©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Dodecapeptide Comprising the Extended Chain-4 Region of the Restriction Endonuclease EcoRI Specifically Binds to the EcoRI Recognition Site (*)

(Received for publication, September 22, 1994; and in revised form, December 18, 1994)

Albert Jeltsch (1) Jürgen Alves (2) Claus Urbanke (2) Günter Maass (2) Heiner Eckstein (3) Zhang Lianshan (3) Ernst Bayer (3) Alfred Pingoud (1)(§)

From the  (1)Institut für Biochemie, FB 15, Justus-Liebig Universität, Heinrich-Buff-Ring 58, 35392 Giessen, Germany, the (2)Zentrum Biochemie, Medizinische Hochschule Hannover, Konstanty-Gutschow-Strasse 8, 30623 Hannover, Germany, and the (3)Institut für Organische Chemie, Universität Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The restriction endonuclease EcoRI binds and cleaves DNA containing GAATTC sequences with high specificity. According to the crystal structure, most of the specific contacts of the enzyme to the DNA are formed by the extended chain region and the first turn of alpha-helix alpha4 (amino acids 137-145). Here, we demonstrate that a dodecapeptide (WDGMAAGNAIER), which is identical in the underlined parts of its sequence to EcoRI amino acids 137-145, specifically binds to GAATTC sequences. The peptide inhibits DNA cleavage by EcoRI but not by BamHI, BclI, EcoRV, HindIII, PacI, and XbaI. DNA cleavage by XbaI is slowed down at sites that partially overlap with EcoRI sites. The peptide inhibits cleavage of GAATTC sites by ApoI, which recognizes the sequence RAATTY. It interferes with DNA methylation by the EcoRI methyltransferase but not by the BamHI methyltransferase. It competes with EcoRI for DNA binding. Based on these results, the DNA binding constant of the peptide to GAATTC sequences was calculated to be 3 times 10^4M. DNA binding is not temperature-dependent, suggesting that binding of the peptide is entropy-driven. As the peptide does not show any nonspecific binding to DNA, its DNA binding specificity is similar to that of EcoRI, in spite of the fact that the affinity is much smaller. These results suggest that contacts to the phosphate groups in EcoRI mainly provide binding affinity, whereas the specificity of EcoRI is based to a large extent on sequence-specific base contacts.


INTRODUCTION

One century ago the concept of complementarity between an enzyme and its substrate was introduced by Emil Fischer (1894) (^1)to explain the specificity of enzymes. This concept has proven to be one of the most successful concepts in enzymology and has been demonstrated to be applicable in numerous cases including very specific enzymes, for example type II restriction endonucleases. These enzymes (for reviews see Heitman (1993) and Roberts and Halford(1993)) recognize palindromic sequences 4-8 bp (^2)in length and cleave the DNA within these sequences. As has been shown, for example, for EcoRI (recognition site, GAATTC), sequences differing in only 1 base pair from the canonical sequence (``star'' sites) are cleaved at least 3 orders of magnitude more slowly (Lesser et al., 1990; Thielking et al., 1990), and sites differing in more than 1 base pair are not cleaved at all (Gardner et al., 1982; Rosenberg and Greene, 1982). Similarly, binding of star sites is impaired by at least 2 orders of magnitude when compared with binding of GAATTC sequences; other sites are bound at least 4 orders of magnitude more weakly than GAATTC (Lesser et al., 1990; Thielking et al., 1990). The structural basis of this high specificity is explained by the x-ray structure analysis of a specific EcoRI-DNA co-crystal (see Fig. 1) (McClarin et al., 1986; Kim et al., 1990). It demonstrated that EcoRI binds as a symmetrical dimer to the palindromic recognition site and identified in the protein-DNA interface many specific contacts between the protein and the DNA (Rosenberg, 1991; Kim et al., 1993). They are formed between the bases of the GAATTC sequence and EcoRI (direct readout) as well as between the phosphate groups of the DNA backbone and the protein (indirect readout). All specific contacts to the bases of the recognition sequence are compiled in Fig. 1B, namely nine hydrogen bonds to the bases of the DNA (two of which are mediated by a water molecule) and five hydrophobic contacts. Additionally, at least nine phosphate contacts are observed. Interestingly, direct readout is almost exclusively due to a short region of EcoRI, the extended chain motif (Met-Ala), which is deeply buried in the major groove of the DNA, and the amino-terminal part of alpha-helix alpha4 (Ile-Arg). This extended chain-alpha4 region (Met-Arg) forms all direct (i.e. not water-mediated) hydrogen bonds and three of five hydrophobic contacts. Taken together, on the basis of the x-ray structure analysis and many biochemical studies (Brennan et al., 1986; Fliess et al., 1986; McLaughlin et al., 1987; Needels et al., 1989; King et al., 1989; Alves et al., 1989a; Heitman and Model, 1990a, 1990b; Osuna et al., 1990; Oelgeschläger et al., 1990; Jeltsch et al. 1993a), it appears as if the specificity of EcoRI is based on an extensive complementarity of the extended chain-alpha4 region and the major groove of the GAATTC sequence. Here we have directly tested this assumption by investigating the DNA binding properties of a short peptide with an amino acid sequence identical to the extended chain-alpha4 region.


Figure 1: A, EcoRI-DNA co-crystal structure (Kim et al., 1990; Brookhaven Data Bank entry 1R1E). The region of the extended chain-alpha4 peptide is highlighted (thick line). B, schematic drawing of the specific base contacts observed in the EcoRI-DNA co-crystal structure (Rosenberg, 1991; Kim et al., 1993). Contacts of one subunit to the GAATTC sequence are indicated, most of which are formed by the extended chain-alpha4 region (Met-Arg).




MATERIALS AND METHODS

Peptide Synthesis

Peptide synthesis was performed on a MilliGen 9050 peptide synthesizer by an automated method as reported earlier (Zhang et al., 1990). An HPLC analysis of the crude product mixture showed only one major peak. A few minor peaks contained less than 5% intensity. The peptide was purified by HPLC (Nucleosil C4, 5 µm) to homogeneity. The identity of the peptide was confirmed by ion spray mass spectroscopy. The concentration of the peptide was determined using a molar extinction coefficient = 5540 M cm, which is a mean value for tryptophan absorbance in polypeptides (Mach et al., 1992).

Oligonucleotide Synthesis

The self-complementary oligodeoxynucleotide oligoRI (TATAGAATTCTAT) was synthesized and purified as described (Alves et al., 1989b).

DNA Preparation

Plasmid DNA was prepared using Midi- or Maxi-preparation kits (QIAGEN Inc.) according to the instructions of the supplier. Supercoiled pUC8 was prepared by CsCl density gradient ultracentrifugation (Sambrook et al., 1989). -DNA was supplied by Boehringer Mannheim. A radioactively labeled 62-bp shift-substrate with one EcoRI site was generated by primer extension in a polymerase chain reaction containing [alpha-P]dATP.

Restriction and Modification Enzymes

EcoRI (recognition site, GAATTC) and EcoRV (GATATC) were homogeneous preparations from overproducing Escherichia coli cells (Geiger et al., 1989; Fliess et al., 1988). BamHI (GGATCC) and MunI (CAATTG) were supplied by the U. S. Biochemical Corp. (Cleveland, OH) and Fermentas (Vilnius, Lithuania), respectively. XbaI (TCTAGA), BclI (TGATCA), and HindIII (AAGCTT) were obtained from Angewandte Gentechnologie Systeme (Heidelberg, Germany). ApoI (RAATTY), PacI (TTAATTAA), EcoRI methyltransferase, and BamHI methyltransferase were from New England Biolabs (Beverly, MA).

Oligodeoxynucleotide Cleavage Experiments

OligoRI cleavage by EcoRI was followed with a continuous spectrophotometric assay that is based on the hyperchromicity of DNA (Waters and Connolly, 1992). Cleavage experiments were carried out in EcoRI buffer (20 mM TrisbulletHCl, pH 7.5, 10 mM MgCl(2), 50 mM NaCl) in microcuvettes (100 µl) in a Hitachi U3210 photometer at the temperatures indicated (4-25 °C). Reaction progress curves were stored and analyzed by linear regression of the initial part of the curve. OligoRI concentrations of 0.5 µM and EcoRI concentrations of 14.8 nM were used. DNA cleavage rates (A/min) were determined from the linear part of the reaction progress curves in the absence or in the presence of 100 µM extended chain-alpha4 peptide.

-DNA Cleavage Experiments

-DNA cleavage experiments in the absence and presence of the extended chain-alpha4 peptide were carried out with EcoRI, EcoRV, BamHI, MunI, HindIII, and BclI in order to determine whether the enzymes are inhibited by the peptide. All reactions were performed in the same buffer (EcoRI buffer: 20 mM TrisbulletHCl, pH 7.5, 10 mM MgCl(2), 50 mM NaCl) to ensure comparable binding conditions for the peptide to the DNA. If not otherwise stated, reactions were carried out at 37 °C. Usually 2 µg of DNA were cleaved with 0.5-3 units of the restriction enzyme. Peptide concentrations were varied between 0 and 500 µM as indicated. After appropriate times aliquots containing 0.25 µg of DNA were withdrawn, stopped by addition of gel loading buffer (0.1 M EDTA, 25% v/v Ficoll, 0.2% w/v bromphenol blue, 0.2% w/v xylene cyanol), and analyzed on 0.8% w/v agarose gels. For the analysis of cleavage rates in the absence and in the presence of peptide, the intermediate patterns of cleavage products of the reactions were compared.

Plasmid-DNA Cleavage Experiments

The plasmid pRIF309+ (5068 bp) was cleaved with EcoRI (three sites, fragments 2547 bp, 1598 bp, and 923 bp), XbaI (two sites, fragments 4153 bp and 915 bp), and PacI (one site). pRVIS1 (4607 bp) was cleaved with XbaI (one site). Plasmid pUC8 (2665 bp) was cleaved with EcoRI (one site) and ApoI (one site). Reactions were carried out in the absence and presence of the extended chain-alpha4 peptide and analyzed as described for the -DNA cleavage reactions.

Mobility-shift Experiments

Gel-shift experiments with EcoRI and a 62-bp DNA fragment containing one EcoRI site were carried out in the absence as well as in the presence of 250 and 500 µM peptide to find out if DNA binding by EcoRI is affected by the addition of extended chain-alpha4 peptide. DNA (100 nM) was mixed with EcoRI in binding buffer (50 mM TrisbulletHCl, pH 7.5, 50 mM NaCl, 10 mM 2-mercaptoethanol, 2 mM spermine, 0.1 mg/ml bovine serum albumin, 2 mM EDTA) and incubated for 30 min at 21 °C. Subsequently, 3 µl of gel loading buffer (50% glycerol, 50 mM TrisbulletHCl, pH 7.5, 50 mM NaCl, 10 mM 2-mercaptoethanol , 2 mM spermine, 0.1 mg/ml bovine serum albumin, 2 mM EDTA, 0.2% w/v bromphenol blue, 0.2% w/v xylene cyanol, 0.2% w/v azorubin) were added to a 5-µl aliquot of the binding mixes, and the samples were subjected to gel electrophoresis on 6% w/v polyacrylamide gels at 150 V in 0.5 TTE (90 mM Tris, 29 mM taurine, 1.25 mM EDTA). Gels were dried, and the radioactivity of the spots was analyzed.

DNA Methylation Kinetics

DNA methylation kinetics in the presence and absence of the extended chain-alpha4 peptide were carried out to determine if the EcoRI methylase, like the EcoRI restriction endonuclease, is inhibited by the peptide. -DNA (2 µg) was mixed with 5 units of EcoRI methylase in 50 mM TrisbulletHCl, pH 8.0, 100 mM NaCl, 10 mM EDTA, 8 µMS-adenosylmethionine in the absence and presence of 500 µM extended chain-alpha4 peptide in 20 µl of reaction volume. After appropriate times, aliquots were withdrawn and added to EcoRI buffer containing EcoRI. In this second reaction non-methylated DNA was digested. In order to ensure digestion even in the presence of peptide, a high concentration (100 units) of EcoRI was used, and the samples were incubated for 45 min at 37 °C. Methylation kinetics with BamHI methylase were performed similarly as described for the EcoRI methylase, except that the analytical digest was carried out with 20 units of BamHI in BamHI cleavage buffer. Control experiments showed that, under these conditions, non-methylated DNA is completely cleaved, regardless of whether the peptide is present in the reaction mixture or not.

Biophysical Methods

Sedimentation equilibrium runs (60,000 rpm; up to 72 h) were carried out in a Spinco model E analytical ultracentrifuge (Beckman) equipped with an electronic spin control, a high-intensity UV illumination system, a photoelectric scanner, and an electronic multiplexer. Concentration profiles were recorded at 280 or 294 nm (depending on the concentration of the peptide) and stored in a computer. Molecular weights were determined from a log(c(r)) versusr^2 plot. Concentrations of the peptide were varied in the runs between 18 and 90 µM. CD spectra of 0.1 mg/ml peptide in EcoRI buffer were recorded at ambient temperature in 0.01-cm cuvettes in a Jobin-Yvon Dichrograph R. J. Mark III calibrated with (+)-camphor-10-sulfonic acid, D-pantolactone, and epiandrosterone. Spectra were recorded in a computer and analyzed numerically in terms of alpha-helix and beta-sheet content (Chen et al., 1972). CD melting curves were recorded at 200 nm from 15 to 80 °C with a temperature rise of 20 °C/h.


RESULTS

The majority of the specific contacts between EcoRI and DNA are formed by amino acids within a short region of the protein, namely the extended chain-alpha4 region (Met-Arg) (Fig. 1). Here we have studied the DNA binding activity of a short dodecameric peptide (H(2)N-WDGMAAGNAIER-COOH), which is identical in sequence to the amino acids Asp-Arg in EcoRI except for the amino-terminal Trp that was added to allow for spectroscopic determination of the concentration of the peptide and for a Leu Gly replacement, which was made in order to increase the solubility of the peptide.

Biophysical Characterization of the Peptide in Solution

The peptide turned out to be soluble in water up to concentrations of 2 mM. It tends, however, to precipitate after repeated freezing/thawing cycles. The analysis of sedimentation equilibrium runs in 20 mM TrisbulletHCl, pH 7.5, 50 mM KCl, 10 mM MgCl(2) resulted in an apparent molecular weight of 1180 ± 70 g mol comparable with the theoretical value of 1290 g mol. Therefore, the peptide is a monomer in solution. CD spectra (250-190 nm) of the peptide in 20 mM TrisbulletHCl, pH 7.5, 50 mM NaCl, 10 mM MgCl(2) demonstrate that the peptide does not adopt a regular structure (alpha-helix or beta-strand) in solution, a conclusion that is supported by the absence of a melting transition between 15 and 80 °C.

Experimental Strategy

To find out whether the peptide can specifically interact with the recognition sequence of EcoRI (GAATTC), we measured the inhibition of the activity of several restriction and modification enzymes by the peptide using various DNA substrates. These inhibition experiments resemble classical footprinting analyses in which the DNA is protected against the attack of a nonspecific nuclease- or DNA-degrading chemical by ligands bound to the DNA. As such, they are indirect measurements that allow one to detect unequivocally even weak binding of the peptide to the DNA. Other standard techniques to detect DNA protein interactions, viz. gel-shift experiments, nitrocellulose filter binding, fluorescence (including fluorescence-detected stopped flow and fluorescence-detected temperature jump), circular dichroism, and NMR spectroscopy, failed to demonstrate DNA binding of the peptide, presumably because the complex is not sufficiently stable and/or because complex formation does not result in a spectroscopic signal change.

Inhibition of DNA Cleavage by EcoRI

First, we measured the influence of the extended chain-alpha4 peptide on the cleavage of a 13-mer oligodeoxynucleotide (oligoRI) by EcoRI. As an example, the reaction progress curves in the absence and presence of 100 µM peptide are shown in Fig. 2. The initial rate of DNA cleavage by EcoRI is reduced by a factor of 1.4 ± 0.1 in the presence of 100 µM peptide. We then used several macromolecular substrates for the cleavage inhibition experiments. It turned out that EcoRI cleavage of -DNA, pUC8-DNA, and pRIF309+ plasmid DNA is strongly inhibited by the addition of peptide at concentrations higher than 50 µM. Examples of the time course of -DNA and pUC8-DNA cleavage by EcoRI in the absence of peptide as well as in the presence of 500 µM peptide are shown in Fig. 3. The relative rates of -DNA and pUC8-DNA cleavage by EcoRI at various peptide concentrations are shown in Fig. 4. These results demonstrate that the peptideinterferes with DNA cleavage by EcoRI, either by binding to the DNA or to the enzyme.


Figure 2: Cleavage of oligoRI by EcoRI in the presence of 100 µM peptide (bulletbulletbulletbullet, right ordinate) as well as in the absence of peptide (-, left ordinate) in EcoRI cleavage buffer at 25 °C. In each case 0.5 µM oligoRI was cleaved with 14.8 nMEcoRI. The initial rates of cleavage (straight line) differ by a factor of 1.4.




Figure 3: A, cleavage of -DNA in the absence (- peptide) of peptide as well as in the presence of 500 µM peptide (+ peptide) in EcoRI cleavage buffer at 37 °C. 2 µg of -DNA were incubated with 0.5 nMEcoRI in 70 µl of reaction volume (c sites = 5.4 nM). After the times given (`, min), aliquots were withdrawn and analyzed electrophoretically. -DNA (48,502 bp) is cleaved by EcoRI into six fragments (21,225, 7,421, 5,804, 5,643, 4,878, and 3,531 bp). B, cleavage of pUC8-DNA in the absence (- peptide) of peptide as well as in the presence of 500 µM peptide (+ peptide) in EcoRI cleavage buffer at 37 °C. 1 µg of pUC8-DNA was incubated with 0.1 nMEcoRI in 100 µl of reaction volume (c sites = 5.7 nM). After the times given (`, min), aliquots were withdrawn and analyzed electrophoretically. The supercoiled pUC8-DNA (sc) is cleaved via an open circle intermediate (oc) to give the linear form (lin).




Figure 4: Rates of -DNA and pUC8-DNA cleavage by EcoRI at various concentrations of peptide. All rates are given relative to the rates of DNA cleavage of identical samples measured in the absence of peptide. Rates were determined in EcoRI buffer at 21 °C. Values given are accurate within ±20%. The line is a theoretical curve corresponding to the best fit of the data (K (K) = 3 times 10^4M). The dashed lines are simulated curves for K = 1.5 times 10^4M (upper) and K = 6 times 10^4M (lower), respectively, and are included to demonstrate that 2-fold variations of the K result in a significantly worse fit.



Inhibition of DNA Cleavage by Other Restriction Enzymes

All results obtained with inhibition studies of different enzymes by the peptide are compiled in Table 1. ApoI (RAATTY), like EcoRI, cleaves GAATTC sequences. The ApoI cleavage of plasmid pUC8 that contains one GAATTC sequence but no additional ApoI sites was inhibited approximately 20-fold in the presence of 500 µM peptide. This value is nearly identical to that obtained for the inhibition of the EcoRI-catalyzed cleavage of pUC8 (17-fold). XbaI (TCTAGA)-catalyzed cleavage of plasmid pRVIS1 was not inhibited by 500 µM peptide in the reaction buffer. In contrast, cleavage of pRIF309+ by XbaI was inhibited by addition of the peptide (Fig. 5). This is an important result, because in pRIF309+ both XbaI sites are part of a polylinker and partially overlap with EcoRI sites (TCTAGAATTC). These findings taken together demonstrate that the inhibition of EcoRI by the peptide is not due to an interaction of the peptide with EcoRI but rather with the DNA.




Figure 5: A, cleavage of pRVIS1 plasmid with XbaI in the absence of peptide (- peptide) as well as in the presence of 500 µM peptide (+ peptide). The supercoiled plasmid (sc) is cleaved via an open circle intermediate (oc) to give the linear DNA (lin). 5 µg of plasmid were incubated with 5 units of XbaI in EcoRI cleavage buffer at 37 °C. After the times given (`, min), aliquots were withdrawn and analyzed. B, cleavage of pRIF309+ plasmid with XbaI in the absence of peptide (- peptide) as well as in the presence of 500 µM peptide (+ peptide). The supercoiled plasmid is cleaved to produce two fragments 4153 and 915 bp in length. 3 µg of plasmid were incubated with 5 units of XbaI in EcoRI cleavage buffer at 37 °C. After the times given (`, min), aliquots were withdrawn and analyzed.



Specificity of the Peptide-DNA Interaction

To determine the specificity of the peptide-DNA interaction we have measured whether the peptide also inhibits several other restriction endonucleases (Table 1). -DNA cleavage by EcoRV (GATATC), BamHI (GGATCC), HindIII (AAGCTT), or BclI (TGATCA) was not influenced by the addition of 500 µM peptide to the reaction mixtures. Similarly, PacI (TTAATTAA) cleavage of pRIF309+ and XbaI (TCTAGA) cleavage of pRVIS1 were not inhibited by the peptide. Consequently, the peptide does not bind to the sequences GATATC, GGATTC, AAGCTT, TAATTA, TGATCA, and TCTAGA, some of which are closely related to GAATTC. In order to test the selectivity with a substrate in which the outer base pairs of the recognition sequence were changed, we measured the DNA cleavage rate of MunI (CAATTG) in the presence of the peptide. In contrast to all other enzymes tested, except for EcoRI and ApoI, MunI turned out to be inhibited by the peptide, albeit more weakly than EcoRI, because 500 µM peptide only resulted in a 1.5-fold inhibition of the cleavage rate. To estimate the binding of the peptide to EcoRI star sequences, we carried out inhibition experiments of EcoRI star cleavage with -DNA as substrate. Star cleavage of EcoRI was induced either by using a Mn-containing cleavage buffer (20 mM TrisbulletHCl, pH 7.5, 50 mM NaCl, 1 mM MnCl(2)) (Hsu and Berg, 1978) or by using very high enzyme concentration in cleavage experiments (up to 1 µM) under normal buffer conditions. Under either condition, star activity of EcoRI was inhibited to the same extent as the canonical cleavage activity (data not shown), demonstrating that the peptide discriminates between GAATTC and star sequences to a similar degree as EcoRI.

Reduction of DNA Binding by EcoRI

As an independent test of the interaction between the peptide and GAATTC, the influence of the peptide on DNA binding by EcoRI was investigated. We employed a gel retardation assay, which is well suited to compare relative binding affinities. DNA binding experiments with EcoRI were carried out in the absence of peptide as well as in the presence of 250 and of 500 µM peptide, respectively, in the binding mixtures. Fig. 6shows an example of the results. Clearly, the peptide competes with EcoRI for binding to the specific DNA sequence, because, at the highest EcoRI concentration used, approximately 85% of the DNA are shifted in the absence of peptide, but only 50% are shifted in the presence of 500 µM peptide.


Figure 6: Gel electrophoretic mobility-shift assay of EcoRI in the absence of peptide (- peptide) as well as in the presence of 250 and 500 µM peptide. DNA was incubated with 0, 15, 25, 50, 75, or 150 nMEcoRI (left to right) at 21 °C in binding buffer.



Inhibition of DNA Methylation by the EcoRI Methylase

DNA methylation activity of the EcoRI methyltransferase was measured in the absence of the extended chain-alpha4 peptide as well as in the presence of 500 µM peptide. As shown in Fig. 7, the activity of the methylase is reduced approximately 4-fold in the presence of peptide, as indicated by the smaller amount of methylated (i.e. uncleavable) DNA when the methylation was carried out in the presence as compared with the absence of peptide. The activity of the BamHI methyltransferase, in contrast, was not affected by the presence of 500 µM peptide in the reaction mixture.


Figure 7: Methylation of -DNA by the EcoRI methyltransferase. 2 µg of -DNA were incubated with 5 units of methylase in reaction buffer at 37 °C in the absence (- peptide) as well as in the presence (+ peptide) of 500 µM peptide. After the times indicated (`, min), aliquots were withdrawn and analyzed by digestion with EcoRI.



Estimation of the Peptide-DNA Binding Constant

Taken together, the data presented so far can only be explained if the peptide specifically interacts with the EcoRI recognition sequence (GAATTC), albeit with a low but clearly detectable affinity. The dependence of the inhibition measured for DNA cleavage and binding by EcoRI on the peptide concentration can be used to calculate the equilibrium constant for the binding of the peptide to the DNA.

The concentration of both EcoRI and the macromolecular DNA substrates used in the cleavage experiments described above was low. Moreover, a high excess of nonspecific competitor sites is present in the reaction mixture when macromolecular DNAs are employed as substrates (Langowski et al., 1980). Under these conditions substrate binding is the rate-limiting step of the reaction (Langowski et al., 1981). Recently, we have shown that EcoRI, when diffusing along the DNA, does not miss a recognition site under conditions similar to those employed here (Jeltsch et al., 1994). This implies that the rate of formation of the enzyme-EcoRI site complex governs the observed reaction rate. Therefore, the overall rate can be described by a second order rate equation, v = kcc(E), where c is the concentration of DNA and c(E) is the concentration of EcoRI. Assuming that EcoRI cannot cleave the DNA in the DNA-peptide complex, under identical conditions the observed reaction rate is proportional to the fraction of the DNA not complexed with peptide,

where v(0) is the initial cleavage velocity in the presence of peptide, v(0) is the initial cleavage velocity in the absence of peptide, c is the concentration of free DNA in the presence of peptide, and c is the total concentration of DNA in the presence of peptide.

The relative rates determined at various peptide concentrations, therefore, can be used directly to calculate the DNA binding constant of the peptide. This analysis was carried out with the computer program TITRAT, which calculates an association constant with a least square fit method using a multistep predictor/corrector module (VA05A) (Powell, 1965). The binding constant turned out to be 3 times 10^4M (Fig. 4). With the same analysis, the equilibrium binding constant for the binding of the peptide to the sequence CAATTG, which was derived from inhibition of MunI-catalyzed DNA cleavage, was estimated to be 1 times 10^3M.

The cleavage of oligoRI was carried out at relatively high substrate concentrations (0.5 µM). Fluorescence-stopped flow studies with oligoRI have shown that substrate binding of EcoRI occurs in a pre-equilibrium kinetically separable from cleavage under these conditions (Alves et al., 1989b). Therefore, the Michaelis-Menten model is applicable to analyzing the kinetics of oligoRI cleavage, v = c(E)kc/K(m) + c, where k is the turnover number.

The effect of the peptide on the rate of DNA cleavage by EcoRI under these conditions is to reduce the concentration of free oligoRI leading to a decrease in rate. The ratio of rates measured in the absence and in the presence of 100 µM peptide (v = v(0)/v(0) = 1/1.4) (Fig. 2) together with K(m) = 80 nM and k = 23 s (Jeltsch et al., 1993b) can be used to deter-mine the concentration of free oligoRI in the presence of peptide (c),

where c is the concentration of oligoRI in the absence of peptide, which is equal to the total concentration of oligoRI. With c, the concentration of oligoRI-peptide complexes and finally K(a) for the peptide-DNA complex can be calculated. With this procedure a K(a) of 3 times 10^4M was obtained in agreement with the result of the analysis of the inhibition of -DNA and pUC8-DNA cleavage.

The DNA binding reaction of EcoRI in the presence of peptide is governed by two coupled equilibria: EcoRI binding to the DNA and peptide binding to the DNA. For a quantitative analysis of the gel electrophoretic mobility-shift assays (Fig. 6), the lanes with the two highest EcoRI concentrations were analyzed to estimate the DNA binding affinity of EcoRI and of the peptide. The fractions of DNA bound by EcoRI in the lanes without peptide yield a binding constant of EcoRI to the specific site of 1.5 times 10^8M under the conditions of the experiments. This value, in combination with the concentration of the EcoRI-DNA complexes in the presence of peptide, can be used to calculate the concentration of the free DNA in these mixtures. Then the concentration of the peptide-DNA complexes and the equilibrium constant for the binding of the peptide to the DNA can be estimated to be 2-4 times 10^4M, which is similar to the values derived in the other analyses.

Temperature Dependence of the Peptide-DNA Interaction

Cleavage of oligoRI by EcoRI in the presence of 100 µM peptide at both 4 and 25 °C resulted in a reduction of the cleavage rate constant by the same factor of 1.4. Similarly, EcoRI cleavage of pUC8-DNA and -DNA was equally inhibited by the peptide at 4, 21, and 37 °C (data not shown).

MunI Peptide DNA Interaction

Recently, it has been suggested on the basis of sequence alignments that the restriction enzyme MunI has a structure similar to that of EcoRI and, in particular, that a SAGRGNAHER region in MunI has a similar function as the extended chain-alpha4 region in EcoRI, namely to recognize the DNA (Siksnys et al., 1994). We have therefore also prepared a MunI peptide (WDPSAGRGNAHER), which corresponds to the amino acid sequence of the putative extended chain region in MunI. This peptide, however, inhibited neither EcoRI nor MunI (data not shown). Although these results do not support the suggestion of structural and functional similarities between the two enzymes, they do not disprove them either. However, these results clearly demonstrate that a peptide of similar length and sequence to the extended chain-alpha4 peptide binds neither to GAATTC nor to CAATTG sequences. It is noteworthy in this context that an EcoRI mutant in which the extended chain-alpha4 region was replaced by the corresponding region of MunI by site-directed mutagenesis of the EcoRI gene was catalytically inactive (Fritz, 1994).


DISCUSSION

Specific interactions of proteins with DNA have been investigated in great detail in several cases. Often specific contacts between the proteins and the DNA are formed by characteristic structural elements (for reviews see Steitz(1990) and Harrison(1991)). Frequently alpha-helices are positioned in the major groove of the DNA, e.g. by helix-turn-helix, basic region helix-loop-helix, basic region leucine zipper, and zinc finger proteins, to provide a structural framework for the recognition interactions (for a recent review see Wolberger(1993)) but beta-sheets, as in the MetJ- and Arc-repressors, are also employed (for a recent review see Rauman et al.(1994)). In the EcoRI-DNA complex, a short segment of the protein comprising amino acids Met-Arg forms nearly all specific contacts to the bases of the recognition sequence. This segment has an extended conformation and is deeply buried in the major groove of the DNA.

Often the DNA-binding regions are stable subdomains that specifically interact with the DNA as demonstrated for basic region leucine zipper, helix-turn-helix, basic region helix-loop-helix, and zinc finger proteins. Indeed many of the available structures of DNA-binding proteins were determined only with the DNA-binding domain or subdomain of the protein. An independent folding of the DNA binding module, however, could not be expected for EcoRI, because the extended chain-alpha4 motif is held in place by several interactions to other parts of the protein. Although the extended chain-alpha4 region of EcoRI, therefore, cannot be considered to be a subdomain, here we demonstrate by several lines of evidence that a dodecameric oligopeptide that contains this sequence specifically binds to GAATTC sequences. These lines of evidence are as follows. (i) The peptide inhibits the EcoRI-catalyzed DNA cleavage of several different substrates (13-mer oligodeoxynucleotide, pUC8-DNA, pRIF309+ plasmid DNA, and -DNA) in a concentration-dependent manner. (ii) The relative cleavage rates of GAATTC sites by EcoRI, which recognizes GAATTC sequences and ApoI (recognition sequence, RAATTY), are equally reduced by the peptide. (iii) DNA cleavage of BamHI (GGATCC), BclI (TGATCA), EcoRV (GATATC), HindIII (AAGCTT), PacI (TTAATTAA), and XbaI (TCTAGA) is not affected by the peptide. (iv) XbaI cleavage at sites that partially overlap with EcoRI sites (TCTAGAATTC) is inhibited by the peptide. (v) DNA methylation by the EcoRI methyltransferase but not by the BamHI methyltransferase is slowed down by the peptide. (vi) The peptide competes with specific DNA binding by EcoRI.

Interestingly, the discrimination of the peptide between GAATTC and CAATTG is less stringent than the discrimination of EcoRI between these sequences. This observation is in accordance with the recognition scheme, which is based on the structure of the specific EcoRI-DNA co-crystal (Rosenberg, 1991), because, in contrast to the AT base pairs, the two symmetry-related GC base pairs are contacted mainly by amino acids outside of the extended chain-alpha4 region rather than by amino acids within this region (Fig. 1). The close contact of Met and Ala to the GC base pair, on the other hand, appears sufficient to discriminate a GC base pair from a TA base pair that contains a methyl group in the major groove of the DNA at this position, because TTAATTAA cleavage by PacI was not inhibited by the peptide.

Specific binding of short peptides to nucleic acids is not a novel phenomenon. For the basic region leucine zipper protein GCN4, it was shown that a small peptide comprising 20 residues specifically interacts with DNA (Talanian et al., 1992). Specific DNA interaction of minor groove-binding peptides is observed with short peptides containing RGR repeats, which resemble minor groove-binding drugs like netropsin or distamycin (Geierstanger et al., 1994). Moreover, a peptide 17 amino acids in length containing the Arg-rich region of the HIV Rev protein is able to bind to the Rev response element in RNA (Tan et al., 1993). Other examples of RNA-binding protein motifs were reviewed recently (Mattaj, 1993). The binding of the extended chain-alpha4 peptide to DNA demonstrated in this work differs from all of these examples, because this peptide does not contain a net positive charge or positive charge clusters, which could support nonspecific binding to DNA via electrostatic contacts to the phosphate backbone. Consequently, in the EcoRI-DNA co-crystal structure, the extended chain-alpha4 region is not involved in phosphate contacts to the DNA.

The binding affinity of the peptide to the GAATTC sequence was determined to be 3 times 10^4M, which corresponds to an interaction energy of, DeltaG(a) = -RT ln K(a) = -25.3 kJ mol (-6.05 kcal mol). The binding constant of the peptide to GAATTC turned out to be temperature-independent between 4 and 37 °C, within the limits of error of our experiments. This result shows that DeltaH(a) of the peptide-DNA association is small and, hence, that the reaction is mainly entropy-driven. This is surprising at first, because the peptide is most likely disordered in solution but presumably well ordered in the complex with the DNA. One has to expect, therefore, the existence of the unfavorable entropy term DeltaS upon complex formation. One might speculate that this term is overcompensated by the favorable term DeltaS that arises, because upon complex formation 1450 Å^2 of solvent-accessible surface are buried. Because 1 Å^2 contributes roughly -100 J mol (Chothia, 1974) to the interaction energy, DeltaG can be estimated to be around -145 kJ mol (-34.7 kcal mol). The peptide contains 43 rotatable bonds that generate rotational isomers (24 freely rotatable backbone bonds and 19 aliphatic C-C, C-N, or C-S bonds of the side chains). Assuming that in solution only three rotational states are populated (i.e. = 60, 180, and 300°) and that rotation is completely frozen in the complex, the contribution of the reduced conformational flexibility to the entropy change of complex formation can be estimated using S = k ln W, where W denotes the number of possible conformations, DeltaS = R ln W/W = R ln 1/(3) = -390 J K. Then, DeltaG can be estimated to be 116 kJ mol (27.8 kcal mol). Although DeltaG and DeltaG are only crude estimates (in DeltaS, for example, an altered flexibility of the DNA is not taken into account), the sum of both terms is close to the DeltaG(a) observed. This estimation shows that the release of ordered water molecules could be the thermodynamic driving force of the peptide-DNA association (Ha et al., 1989). What then is the function of the specific hydrogen bonds? Before complex formation all hydrogen bond donors and acceptors of the peptide and the DNA interact with water molecules. If both surfaces match perfectly, all hydrogen bond donors and acceptors are saturated after complex formation, too, resulting in a very small net enthalpy change. If, however, the surfaces are not chemically complementary to each other, some hydrogen bond donors or acceptors would remain free but without access to water in the DNA-protein interface yielding a large and positive DeltaH. This would prevent association to such (i.e. nonspecific) sites, because the overall DeltaG of the complex formation would become positive.

The binding affinity of the peptide to CAATTG sequences has been estimated to be 10^3M. A nonspecific binding affinity to other sequences was not detectable. Given the sensitivity of the experiments, the binding affinity to nonspecific sites can be estimated to be below 10^2M. Because this peptide contains neither a positive net charge nor positive charge clusters, there is no structural basis for a nonspecific binding of the peptide to DNA. The specificity of the peptide in the discrimination of GAATTC and nonspecific sequences, hence, is in the order of 10^3-10^4. This value is similar to the discrimination factor of EcoRI in binding GAATTC and nonspecific sequences (e.g.K(a)(GAATTC)/K(a)(CTTAAG) = 7 times 10^3) (Lesser et al., 1990) measured, however, in the absence of Mg. This comparison demonstrates that the extended chain-alpha4 region in EcoRI provides the major contribution to the binding specificity of the enzyme. This conclusion is further supported by the finding that cleavage at EcoRI star sites is similarly inhibited by the peptide as cleavage at canonical sites.


CONCLUSIONS

Most of the specific contacts of EcoRI to the bases of its recognition sequence (GAATTC) are formed by a short continuous peptide sequence, the extended chain-alpha4 motif. As demonstrated by the crystal structure (Kim et al., 1990), this amino acid motif is largely complementary to the major groove of the GAATTC sequence, which enables it to form a variety of specific contacts to the DNA; 8 amino acid residues (Met-Arg) are involved in 10 specific interactions with the bases of the recognition sequence. Here, we have demonstrated that a dodecapeptide containing the sequence of the extended chain-alpha4 motif specifically binds to GAATTC sequences. Therefore, binding specificity of EcoRI is based mainly on the specific contacts between a small amino acid sequence motif and the bases of the DNA, whereas binding affinity is provided by contacts between amino acid residues dispersed over the entire DNA binding site of the protein and the phosphate groups of the DNA. Our data suggest that, at least for the EcoRI restriction endonuclease, direct readout is more important to ensure binding specificity than indirect readout. It must be kept in mind, however, that cleavage specificity of restriction enzymes is only in part due to binding specificity. It might well be that in the transition state contacts to the phosphate groups play an important role and determine whether a sequence is cleaved or not (Koziolkiewicz and Stec, 1992; Jeltsch et al., 1993c).


FOOTNOTES

(^1)
``Um ein Bild zu gebrauchen, will ich sagen, daß Enzym und Glucosid wie Schloss und Schüssel zueinander passen müssen, um eine chemische Wirkung aufeinander ausüben zu können.''-Emil Fischer, 1894.

(^2)
The abbreviations used are: bp, base pair(s); K, association constant; G, Gibbs free energy of association; H, enthalpy of association; HPLC, high pressure liquid chromatography; oligoRI, d(TATAGAATTCTAT).

*
This work was supported by the Deutsche Forschungsgemeinschaft (Pi 122/5-3 and Ma 465/17-1) and the Fonds der Chemischen Industrie (to A. J.). 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.

§
To whom correspondence should be addressed. Tel.: 49-641-702-5824; Fax: 49-641-702-5821.


ACKNOWLEDGEMENTS

We thank Dr. D. E. Wemmer and co-workers for efforts to demonstrate binding of the peptide to DNA by NMR techniques. We gratefully acknowledge technical assistance by H. Büngen and U. Kaysser.


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