,586(12):1736-1741
The restriction endonuclease PvuII has been introduced as a sequence-specific cleavage module in highly-specific nucleases for gene targeting. Here, a structural reorganization of the single-chain variant of PvuII (scPvuII) was performed by circular permutation as a proof-of-concept in order to find out whether the relocated, new termini next to structural elements important for DNA recognition and catalysis could be used for the fusion with other regulatory protein domains. Three circularly permuted variants of scPvuII were obtained that all maintain the specific endonucleolytic activity of scPvuII.  相似文献   

14.
The structure of the rare-cutting restriction endonuclease NotI, which recognizes the 8 bp target 5'-GCGGCCGC-3', has been solved with and without bound DNA. Because of its specificity (recognizing a site that occurs once per 65 kb), NotI is used to generate large genomic fragments and to map DNA methylation status. NotI contains a unique metal binding fold, found in a variety of putative endonucleases, occupied by an iron atom coordinated within a tetrahedral Cys4 motif. This domain positions nearby protein elements for DNA recognition, and serves a structural role. While recognition of the central six base pairs of the target is accomplished via a saturated hydrogen bond network typical of restriction enzymes, the most peripheral base pairs are engaged in a single direct contact in the major groove, reflecting reduced pressure to recognize those positions. NotI may represent an evolutionary intermediate between mobile endonucleases (which recognize longer target sites) and canonical restriction endonucleases.  相似文献   

15.
The type IIs restriction endonuclease MboII recognizes nonsymmetrical GAAGA sites, cutting 8 (top strand) and 7 (bottom strand) bases to the right. Gel retardation showed that MboII bound specifically to GAAGA sequences, producing two distinct complexes each containing one MboII and one DNA molecule. Interference analysis indicated that the initial species formed, named complex 1, comprised an interaction between the enzyme and the GAAGA target. Complex 2 involved interaction of the protein with both the GAAGA and the cutting sites. Only in the presence of divalent metal ions such as Ca(2+) is the conversion of complex 1 to 2 rapid. Additionally, a very retarded complex was seen with Ca(2+), possibly a (MboII)(2)-(DNA)(2) complex. Plasmids containing a single GAAGA site were hydrolyzed slowly by MboII. Plasmids containing two sites were cut far more rapidly, suggesting that the enzyme requires two recognition sites in the same DNA molecule for efficient hydrolysis. MboII appears to have a mechanism similar to the best characterized type IIs enzyme, FokI. Both enzymes initially bind DNA as monomers, followed by dimerization to give an (enzyme)(2)-(DNA)(2) complex. Dimerization is efficient only when the two target sites are located in the same DNA molecule and requires divalent metal ions.  相似文献   

16.
The structure of I-HmuI, which represents the last family of homing endonucleases without a defining crystallographic structure, has been determined in complex with its DNA target. A series of diverse protein structural domains and motifs, contacting sequential stretches of nucleotide bases, are distributed along the DNA target. I-HmuI contains an N-terminal domain with a DNA-binding surface found in the I-PpoI homing endonuclease and an associated HNH/N active site found in the bacterial colicins, and a C-terminal DNA-binding domain previously observed in the I-TevI homing endonuclease. The combination and exchange of these features between protein families indicates that the genetic mobility associated with homing endonucleases extends to the level of independent structural domains. I-HmuI provides an unambiguous structural connection between the His-Cys box endonucleases and the bacterial colicins, supporting the hypothesis that these enzymes diverged from a common ancestral nuclease.  相似文献   

17.
Non-specific binding of restriction endonuclease EcoR1 to DNA.   总被引:5,自引:5,他引:5       下载免费PDF全文
Restriction endonuclease, EcoRl cleaves the DNA sequence (see formula in text) at the points indicated. Under certain conditions, EcoRl activity is observed when (see formula in text) is cut. Mg2+ is required for both activities. We find that in addition to binding to the above sites, EcoRl will also bind, although less strongly, to DNA containing neither site. Methyl acetimidate, which reacts specifically with lysine residues, inactivates the enzyme. This specific effect can be prevented by SV40 DNA and lambda DNA which contain EcoRl and EcoRl sites, by 0X174 DNA, which has only EcoRl sites and also by Polyd(AT) and polyd(GC) containing neither site. Protection occurs in the absence or presence of magnesium. The significance of this non-specific binding, both for the use and mechanism of EcoRl will be discussed.  相似文献   

18.
19.
The PvuII endonuclease (PvuIIR) is a restriction enzyme from a type II restriction-modification system of Proteus vulgaris coded on plasmid pPvu1. The protein recognizes the DNA sequence 5' CAG'CTG 3' and shows no sequence homology to other restriction enzymes. This makes PvuIIR an interesting subject for structural determination. A purification procedure was developed that yields milligram quantities of the PvuIIR from plasmids expressed in the Escherichia coli strain HB101. The protein was crystallized using ammonium sulphate as precipitant. The crystals are orthorhombic, space group P2(1)2(1)2 with cell dimensions: a = 84.2 A, b = 106.2 A, c = 46.9 A. The asymmetric unit contains one PvuIIR dimer. Diffraction extends to 2.3 A, so the crystals may permit structural determination at atomic resolution.  相似文献   

20.
On the basis of structural work, metal ions are proposed to play a catalytic role in reactions mediated by many phosphoryl transfer enzymes. To gain dynamic support for such mechanisms, the role of metal ion cofactors in phosphate diester hydrolysis catalysed by a flap endonuclease has been studied. The pH maximal rate profiles were measured in the presence of various metal ion cofactors; in each case, a single ionic form of the enzyme/cofactor accounts for the pH dependence. The kinetic pK(a)s display good correlation with the acidity of the corresponding hexahydrated metal ions, which strongly suggests a role for metal-bound hydroxide, or its equivalent ionic species, in the reaction. Comparing rates of reaction in the pH-independent regions, a small negative beta(nuc) value is observed. This suggests that expected trends in the nucleophilicity of the various metal-bound hydroxides are balanced by a second form of metal ion catalysis that is related to the acidity of the hexahydrated metal ion. This is likely to be either electrophilic catalysis or leaving group activation.  相似文献   

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