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1.
Dupureur CM  Conlan LH 《Biochemistry》2000,39(35):10921-10927
In efforts to understand the mechanisms of many nucleic acid enzymes, the first site-directed mutations are made at conserved acidic active residues. Almost without exception, the low or null activities of the resulting variants are attributed to the importance of the acidic residue(s) to the ligation of required metal ions. Using (25)Mg NMR spectroscopy as a direct probe of metal ion binding and the homodimeric PvuII restriction endonuclease as a model system, this interpretation is examined and clarified. Our results indicate that Mg(II) binds wild-type PvuII endonuclease in the absence of DNA with a K(d,app) of 1.9 mM. Hill analysis yields an n(H) coefficient of 1.4, a value consistent with the binding of more than one Mg(II) ion per monomer active site. Variable pH studies indicate that two ionizable groups are responsible for Mg(II) binding by wild-type PvuII endonuclease near physiological pH. The pK(a,app) for these ionizations is 6.7, a value which is unusual for acidic residues but consistent with data obtained for critical groups in MunI endonuclease and a number of other hydrolases. To assign residues critical to ligating Mg(II), binding measurements were performed on the low activity catalytic site mutants E68A and D58A. As expected, E68A binds Mg(II) ions very weakly (K(d,app) approximately 40 mM), implicating Glu68 as critical to Mg(II) binding. Interestingly, while D58A has only residual specific activity, it retains an affinity for Mg(II) with a K(d,app) of 3.6 mM and exhibits a Hill coefficient of 0.7. Moreover, in this variant, multiple ionizable groups with pK(a,app) of 7.2 are involved in Mg(II) binding, suggesting a shuffling of Mg(II) ligands in the active site. These data indicate that Asp58 is important for the critical positioning of metal ion(s) required for catalysis.  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.
Dupureur CM 《Biochemistry》2005,44(13):5065-5074
Sequence specific DNA binding proteins are thought to adopt distinct conformations when binding to target (cognate) and nontarget (noncognate) sequences. There is both biochemical and crystallographic evidence that this behavior is important in mediating sequence recognition by the Mg(II)-dependent type II restriction enzymes. Despite this, there are few systematic comparisons of the structural behavior of these enzymes in various complexes. Here, (1)H-(15)N HSQC NMR spectroscopy is applied to PvuII endonuclease (2 x 18 kDa) in an effort to better understand the relationship between sequence recognition and enzyme conformational behavior. Spectra of the free enzyme collected in the absence and presence of metal ions indicate that while there is a modest backbone conformational response upon binding Ca(II), this does not occur with Mg(II). Substrate binding itself is accompanied by very dramatic spectral changes consistent with a large-scale conformational response. HSQC spectra of the enzyme bound to cognate (specific) and noncognate (nonspecific) oligonucleotides in the presence of Ca(II) are dramatically distinct, revealing for the first time the structural uniqueness of a PvuII cognate complex in solution. The strong correlation between NMR spectral overlap and crystallographic data (C(alpha) rmsd) permits characterization of the nonspecific PvuII complex as being more similar to the free enzyme than to the specific complex. Collectively, these data support the notion that it is the DNA, not the metal ion, which promotes a unique conformational response by the enzyme. It therefore follows that the principle role of metal ions in complex formation is one of driving substrate affinity and stability rather than conformationally priming the enzyme for substrate binding and sequence recognition. These results not only provide valuable insights into the mechanism of protein-DNA interactions but also demonstrate the utility of NMR spectroscopy in structure-function studies of these representative nucleic acid systems.  相似文献   

4.
Bowen LM  Muller G  Riehl JP  Dupureur CM 《Biochemistry》2004,43(48):15286-15295
Type II restriction enzymes are homodimeric systems that bind four to eight base pair palindromic recognition sequences of DNA and catalyze metal ion-dependent phosphodiester cleavage. While Mg(II) is required for cleavage in these enzymes, in some systems Ca(II) promotes avid substrate binding and sequence discrimination. These properties make them useful model systems for understanding the roles of alkaline earth metal ions in nucleic acid processing. We have previously shown that two Ca(II) ions stimulate DNA binding by PvuII endonuclease and that the trivalent lanthanide ions Tb(III) and Eu(III) support subnanomolar DNA binding in this system. Here we capitalize on this behavior, employing a unique combination of luminescence spectroscopy and DNA binding assays to characterize Ln(III) binding behavior by this enzyme. Upon excitation of tyrosine residues, the emissions of both Tb(III) and Eu(III) are enhanced severalfold. This enhancement is reduced by the addition of a large excess of Ca(II), indicating that these ions bind in the active site. Poor enhancements and affinities in the presence of the active site variant E68A indicate that Glu68 is an important Ln(III) ligand, similar to that observed with Ca(II), Mg(II), and Mn(II). At low micromolar Eu(III) concentrations in the presence of enzyme (10-20 microM), Eu(III) excitation (7)F(0) --> (5)D(0) spectra yield one dominant peak at 579.2 nm. A second, smaller peak at 579.4 nm is apparent at high Eu(III) concentrations (150 microM). Titration data for both Tb(III) and Eu(III) fit well to a two-site model featuring a strong site (K(d) = 1-3 microM) and a much weaker site (K(d) approximately 100-200 microM). Experiments with the E68A variant indicate that the Glu68 side chain is not required for the binding of this second Ln(III) equivalent; however, the dramatic increase in DNA binding affinity around 100 microM Ln(III) for the wild-type enzyme and metal-enhanced substrate affinity for E68A are consistent with functional relevance for this weaker site. This discrimination of sites should make it possible to use lanthanide substitution and lanthanide spectroscopy to probe individual metal ion binding sites, thus adding an important tool to the study of restriction enzyme structure and function.  相似文献   

5.
Bowen LM  Dupureur CM 《Biochemistry》2003,42(43):12643-12653
Restriction enzymes are important model systems for understanding the mechanistic contributions of metal ions to nuclease activity. These systems are unique in that they combine distinct functions which have been shown to depend on metal ions: high-affinity DNA binding, sequence-specific recognition of DNA, and Mg(II)-dependent phosphodiester cleavage. While Ca(II) and Mn(II) are commonly used to promote DNA binding and cleavage, respectively, the metal ion properties that are critical to the support of these functions are not clear. To address this question, we assessed the abilities of a series of metal ions to promote DNA binding, sequence specificity, and cleavage in the representative PvuII endonuclease. Among the metal ions tested [Ca(II), Sr(II), Ba(II), Eu(III), Tb(III), Cd(II), Mn(II), Co(II), and Zn(II)], only Mn(II) and Co(II) were similar enough to Mg(II) to support detectable cleavage activity. Interestingly, cofactor requirements for the support of DNA binding are much more permissive; the survey of DNA binding cofactors indicated that Cd(II) and the heavier and larger alkaline earth metal ions Sr(II) and Ba(II) were effective cofactors, stimulating DNA binding affinity 20-200-fold. Impressively, the trivalent lanthanides Tb(III) and Eu(III) promoted DNA binding as efficiently as Ca(II), corresponding to an increase in affinity over 1000-fold higher than that observed under metal-free conditions. The trend for DNA binding affinity supported by these ions suggests that ionic radius and charge are not critical to the promotion of DNA binding. To examine the role of metal ions in sequence discrimination, we determined specificity factors [K(a)(specific)/K(a)(nonspecific)] in the presence of Cd(II), Ba(II), and Tb(III). Most interestingly, all of these ions compromised sequence specificity to some degree compared to Ca(II), by either increased affinity for a noncognate sequence, decreased affinity for the cognate sequence, or both. These results suggest that while amino acid-base contacts are important for specificity, the properties of metal ion cofactors at the catalytic site are also critical for sequence discrimination. This insight is invaluable to our efforts to understand and subsequently design sequence-specific nucleases.  相似文献   

6.
The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 A from both metal ions in the enzyme-substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H-15N heteronuclear single quantum coherence spectra of enzyme-Ca(II) and enzyme-Ca(II)-DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.  相似文献   

7.
The activities of restriction enzymes are important examples of Mg(II)-dependent hydrolysis of DNA. While a number of crystallographic studies of enzyme-DNA complexes have also involved metal ions, there have been no solution studies exploring the relationship between enzyme conformation and metal-ion binding in restriction enzymes. Using PvuII restriction endonuclease as a model system, we have successfully developed biosynthetic fluorination and NMR spectroscopy as a solution probe of restriction-enzyme conformation. The utility of this method is demonstrated with a study of metal-ion binding by PvuII endonuclease. Replacement of 74% (+/- 10%) of the Tyr residues in PvuII endonuclease by 3-fluorotyrosine produces an enzyme with Mg(II)-supported specific activity and sequence specificity that is indistinguishable from that of the native enzyme. Mn(II) supports residual activity of both the native and fluorinated enzymes; Ca(II) does not support activity in either enzyme, a result consistent with previous studies. 1H- and 19F-NMR spectroscopic studies reveal that while Mg(II) does not alter the enzyme conformation, the paramagnetic Mn(II) produces both short-range spectral broadening and longer range changes in chemical shift. Most interestingly, Ca(II) binding perturbs a larger number of different resonances than Mn(II). Coupled with earlier mutagenesis studies that place Ca(II) in the active site [Nastri, H. G., Evans, P.D., Walker, I.H. & Riggs, P.D. (1997) J. Biol. Chem. 272, 25761-25767], these data suggest that the enzyme makes conformational adjustments to accommodate the distinct geometric preferences of Ca(II) and may play a role in the inability of this metal ion to support activity in restriction enzymes.  相似文献   

8.
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   

9.
We have determined the crystal structure of the PvuII endonuclease in the presence of Mg(2+). According to the structural data, divalent metal ion binding in the PvuII subunits is highly asymmetric. The PvuII-Mg(2+) complex has two distinct metal ion binding sites, one in each monomer. One site is formed by the catalytic residues Asp58 and Glu68, and has extensive similarities to a catalytically important site found in all structurally examined restriction endonucleases. The other binding site is located in the other monomer, in the immediate vicinity of the hydroxyl group of Tyr94; it has no analogy to metal ion binding sites found so far in restriction endonucleases. To assign the number of metal ions involved and to better understand the role of Mg(2+) binding to Tyr94 for the function of PvuII, we have exchanged Tyr94 by Phe and characterized the metal ion dependence of DNA cleavage of wild-type PvuII and the Y94F variant. Wild-type PvuII cleaves both strands of the DNA in a concerted reaction. Mg(2+) binding, as measured by the Mg(2+) dependence of DNA cleavage, occurs with a Hill coefficient of 4, meaning that at least two metal ions are bound to each subunit in a cooperative fashion upon formation of the active complex. Quenched-flow experiments show that DNA cleavage occurs about tenfold faster if Mg(2+) is pre-incubated with enzyme or DNA than if preformed enzyme-DNA complexes are mixed with Mg(2+). These results show that Mg(2+) cannot easily enter the active center of the preformed enzyme-DNA complex, but that for fast cleavage the metal ions must already be bound to the apoenzyme and carried with the enzyme into the enzyme-DNA complex. The Y94F variant, in contrast to wild-type PvuII, does not cleave DNA in a concerted manner and metal ion binding occurs with a Hill coefficient of 1. These results indicate that removal of the Mg(2+) binding site at Tyr94 completely disrupts the cooperativity in DNA cleavage. Moreover, in quenched-flow experiments Y94F cleaves DNA about ten times more slowly than wild-type PvuII, regardless of the order of mixing. From these results we conclude that wild-type PvuII cleaves DNA in a fast and concerted reaction, because the Mg(2+) required for catalysis are already bound at the enzyme, one of them at Tyr94. We suggest that this Mg(2+) is shifted to the active center during binding of a specific DNA substrate. These results, for the first time, shed light on the pathway by which metal ions as essential cofactors enter the catalytic center of restriction endonucleases.  相似文献   

10.
The PspGI restriction–modification system recognizes the sequence CCWGG. R.PspGI cuts DNA before the first C in the cognate sequence and M.PspGI is thought to methylate N4 of one of the cytosines in the sequence. M.PspGI enhances fluorescence of 2-aminopurine in DNA if it replaces the second C in the sequence, while R.PspGI enhances fluorescence when the fluorophore replaces adenine in the central base pair. This strongly suggests that the methyltransferase flips the second C in the recognition sequence, while the endonuclease flips both bases in the central base pair out of the duplex. M.PspGI is the first N4-cytosine MTase for which biochemical evidence for base flipping has been presented. It is also the first type IIP methyltransferase whose catalytic activity is strongly stimulated by divalent metal ions. However, divalent metal ions are not required for its base-flipping activity. In contrast, these ions are required for both base flipping and catalysis by the endonuclease. The two enzymes have similar temperature profiles for base flipping and optimal flipping occurs at temperatures substantially below the growth temperature of the source organism for PspGI and for the catalytic activity of endonuclease. We discuss the implications of these results for DNA binding by these enzymes and their evolutionary origin.  相似文献   

11.
12.
The active sites of Mg(II)-dependent nucleases feature a cluster of conserved charged residues which includes both acidic (Asp and Glu) and basic (Lys) side chains. In restriction enzymes, these side chains are part of the conserved PD...(D/E)XK functional sequence motif which has been implicated as being important in metal ion binding and catalytic steps. Recent work revealing the unusual behavior of the active site variant D58A of the representative PvuII endonuclease prompted speculation that the array of charged groups in the nuclease active site may also be linked to conformational behavior [Dupureur, C. M., and Conlan, L. H. (2000) Biochemistry 39, 10921-10927]. To address this issue, we analyzed the conformational behavior of active site variants of PvuII endonuclease using both NMR spectroscopic and thermodynamic methods. NMR spectroscopic analysis via (19)F and (1)H-(15)N HSQC experiments indicates that a number of side chain and backbone amide groups are perturbed upon Ala substitution at conserved active site residues Asp58, Glu68, and Lys70. Spectral changes are particularly pronounced for the lowest-activity mutants (D58A and K70A). These changes are accompanied by perturbations in conformational stability. Ala substitution at each of these positions results in 2-5 kcal/mol of stabilization over the wild-type enzyme at pH 7.7, changes which constitute increases in DeltaG(d)(H2O) of 20-50%. The pH dependencies of mutant enzyme stabilities are distinct from those of the wild type, results which confirm that these ionizable groups strongly influence stability. Wild-type enzyme stability is correlated with the ionization of groups shown to be important to metal ion binding and orientation. Correlations between spectral changes and conformational stability indicate that the latter measurements may prove useful in the evaluation of site-directed mutant restriction enzymes. More importantly, these results indicate that structure-function relationships in restriction enzyme active sites can be complex, and that the ensemble of conserved charged residues which mediate DNA hydrolysis in Mg(II)-dependent nucleases constitutes a critical link between function and conformation.  相似文献   

13.
The crystal structure of EcoRV endonuclease bound to non-cognate DNA at 2.0 angstroms resolution shows that very small structural adaptations are sufficient to ensure the extreme sequence specificity characteristic of restriction enzymes. EcoRV bends its specific GATATC site sharply by 50 degrees into the major groove at the center TA step, generating unusual base-base interactions along each individual DNA strand. In the symmetric non-cognate complex bound to GAATTC, the center step bend is relaxed to avoid steric hindrance caused by the different placement of the exocyclic thymine methyl groups. The decreased base-pair unstacking in turn leads to small conformational rearrangements in the sugar-phosphate backbone, sufficient to destabilize binding of crucial divalent metal ions in the active site. A second crystal structure of EcoRV bound to the base-analog GAAUTC site shows that the 50 degrees center-step bend of the DNA is restored. However, while divalent metals bind at high occupancy in this structure, one metal ion shifts away from binding at the scissile DNA phosphate to a position near the 3'-adjacent phosphate group. This may explain why the 10(4)-fold attenuated cleavage efficiency toward GAATTC is reconstituted by less than tenfold toward GAAUTC. Examination of DNA binding and bending by equilibrium and stopped-flow florescence quenching and fluorescence resonance energy transfer (FRET) methods demonstrates that the capacity of EcoRV to bend the GAATTC non-cognate site is severely limited, but that full bending of GAAUTC is achieved at only a threefold reduced rate compared with the cognate complex. Together, the structural and biochemical data demonstrate the existence of distinct mechanisms for ensuring specificity at the bending and catalytic steps, respectively. The limited conformational rearrangements observed in the EcoRV non-cognate complex provide a sharp contrast to the extensive structural changes found in a non-cognate BamHI-DNA crystal structure, thus demonstrating a diversity of mechanisms by which restriction enzymes are able to achieve specificity.  相似文献   

14.
Most nucleases rely on divalent cations as cofactors to catalyze the hydrolysis of nucleic acid phosphodiester bonds. Here both equilibrium and kinetic experiments are used to test recently proposed models regarding the metal ion dependence of product release and the degree of cooperativity between metal ions bound in the active sites of the homodimeric PvuII endonuclease. Equilibrium fluorescence anisotropy studies indicate that product binding is dramatically weakened in the presence of metal ions. Pre-steady state kinetics indicate that product release is at least partially rate limiting. Steady state and pre-steady state data fit best to models in which metals remain bound to the enzyme after the release of product. Finally, analysis of cooperative and independent binding models for metal ions indicates that single turnover kinetic data are consistent with little to no positive cooperativity between the two metal ions binding each active site.  相似文献   

15.
16.
Reid SL  Parry D  Liu HH  Connolly BA 《Biochemistry》2001,40(8):2484-2494
Oligonucleotides labeled with hexachlorofluorescein (hex) have enabled the interaction of the restriction endonuclease EcoRV with DNA to be evaluated using fluorescence anisotropy. The sensitivity of hex allowed measurements at oligonucleotide concentrations as low as 1 nM, enabling K(D) values in the low nanomolar range to be measured. Both direct titration, i.e., addition of increasing amounts of the endonuclease to hex-labeled oligonucleotides, and displacement titration, i.e., addition of unlabeled oligonucleotide to preformed hex-oligonucleotide/EcoRV endonuclease complexes, have been used for K(D) determination. Displacement titration is the method of choice; artifacts due to any direct interaction of the enzyme with the dye are eliminated, and higher fluorescent-labeled oligonucleotide concentrations may be used, improving signal-to-noise ratio. Using this approach (with three different oligonucleotides) we found that the EcoRV restriction endonuclease showed a preference of between 1.5 and 6.5 for its GATATC target sequence at pH 7.5 and 100 mM NaCl, when the divalent cation Ca2+ is absent. As expected, both the presence of Ca2+ and a decrease in pH value stimulated the binding of specific sequences but had much less effect on nonspecific ones.  相似文献   

17.
The PvuII restriction endonuclease has been converted from its natural homodimeric form into a single polypeptide chain by tandemly linking the two subunits through a short peptide linker. The arrangement of the single-chain PvuII (sc PvuII) is (2-157)-GlySerGlyGly-(2-157), where (2-157) represents the amino acid residues of the enzyme subunit and GlySerGlyGly is the peptide linker. By introducing the corresponding tandem gene into Escherichia coli, PvuII endonuclease activity could be detected in functional in vivo assays. The sc enzyme was expressed at high level as a soluble protein. The purified enzyme was shown to have the molecular mass expected for the designed sc protein. Based on the DNA cleavage patterns obtained with different substrates, the cleavage specificity of the sc PvuII is indistinguishable from that of the wild-type (wt) enzyme. The sc enzyme binds specifically to the cognate DNA site under non-catalytic conditions, in the presence of Ca2+, with the expected 1:1 stoichiometry. Under standard catalytic conditions, the sc enzyme cleaves simultaneously the two DNA strands in a concerted manner. Steady-state kinetic parameters of DNA cleavage by the sc and wt PvuII showed that the sc enzyme is a potent, but somewhat less efficient catalyst; the k(cat)/K(M) values are 1.11 x 10(9) and 3.50 x 10(9) min(-1) M(-1) for the sc and wt enzyme, respectively. The activity decrease is due to the lower turnover number and to the lower substrate affinity. The sc arrangement provides a facile route to obtain asymmetrically modified heterodimeric enzymes.  相似文献   

18.
Structure of PvuII endonuclease with cognate DNA.   总被引:11,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   

19.
W Cao 《Biochemistry》1999,38(25):8080-8087
Restriction endonucleases achieve sequence-specific recognition and strand cleavage through the interplay of base, phosphate backbone, and metal cofactor interactions. In this study, we investigate the binding kinetics of TaqI endonuclease using the wild-type enzyme and a binding proficient, catalysis deficient mutant TaqI-D137A both in the absence of a metal cofactor and in the presence of Mg2+ or Ca2+. As demonstrated by gel mobility shift analyses, TaqI endonuclease requires a metal cofactor for achieving high-affinity specific binding to its cognate sequence, TCGA. In the absence of a metal cofactor, the enzyme binds all DNA sequences (TaqI cognate site, star site, and nonspecific site) with essentially equal affinity, thereby exhibiting little discrimination. The dissociation constant of the cognate sequence in the presence of Mg2+ at 60 degrees C is 0. 26 nM, a value comparable to our previously reported Km of 0.5 nM measured under steady-state conditions. The TaqI-TCGA-Mg2+ complex is stable, with a half-life of 21 min at 60 degrees C. The boundary of the protein-DNA interface is approximated to be about 18 bp as determined by DNase I footprinting. Data from this study support the notion that a metal cofactor plays a critical role for achieving sequence-specific discrimination in a subset of nucleases, including TaqI, EcoRV, and others.  相似文献   

20.
A group of highly efficient Zn(II)-dependent RNA-cleaving deoxyribozymes has been obtained through in vitro selection. They share a common motif with the ‘8–17’ deoxyribozyme isolated under different conditions, including different design of the random pool and metal ion cofactor. We found that this commonly selected motif can efficiently cleave both RNA and DNA/RNA chimeric substrates. It can cleave any substrate containing rNG (where rN is any ribonucleotide base and G can be either ribo- or deoxyribo-G). The pH profile and reaction products of this deoxyribozyme are similar to those reported for hammerhead ribozyme. This deoxyribozyme has higher activity in the presence of transition metal ions compared to alkaline earth metal ions. At saturating concentrations of Zn2+, the cleavage rate is 1.35 min–1 at pH 6.0; based on pH profile this rate is estimated to be at least ~30 times faster at pH 7.5, where most assays of Mg2+-dependent DNA and RNA enzymes are carried out. This work represents a comprehensive characterization of a nucleic acid-based endonuclease that prefers transition metal ions to alkaline earth metal ions. The results demonstrate that nucleic acid enzymes are capable of binding transition metal ions such as Zn2+ with high affinity, and the resulting enzymes are more efficient at RNA cleavage than most Mg2+-dependent nucleic acid enzymes under similar conditions.  相似文献   

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