©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease
A STUDY USING OLIGODEOXYNUCLEOTIDE PHOSPHOROTHIOATES (*)

(Received for publication, November 3, 1995; and in revised form, January 31, 1996)

Harry Thorogood (1) Jane A. Grasby (2)(§) Bernard A. Connolly (1)(¶)

From the  (1)Department of Biochemistry and Genetics, The University of Newcastle, Newcastle upon Tyne NE2 4HH and the (2)Department of Biochemistry, University of Southampton, Southampton SO9 3TU, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
CONCLUSION
FOOTNOTES
REFERENCES

ABSTRACT

A set of phosphorothioate-containing oligonucleotides based on pGACGATATCGTC, a self-complementary dodecamer that contains the EcoRV recognition sequence (GATATC), has been prepared. The phosphorothioate group has been individually introduced at the central nine phosphate positions and the two diastereomers produced at each site separated and purified. The K and V(max) values found for each of these modified DNA molecules with the EcoRV restriction endonuclease have been determined and compared with those seen for the unmodified all-phosphate-containing dodecamer. This has enabled an evaluation of the roles that both of the non-esterified oxygen atoms in the individual phosphates play in DNA binding and hydrolysis by the endonuclease. The results have also been compared with crystal structures of the EcoRV endonuclease, complexed with an oligodeoxynucleotide, to allow further definition of phosphate group function during substrate binding and turnover. For further study, see the related article ``Probing the Indirect Readout of the Restriction Enzyme EcoRV: Mutational Analysis of Contacts to the DNA Backbone'' (Wenz, A., Jeltsch, A., and Pingoud, A. (1996) J. Biol. Chem. 271, 5565-5573).


INTRODUCTION

The specific recognition of DNA sequences by proteins involves the formation of a very precise and intimate interface between the two macromolecules. Direct contacts are observed between the protein and both the bases and phosphates, and in addition complicated networks bind the interacting elements together(1, 2, 3, 4) . Much of the emphasis has been on the study of contacts between the protein and the DNA bases. This, known as direct readout, is thought to contribute much of the selectivity(5, 6, 7) . The phosphate backbone has received less attention. However, as well as a passive role in endowing a basal DNA binding affinity to a DNA-binding protein, it is clear that the phosphates normally play additional, more active, roles in the generation of specificity. Work with the trp repressor has led to the concept of indirect readout(8) . Most simply, this takes place when a protein binds specifically to the phosphates in a DNA sequence that has an unusual conformation, with an altered phosphate backbone, that differs from ``ideal'' B-DNA. In addition, almost all DNA sequences are distorted on binding to proteins and this is often required to match up protein-phosphate contacts. Thus phosphates can play a role in selectivity following any DNA distortion that takes place on binding. This coupling of the recognition of specific bases, DNA distortion, and specific phosphate binding occurs with the restriction endonuclease EcoRI(9, 10) . The importance of DNA phosphate-protein contacts provides a compelling reason for their study. One of the commonest methods for evaluating protein-substrate interactions is to make alterations to the partners involved and observe the consequences. This has been widely carried out for protein amino acids using site-directed mutagenesis and for DNA with modified bases(11, 12, 13, 14, 15, 16) . For DNA phosphates the most useful analogues are the phosphorothioates (17, 18, 19, 20, 21) , illustrated in Fig. 1, which exist as a pair of diastereomers. Phosphorothioates probably represent the most conservative change that can be made to a phosphate. The sulfur atom is slightly bigger than the oxygen it replaces and the P-S bond length a little longer than the P-O(22) . Phosphorothioates are also slightly more acidic than phosphates (23) and may be differently solvated. With phosphates the negative charge is evenly distributed over the two non-bridging oxygens, but in the case of phosphorothioates, current evidence favors negative charge localization on sulfur(22, 24, 25) . Importantly Mg, an essential cofactor for the EcoRV endonuclease, strongly favors co-ordination to oxygen atoms in phosphorothioates(26, 27) . Despite these differences, the single known structure of a phosphorothioate-containing oligonucleotide shows minimal structural perturbations as compared to the all-phosphate parent(28) . Oligonucleotide phosphorothioates have been used to determine the stereochemical course of the reactions catalyzed by EcoRI (29) and EcoRV (30) and to probe details of protein-DNA interactions(31, 32, 33, 34) .


Figure 1: The structure of oligodeoxynucleotide phosphorothioates, which exist as a pair of diastereomers.



The EcoRV endonuclease is a well characterized enzyme (35, 36) for which crystallographic data is available(37, 38, 39) . A large body of kinetic and binding data has revealed that, in the absence of Mg, the endonuclease binds to all DNA sequences with equal affinity(40, 41, 42, 43) . However, specific contacts, made only to the bases in the cognate GATATC sequence cause severe distortion of the DNA, and this concomitantly creates a high affinity Mg binding site, allowing hydrolysis to take place. The observation that specific binding to GATATC sequences occurs in the presence of the essential metal ion, and that the DNA in these complexes is bent to the same extent as is observed in the crystal structure, lends support to the above model(44, 45, 46) . Recent data (39, 47, 48) has suggested that the endonuclease might use two metal ions for catalysis.

The endonuclease DNA contacts seen with cognate but not non-cognate sequences provide the energy required for the energetically unfavorable distortion of the bound DNA. This distortion is essential for hydrolysis, and so it is these interactions that are ultimately responsible for the discrimination of geq10^6(40) shown by the enzyme. The interactions, between the protein and the GATATC bases, have been probed using alternative sequences(40, 42, 49) , base analogues(13, 14, 15, 50, 51, 52, 53) , and site-directed mutagenesis(43, 54) , and many have been shown to be essential for efficient catalysis. The endonuclease also makes extensive contacts to the phosphate backbone (38, 39) . However, there has been little systematic investigation into the role that these interactions play in DNA recognition. M13 DNA, containing R(p)-phosphorothioates, showed that GATsATC sequences were very refractory to hydrolysis and substitution elsewhere tended to reduce cutting rates(32) . Very recently, the phosphorothioate oligonucleotides used in this study have been used to show the presence of a metal ion binding site distinct from the catalytic center(55) . In this publication, we examine the effects of both isomers of phosphorothioates within and immediately flanking the EcoRV restriction site on endonuclease-catalyzed hydrolysis and relate the results found to the available crystal structures.


EXPERIMENTAL PROCEDURES

EcoRV Endonuclease Purification

The purification of the EcoRV restriction endonuclease from overproducing Escherichia coli strains (56) by a slight modification of the method originally described (57) has been published(30) . SDS-gel electrophoresis and Coomassie Blue staining showed >95% purity. The concentration of stock solutions was determined using an E value of 18.0(57) .

Oligodeoxynucleotide Preparation

All oligonucleotides were prepared on a 1-µmol scale using phosphoramidite chemistry using an Applied Biosystems 381A DNA synthesizer. Reagents were purchased from Cruachem Ltd. (Glasgow, Scotland). Phosphorothioates were introduced using a 0.05 M acetonitrile solution of the sulfurizing agent 3H-1,2-benzodithiol-3-one 1,1-dioxide (58, 59) which was purchased from Cambio Ltd. (Cambridge, United Kingdom). The synthesis were interrupted immediately prior to the iodine oxidation step of the appropriate intermediate phosphite. The resin was washed with acetonitrile (2 min) and the sulfurizing agent applied for 20 s. After a 30-s pause, the resin was washed with acetonitrile (2 min) and normal synthesis resumed. 5`-Phosphates were added during the synthesis using the reagent 5`-phosphate-on (Cruachem Ltd.). All synthesis were performed trityl-off and the oligonucleotides removed from the resin and deblocked with ammonia in the normal way.

Oligonucleotide Purification and Phosphorothioate Diastereomer Separation

Purification and diastereomer separation was achieved by reverse phase HPLC (^1)on Apex-I octadecylsilyl (C-18) columns (5-µm particle size, 25 times 0.45 cm) purchased from Jones Chromatography (Llanbradach, Wales). For most oligonucleotides, triethylammonium-acetate buffers with an acetonitrile gradient were used (buffer A, 0.1 M acetic acid adjusted to pH 6.5 with triethylamine containing 2.5% acetonitrile; buffer B, 0.1 M acetic acid adjusted to pH 6.5 with triethylamine containing 65% acetonitrile). Gradients consisting of t = 0, 0% B, t = 5 min 0% B, t = 30 min 20% B were used. For the separation of the isomers of pGACGsATATCGTC and pGACGATAsTCGTC, buffers based on morpholine-acetate were used (buffer C, 0.1 M acetic acid adjusted to pH 6.5 with morpholine containing 2.5% acetonitrile; buffer D, 0.1 M acetic acid adjusted to pH 6.5 with morpholine containing 50% acetonitrile). The same gradient profile as above was used. All columns were run at 50 °C at 1 ml min. Following purification all oligonucleotides were desalted using disposable NAP-25 gel filtration columns (Pharmacia, St. Albans, United Kingdom). The concentrations of the oligonucleotides were determined using an E of 1.66 times 10^5M cm for the double-stranded form(13) .

Assignment of the Absolute Configuration of the Oligonucleotide Phosphorothioates by Base Composition Analysis

About 1 OD of the purified phosphorothioate was treated either with 10 µg of snake venom phosphodiesterase and 5 µg of alkaline phosphatase in 100 µl of 50 mM Hepes (pH 7.5), 100 mM NaCl, and 10 mM MgCl(2); or with 10 µg of nuclease P1 and 5 µg of alkaline phosphatase in the same buffer. After a 2-h incubation at 30 °C, the products formed were analyzed by reverse phase HPLC (columns and HPLC buffers A and B as above) using a gradient t = 0 min 0% B, t = 25 min 25% B, t = 35 min 50% B. Columns were run at 1 ml min and room temperature. The standard deoxynucleosides eluted in the order dC, dG, T, dA. dCMPs eluted between dC and dG, dGMPs and dTMPs between dG and T, and dAMPs between T and dA. All undigested dinucleotides, NsN, eluted after dA. The products formed in the enzymatic digests were identified by co-elution with standards. The dNTPs standards were a kind gift from Prof. F. Eckstein (Max Plank Institut für experimentelle Medizin, Göttingen, Germany). A few NsN standards were also obtained from this source, but most of the dinucleotide assignments are tentative due to a lack of reference material.

Cleavage of Oligonucleotide Phosphorothioates

To a 12 µM solution of oligonucleotide in 300 µl of 50 mM Hepes, pH 7.5, 100 mM NaCl, and 10 mM MgCl(2) was added EcoRV restriction endonuclease to a final concentration of 0.9 µM. The mixture was incubated at 20 °C and samples analyzed by HPLC at 1, 3, and 24 h. In cases where the digestion was incomplete, an additional aliquot of the endonuclease was added and the digestion continued for another 24 h. The HPLC protocol used the triethylammonium-acetate buffer system detailed under ``Oligonucleotide Purification and Phosphorothioate Separation.'' On completion of hydrolysis, the product peaks were purified by this HPLC method and analyzed for base composition as above.

K(m) and V(max) Determination

Kinetic constants were evaluated using a continuous UV assay (60) at 30 °C. A buffer consisting of 50 mM Hepes, pH 7.5, containing 55 mM NaCl and 25 mM MgCl(2) was used with volumes of either 1 ml (1-cm path length) or 2 ml (2-cm path length). Oligonucleotide concentrations varied between 0.15 and 7.5 µM. The reaction was initiated by the addition of endonuclease (concentrations between 5 and 50 nM), and in almost every case the oligonucleotide concentration was geq10 times the level of enzyme. The increase in absorbance at 254 nm was monitored using a Uvikon 930 spectrophotometer and the change in absorbance related to the amount of oligonucleotide hydrolyzed using the relationship: Deltaabsorbance (254 nm)/nmol oligonucleotide hydrolyzed = 0.034(60) . V(max) and K(m) values were evaluated from plots of [S]/v against [S], using eight different concentrations of the substrate. Each determination was carried out at least four times.


RESULTS

Synthesis of Phosphorothioate-containing Oligonucleotides

The dodecamers listed in Table 1were prepared using phosphoramidite chemistry and replacing the normal iodine/water oxidation step with a sulfurization using 3H-1,2-benzodithiol-3-one 1,1-dioxide(58, 59) . In all cases the 5`-phosphate was attached to the oligonucleotide as part of the chemical synthesis. Following deblocking of the oligonucleotides, analysis by reverse phase HPLC showed the presence of the two phosphorothioate diastereomers, which usually comprised more than 80% of the UV absorbing material (Fig. 2). The most demanding step in the synthesis was the separation of the two diastereomers. All the diastereomer pairs could be separated by reverse phase HPLC on C-18 columns, but the ease with which this could be achieved showed great variability. This has been observed in several previous studies(19, 61) . High temperatures (50 °C) were necessary for good separation, probably by melting the self-complementary strands. Most of the phosphorothioate isomers could be separated using acetonitrile gradients in triethylammonium acetate, pH 6.5, buffers. A few (pGACGsATATCGTC and pGACGATAsTCGTC) were poorly resolved in this buffer, and in these cases replacing the triethylammonium acetate with morpholine acetate, pH 6.5, resulted in better separation. Variations were also made to other HPLC parameters that included altering the organic component (methanol, ethanol, 2-propanol), changing the column material (C-4, C-8, phenyl) and using different pH values but none of these improved separation. Negatively charged oligodeoxynucleotides run as their salts during reverse phase purification and this may explain why the counter ion (triethylamine or morpholine) can alter separation parameters. However, the resolution of oligonucleotide phosphorothioates on C-18 HPLC is highly unpredictable. We are unable to forecast whether a particular pair of phosphorothioate isomers will be easy or difficult to resolve or to state the counter ion that will give the best separation for an individual case. Following optimization of the separation each individual phosphorothioate could be obtained at purities of greater than 97% with less than 3% contamination with the other isomer (Fig. 2).




Figure 2: The reverse phase HPLC trace of crude pGACGATATCsGTC (blue line). The two diastereomers are clearly visible as the two largest peaks between 10 and 12 min. The R(p) isomer elutes before the S(p). The traces of the purified diastereomers following HPLC separation are also shown. Green line, R(p) isomer; red line, S(p) isomer.



Many examples (17, 19, 30, 61) have shown that R(p)-oligonucleotide phosphorothioates elute before the S(p) on reverse phase columns using triethylammonium acetate buffers. This was found here in every case, and the faster elution of R(p)-isomers was also maintained when the buffer was altered to morpholine acetate. The configuration of fully purified oligonucleotide phosphorothioates can be unequivocally assigned using digestion with enzymes of known stereospecificities. Snake venom phosphodiesterase cuts oligonucleotide phosphorothioates having the R(p) conformation but does not digest those of S(p)(62) . These stereospecificities are reversed with nuclease P1(63) . Therefore the phosphorothioates were separately digested with nuclease P1 and snake venom phosphodiesterase and the resulting deoxynucleoside products were analyzed by reverse phase HPLC(17, 30) . When ``early'' isomers were treated with the snake venom enzyme, a peak corresponding to dNMPs was observed by HPLC, whereas reaction with P1 gave a NsN unhydrolyzed dinucleotide peak (not shown). ``Late'' isomers gave NsN with venom phosphodiesterase and dNMPs with nuclease P1 (not shown). This confirms that the early eluting isomers have the R(p) configuration and the late the S(p).

Hydrolysis of Phosphorothioate Oligonucleotides by the EcoRV Endonuclease

Prior to the determination of kinetic constants, all the oligonucleotides were incubated with a large quantity of the endonuclease (12 µM oligonucleotide, 0.9 µMEcoRV endonuclease) and any reaction monitored by HPLC after 1, 3, and 24 h. Most of the oligonucleotides were completely hydrolyzed to two new products (not shown). In some cases this required only 1 h, whereas in others the full 24 h was needed. Purification and base composition analysis of the two products, showed that, in every case, the endonuclease had cut at its true site (GATATC). These results show that the presence of a phosphorothioate does not change the cutting site of the enzyme. Furthermore, even at high endonuclease levels, no further decomposition of the products took place, indicating a high level of purity and the lack of any contaminating nucleases. R(p)-pGACGATsATCGTC was only partially hydrolyzed after 24 h, but the addition of another aliquot of enzyme and a second 24-h incubation gave complete cutting. We have previously used R(p)-GACGATsATCGTC to determine the stereochemical course of the endonuclease-catalyzed reaction and shown it to be a slow substrate(30) . We did not observe any products when the S(p) isomers of both pGACGATsATCGTC and pGACGATAsTCGTC were treated with two aliquots of the endonuclease. Thus both of these appear to be non-substrates and, based on the detection limits of this assay, are cut at leq0.1% of the rate shown by the parent, all-oxygen-containing oligonucleotide.

Kinetic Constants for the Hydrolysis of the Phosphorothioates by the Endonuclease

The K(m) and V(max) values for the phosphorothioates and the all-phosphate parent were determined using a continuous UV absorbance assay(60) . This is based on the hyperchromic effect and the increase in absorbance at 260 nm when a double-stranded dodecamer is cut to single-stranded hexameric products. Kinetic constants could not be determined for both the isomers of pGACGATsATCGTC and the S(p) isomer of pGACGATAsTCGTC, because of the very low, or zero, hydrolysis rates. For all the other oligonucleotides, the determination of K(m) and V(max) values, using plots of [substrate]/velocity against [substrate], was relatively straightforward, and a representative example is given in Fig. 3. The data set is summarized in Table 1and the (V(max)/K(m))/(V(max)/K(m)) shown pictorially in Fig. 4. Phosphorothioate substitution invariably caused a reduction in V(max) values, and the spread that was obtained ranged from very similar to the control (i.e. only a slight reduction) to zero or near zero. These reductions are relatively easy to explain in terms of disturbance to endonuclease-phosphate contacts and further alterations to the protein-DNA interface leading to poor assembly of a catalytically competent complex (see ``Discussion''). Most of the phosphorothioates bound more tightly to the endonuclease as shown by the decrease in K(m) values, which in one case (S(p)-pGACGAsTATCGTC) was reduced 10-fold. However, smaller reductions were much more common. Only three sulfur-containing oligonucleotides bound more weakly to the enzyme, but in all these cases the K(m) increase was very small. Better binding of phosphorothioates is more difficult to rationalize than drops in V(max), but similar results have been seen with the EcoRI endonuclease (33) and the R17 coat protein with phosphorothioate-substituted RNA(64) . A possible rationalization involves the preferred negative charge location on sulfur in phosphorothioates. Charge localization might strengthen binding when the sulfur is involved in an ionic interaction with a positively charged amino acid. However, a comparison of the results given in Table 1with the actual protein-DNA contacts (Fig. 5) shows this simple explanation does not hold. The results with EcoRI were explained by subtle adaptations to the protein-DNA interface, which improved interactions at other sites. Base analogues may also improve interaction, as seen with the trp repressor (65) and the EcoRI endonuclease (12) . In these two cases, the base analogue used appeared to facilitate DNA distortion necessary for tight binding. Both of these explanations could explain the results we observe, although there is no strong evidence that phosphorothioate DNA is especially flexible. The discrimination between substrates is best measured using V(max)/K(m) (the specificity constant) and, as shown in Table 1and Fig. 4, these showed a considerable spread of values when referenced to the control. Two of the modified oligonucleotides, the R(p)-isomers of pGAsCGATATCGTC and pGACsGATATCGTC, had larger V(max)/K(m) values mainly due to their lowered K(m) values. The other phosphorothioates had specificity constants that ranged in value from that seen with the parent down to zero. In general the six phosphates GACGpApTpApTpCpGTC, within and immediately 3` to the recognition site, seem to be important in the endonuclease-catalyzed hydrolysis. Phosphates farther out, in either direction, play little role.


Figure 3: Determination of the Kand V values for both the R(p) and the S(p) diastereomers of pGACGATATCsGTC. All the other phosphorothioates gave results of similar quality.




Figure 4: A summary of the specificity constants (V/K) for all the phosphorothioate-containing oligonucleotides. These are referenced to the all-phosphate-containing dodecamer control, for which this value is set to 100%.




Figure 5: The interactions seen between the DNA phosphate groups and the EcoRV restriction endonuclease. This is taken from the complex of EcoRV with AAAGATATCTT that contains Mg bound to one of the DNA strands (1RVB co-crystal structure in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY)(39) . ASN185 is asparagine 185 from one subunit of the dimeric endonuclease (the A subunit) and ASN*185 asparagine from the other (B) subunit etc. MC, main chain; SC, side chain. bullet, water molecule. All the contacts illustrated are leq3.5 Å in length. The sequence used for crystallography has been substituted, at the appropriate positions, by the sequence used in this study, and the GATATC recognition site is shown in uppercase letters. Only the contacts made to the phosphates that have been investigated in this paper are shown.




CONCLUSION

Phosphorothioates as Phosphate Analogues

Investigators using analogues aim to delete a protein-substrate contact, most simply by the replacement of an interacting group with a hydrogen atom. In some cases this can be cleanly achieved, and good examples include the replacement of serine or cysteine with alanine and tyrosine with phenylalanine in site-directed mutagenesis. With oligonucleotides the use of the 7-deaza derivatives of dA and dG, where the 7-ring nitrogen of the purines is replaced by a CH function, also approaches this ideal(12) . However, most functions are not amenable to this straightforward manipulation, and the phosphate group of DNA falls into this category. DNA-binding proteins make both salt bridges and hydrogen bond interactions to the two non-bridging oxygen atoms of the phosphate group. Oligonucleotide derivatives in which one non-bridging oxygen is replaced with hydrogen (H-phosphonates, O=P[OR](2)-H) have been used in some applications (66) but are difficult to prepare, in chirally pure form, and additionally exist in equilibrium with trivalent tautomers (HO-P[OR](2)). It should also be noted that these derivatives, and indeed all phosphate analogues, not only perturb the non-bridging oxygen substituted but also alter the nature of the other non-bridging oxygen. In the absence of simple, replacement derivatives, other analogues must be used. Perhaps the most suitable are the phosphorothioates, which represent a very subtle phosphate modification with changes to size, acidity, solvation, charge distribution, and metal ion binding, as mentioned in the Introduction. However, the advantage of completely deleting an interaction is that it is possible to predict the consequence, providing that no additional effects come into play. Thus a single phosphate contact has been estimated to contribute 1.3 kcal/mol to a protein-DNA complex(33, 67) , and this should result in a 10-fold reduction in V(max)/K(m) (or any other appropriate metric) when deleted(68) . A knowledge of expected energetic consequences is important, as deviations from it are of great interest and usually indicate further adjustments to the protein-DNA interface. The similarity between phosphate and phosphorothioates mean that protein-DNA interactions are much more likely to be weakened rather than completely abolished. Thus one might not expect the full penalty of 1.3 kcal/mol. As pointed out previously(33) , the exact consequence of phosphorothioate substitution will depend on the nature of the protein-phosphate interaction under consideration. In cases of an ionic interaction between a flexible lysine or arginine side chain and one non-bridging oxygen, these authors predict very little effect for phosphorothioate substitution. Even in the most critical examples, where both non-bridging oxygen atoms form very precise and constrained hydrogen bonds with the protein, an energetic penalty of 0.7 kcal/mol, rather than 1.3 kcal/mol, was observed. This would be associated with a drop in V(max)/K(m) to about 30% of the wild type rate.

In addition to the above difficulties, the tacit assumption that the analogue does not change the overall global structure of the macromolecule is usually made. This is hard to establish unequivocally. Only one crystal structure, for all R(p)-GsCGsCGsC, of an oligonucleotide phosphorothioate is known(28) . Comparison with (GC)(n) runs of other sequences indicates that the phosphorothioates cause no structural perturbations in this instance. However, it remains to be established whether this will hold generally for other phosphorothioate-containing oligonucleotides. Many synthetic oligonucleotides containing one or a small number of phosphorothioates, analogous to those used in this study, have been prepared. However, very little structural characterization has been reported. The low resolution methods used (usually based on T(m) or circular dichroism spectroscopy) have shown near identity with parent, all-phosphate oligomers. It is also possible to model phosphorothioates into ideal B-DNA without altering structural parameters. In R(p)-isomers the sulfur points into the major grove, and for S(p) it is located directly on the face of the sugar phosphate backbone. Thus we have made the usual simplifying assumption that phosphorothioates do not alter overall DNA structure.

Endonuclease DNA-Phosphate Contacts Observed by Crystallography

Several EcoRV endonuclease-oligonucleotide co-crystal structures have been solved (38, 39) . The highest resolution structures contain an 11`mer, AAAGATATCTT, bound to the enzyme and three different complexes, enzyme-DNA, enzyme-DNA-Mg, and enzyme-product, have been elucidated. We have based the discussion of our results on the endonuclease-DNA-Mg complex, as this best represents the catalytically significant ternary complex. It contains the greatest number of protein-phosphate contacts and these are highly organized, i.e. involved in extensive networks. The relevant enzyme-phosphate contacts seen in this structure are illustrated in Fig. 5. As can be seen, the catalytically essential cofactor Mg is only bound to one of the strands. Additionally the contacts between the two macromolecules differ slightly between the two DNA strands, and slight variations in these interactions are also seen in the other two complexes with this 11`-mer and in the earlier structures with different oligonucleotides. It is important to realize that the Mg-containing strand, shown in Fig. 5, is not cut in the crystal, despite having all the components required for hydrolysis in solution. Thus further conformational changes, and associated changes to endonuclease-phosphate contacts, may still be required to reach the transition state.

Relationship of Results Seen with Phosphorothioates to Crystal Structure

For three positions GApCGATATCGTC, GACpGATATCGTC, and GACGATATCGpTC, which all flank the GATATC recognition site, the introduction of a phosphorothioate has little effect and both diastereomers are cleaved almost as well or better than the parent. Examination of Fig. 5shows that these phosphates interact rather weakly with the endonuclease. The phosphate GACpGATATCGTC makes no contacts, either directly or via water, to the protein. With GACGATATCGpTC one of the phosphate oxygens, the pro-S, interacts with the flexible side chain of Gln, but no contacts are seen to the pro-R. More extensive protein-DNA interactions are seen with GApCGATATCGTC with a direct interaction with Ser and some water-mediated contacts to other amino acids. Most importantly these three phosphates do not take part in the extensive network that links the more critical phosphates to important catalytic and recognition elements of the endonuclease. It is likely that the combination of a low level of direct interactions and a lack of extensive networking accounts for the tolerance of these positions to phosphorothioate substitution.

The three phosphates GACGpATATCGTC, GACGApTATCGTC, and GACGATpATCGTC, which fall within the recognition site and include the scissile phosphodiester can be considered together. These phosphates are characterized by extensive direct contacts to the protein and participation in an extended interconnected network of hydrogen bonds. Much of this network is mediated by highly structured and ordered water molecules(39) . This network performs the following roles: 1) it interconnects the three phosphates, 2) it joins the phosphates to the amino acids in the recognition (R)-loop (these comprise amino acids 182-187, which interact with the GATATC bases via the major groove), 3) it links the phosphates to the Q-loop (this is centered on amino acid 70 and interacts with the DNA via the minor groove), and 4) it assembles the catalytic components (Mg and three acidic residues: Glu, Asp, and Asp) in a manner competent for catalysis. The pro-S oxygen of GACGpATATCGTC makes the fewest contacts, an isolated charge/charge interaction with the flexible side chain of Lys, and this is the only position where sulfur substitution gives a reasonable substrate. Alterations to all the other five oxygen atoms give poor substrates. This undoubtedly arises from disturbances to the elaborate network illustrated in Fig. 5, which gives the impression of a very high degree of co-operativity, whereby these three phosphates are connected to the amino acids responsible for both specific base recognition and for catalysis. Thus changes to these phosphate oxygens, even the very minor one of sulfur substitution, are likely to lead to movements throughout the hydrogen-bonded network and to the weakening of further protein-DNA interactions. This concerted breakdown of the macromolecular interface will reduce the binding energy available for DNA distortion and thus prevent efficient catalysis. Two further points deserve mentioning. First, by comparing the Mg- and Mn-catalyzed cleavage of the phosphorothioates, an additional metal ion, remote from the active site, was proposed to bind to the GACGpATATCGTC phosphate(55) . The exact role of this metal ion in selectivity remains obscure, but it could certainly be incorporated as an additional structural element in the network illustrated. Second, in the case of the scissile bond GACGATpATCGTC, the endonuclease is required to cleave a phosphorothioate diester rather than a phosphodiester. These two diesters show approximately similar hydrolysis rates, using model compounds in solution(69) , and so the very low enzyme-catalyzed rate cannot be due to intrinsic lower chemical reactivity. Rather, as above, it is likely to arise because of network disturbance and in the case of the S(p)-phosphorothioate (a non-substrate) to reduced Mg binding. This atom provides one of the ligands for the metal ion, and it is well documented that phosphorothioates bind Mg more poorly than do phosphates(26, 27) .

The S(p) phosphorothioate of GACGATApTCGTC is also not a substrate, whereas the R(p) is well cut. A similar result has been observed with the EcoRI endonuclease (34) . Based on these results, and a number of other observations, it has been proposed that the pro-R oxygen of this phosphate is the base that deprotonates the hydrolytic water molecule for both nucleases(66, 70) . The location of the negative charge on sulfur means that, with the R(p) phosphorothioate an S atom replaces the pro-R oxygen, and this is able to deprotonate the attacking water molecule, giving turnover. In contrast with the S(p) phosphorothioate, an uncharged double-bonded oxygen is placed in the R(p) position. This cannot abstract a proton from the water, and so no hydrolysis is seen. This proposal has some difficulties, such as phosphodiesters having (at least in free solution) the wrong pK(a) value to deprotonate water. Nevertheless, it is clear from our results that the pro-R oxygen of this phosphate has a very important function. However, it is possible that the loss of catalysis could also be due to disruptions to the interactions shown in Fig. 5. These differ slightly between the two strands and involve direct contacts to the side chains of Lys and Thr, as well as a set of water mediated contacts that interconnects this phosphate with the adjacent scissile phosphate and the catalytic apparatus.

Both phosphorothioates of GACGATATpCGTC were poor substrates with an extremely low rate being observed with the R(p) isomer. The pro-R oxygen of GACGATATpCGTC contacts the side chain of Thr and is also in contact, via two water molecules, with the important pro-R oxygen of the neighboring 5` phosphate. One assumes that the large reductions in rate seen with the R(p) phosphorothioate of GACGATATpCGTC arise from both alterations to its immediate protein contacts and to disturbances to the critical preceding phosphate. The pro-S oxygen of GACGATATpCGTC makes a hydrogen bond to the side chain of Thr. In a related article(75) , it is shown, using site-directed mutagenesis, that Thr is the most critical of all phosphate binding residues. As pointed out in this report, Thr is at one end of an alpha-helix that also contains a critical catalytic residue Glu. Furthermore, Fig. 5shows that Thr interacts with Gln on the other subunit. This Gln is in the Q loop and approaches the minor groove of the DNA. The two oxygen atoms of GACGATATCpGTC interact with the side chains of Tyr and Arg, and this explains simply the phosphorothioate results. At least one of the contacts will be weakened by phosphorothioate substitution. We also note that the pro-S oxygen forms a solvent-mediated contact with the side chain of Gln. The main chain carbonyl oxygen of this amino acid is a Mg ligand in the enzyme product complex(39) .

Role of Phosphates in the EcoRV Endonuclease-catalyzed Reaction

The distinguishing feature of all restriction endonucleases is their extraordinary accuracy, and the key to this is shown in Fig. 5. The protein does not make isolated, non-interacting, contacts to the bases in its recognition sequence and the sugar-phosphate backbone. Rather, all the protein-DNA contacts are intimately connected to each other and additionally to the metal ion binding site and to important catalytic residues. Previously it has been shown that DNA recognition is closely coupled to both metal ion binding (42) and catalysis(43) , and this study demonstrates that phosphate groups are also critically involved in these processes.

The important phosphates, as assessed using phosphorothioates, comprise GACGpApTpApTpCpGTC. This agrees very well with the site-directed mutagenesis results reported in a related paper(75) . Furthermore, a confirmation of the critical nature of these phosphates has come from an ethylation interference study with the RV endonuclease. (^2)At present we are unable to comment in depth as to what each phosphate contributes, quantitatively, to catalysis and specificity. This is because the steady state kinetics we have used only report on the slowest step of the reaction, which for these oligonucleotides is a mixture of the cleavage step and product release(47) . Thus the results in Table 1report on the important cutting step but may not provide a full and accurate description of it. In addition, the symmetrical oligonucleotides used place two phosphorothioates in the double-stranded 12`-mer, and in some cases situations like this can give rise to non-additive effects(33) . Nevertheless, we note that many of the phosphorothioates have V(max)/K(m) values that are much less than 30% of the rate seen with the control. This occurs not only for phosphates that may be directly involved in catalysis (the scissile phosphate and the potential phosphate base removed one step in the 3` direction) but also for several others, removed from the cleavage site, such as GACGpATATCGTC, GACGATATpCGTC, and GACGATATCpGTC. It has previously been suggested that a 0.7 kcal/mol penalty, i.e. a drop in V(max)/K(m) to 30% of the control value, is the most one might expect from a single phosphorothioate substitution(12, 33) . In our case we have two phosphorothioates, one per strand, and this would translate to a reduction to 15%. We propose that the larger V(max)/K(m) reductions seen, arise from an initial perturbation of the protein-phosphate interaction, which is followed by changes in the network of hydrogen bonds outlined in Fig. 5. These concerted alterations to the protein-DNA interface cause the disruption of additional protein-DNA contacts, and so result in poorer turnover than might be expected from simple phosphorothioate substitution. Of the important phosphates, only GACGApTATCGTC gives V(max)/K(m) reductions consistent with straightforward phosphorothioate replacement. However, these preliminary suggestions await confirmation using more sophisticated kinetic measurements.

Under physiological conditions the EcoRV endonuclease must distinguish between GATATC and all other sequences. One-base pair alterations to the GATATC recognition site give substrates that, under optimal conditions, are cut at vanishingly small rates(40, 42, 49) . Using base analogues, we and others have shown that even small changes to the bases in the recognition site frequently give very poor substrates(13, 14, 15, 50, 51, 52) . Often the reduction in turnover is much greater than can be accounted for in terms of simple loss of protein-DNA contacts, exactly as is seen with some of the phosphates. Just as alterations to particular phosphates lead to much poorer substrates than expected, because of weakening of other protein-DNA interactions, it is clear that changes to the GATATC sequence do not simply result in the deletion of the contact in question. Instead, concerted movements lead to further misalignments that include incorrect protein-phosphate interactions. This paper begins to identify the phosphates involved in this process. Only with a fully cognate GATATC sequence are the proper protein-phosphate contacts made, and only this provides enough energy to bend the DNA, create the metal binding site, and give rapid hydrolysis.

Relationship to Other Endonucleases

The idea that protein-phosphate interactions change with non-cognate sequences and that this contributes to selectivity is not new, and certainly we are not the first to point it out. Using ethylation interference methods with the EcoRI endonuclease(9, 10, 12) , it has been demonstrated that the phosphates contacted are dependent on base sequence. With the cognate (GAATTC) sequence, a particular set of endonuclease-phosphate contacts is seen and this pattern changes with non-canonical sequences. These alterations in phosphate contacts contribute significantly to the selection of cognate sites. Furthermore, using single-turnover kinetics and binding studies with oligonucleotides containing a single phosphorothioate of defined stereochemistry, this group (33) also demonstrated that phosphates played an important role in transition state stabilization and began to dissect out the exact energetic contributions of the substitution. The importance of phosphates, away from the cleavage, has also been shown for this endonuclease by steady state methods(34) . A related study with TaqI endonuclease (67) used S-methyl phosphorothioate triesters, formed by the reaction of oligonucleotide phosphorothioates with methyl iodide. This study both identified important TaqI phosphate contacts but also led to the conclusion that sequence-specific phosphate contacts were utilized in the transition state to amplify selectivity and that base and phosphate recognition were tightly linked. Thus, with EcoRV, EcoRI, TaqI, and probably all restriction endonucleases, indirect readout of phosphates, in a specific manner that is critically dependent on the presence of the cognate site, plays a vital role in generating high selectivity. There is no doubt that these protein-phosphate interactions deserve further consideration and analogues such as the phosphorothioates and S-methylphosphorothioates as well as O-alkyl phosphotriesters (34, 71, 72) and methylphosphonates (73, 74) are available for this purpose. At present, the most difficult step is the resolution and purification of the two diastereomers seen with all these analogues, and this is what is currently limiting further enzymological studies.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Chemistry, University of Sheffield, Sheffield S3 7HF, United Kingdom.

To whom correspondence should be addressed. Tel.: 44-191-222-7371; Fax: 44-191-222-7424; b.a.connolly{at}ncl.ac.uk.

(^1)
The abbreviation used is: HPLC, high performance liquid chromatography.

(^2)
L. Jen-Jacobson, personal communication.


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