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Nucleic Acids Research, 2000, Vol. 28, No. 5 1085-1091
© 2000 Oxford University Press

Identification of a base-specific contact between the restriction endonuclease SsoII and its recognition sequence by photocross-linking

Elena A. Kubareva, Hubert Thole1, Anna S. Karyagina2, Tatiana S. Oretskaya, Alfred Pingoud3 and Vera Pingoud3,*

A. N. Belozersky Institute of Physical and Chemical Biology and Chemistry Department, Moscow State University, Moscow 119899, Russia, 1Zentrum Kinderheilkunde, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, D-30623 Hannover, Germany, 2Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya str. 10, Moscow 119121, Russia and 3Institut für Biochemie, Fachbereich 08, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany

Received November 29, 1999; Revised and Accepted January 13, 2000.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A target sequence-specific DNA binding region of the restriction endonuclease SsoII was identified by photocross-linking with an oligodeoxynucleotide duplex which was substituted with 5-iododeoxy­uridine (5-IdU) at the central position of the SsoII recognition site (CCNGG). For this purpose the SsoII–DNA complex was irradiated with a helium/cadmium laser (325 nm). The cross-linking yield obtained was ~50%. In the presence of excess unmodified oligodeoxynucleotide or with oligode­oxynucleotides substituted with 5-IdU elsewhere, no cross-linking was observed, indicating the specificity of the cross-linking reaction. The cross-linked SsoII-oligodeoxynucleotide complex was digested with chymotrypsin, a cross-linked peptide–oligodeoxy­nucleotide complex isolated and the site of cross-linking identified by Edman sequencing to be Trp61. In line with this identification is the finding that the W61A variant cannot be cross-linked with the IdU-substituted oligodeoxynucleotide, shows a decrease in affinity towards DNA and is inactive in cleavage. It is concluded that the region around Trp61 is involved in specific binding of SsoII to its DNA substrate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Type II restriction endonucleases are homodimeric enzymes that recognize a palindromic sequence of double-stranded DNA and, in the presence of Mg2+, specifically cleave the DNA within or close to that sequence (reviewed in 1,2). More than 3000 restriction endonucleases have been characterized (3) and many sequenced. With few exceptions, the amino acid sequences turned out to have little if any similarity, which originally was interpreted to mean that the members of this huge and diverse family of enzymes are evolutionarily unrelated (4). Only by correlating genotype (amino acid sequence) with phenotype (recognition sequence), could evidence be obtained for an evolutionary relationship among type II restriction endonucleases (5). This became more evident after determination of the crystal structures of now altogether seven type II restriction enzymes (reviewed in 6,7), which all have a structurally similar core that consists of a five-stranded ß-sheet flanked by two {alpha}-helices (8). Intriguingly, a type IIS restriction endonuclease, FokI (9), an endonuclease involved in repair, MutH (10) and an exonuclease, {lambda}-exonuclease (11), have a similar catalytic core as the seven type II restriction enzymes for which crystal structures were determined [EcoRI (12), EcoRV (13), PvuII (14,15), BamHI (16), Cfr10I (17), BglI (18), MunI (19)] suggesting that all these nucleases diverged from a common ancestor. This catalytic core contains a sequence motif, the PD/E...D/EXK motif (20,21) which is present in many restriction enzymes and, in quite a number of these, has been confirmed to be involved in catalysis by a mutational analysis (22). A recent addition to the list of restriction enzymes containing a bona fide PD/E...D/EXKmotif is the type IIE restriction enzyme EcoRII (23), which recognizes the sequence CCWGG. For EcoRII it was shown using a peptide scanning technique that it interacts with its DNA target via at least two regions, one comprising the right half of the PD/E...D/EXK motif and the other located approximately 200 amino acid residues away and containing the KXRXXK motif present in some restriction enzymes, among them SsoII (24) which can be considered to be an isochizomer of EcoRII with a slightly more relaxed recognition sequence, namely CCNGG. Both EcoRII and SsoII cleave DNA at the phosphodiester bond located at the 5'-side of the recognition site and produce sticky ends with a 5 bp overhang. Different from EcoRII which belongs to the type IIE restriction endonucleases characterized by an essential interaction with two copies of the recognition site (25–27), SsoII is not dependent on binding of a second recognition site in order to catalyze DNA cleavage. Thus, in spite of sequence similarities, the isoschizomers EcoRII and SsoII belong to different subfamilies within the family of type II restriction enzymes, which merits a detailed comparison. To this end we have begun to map the DNA binding site of SsoII. We present here the results of a photocross-linking study, which demonstrate that SsoII can be specifically cross-linked via Trp61 to the central base of its recognition sequence. This cross-link identifies a region in SsoII involved in DNA binding.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Purification of recombinant His6-tagged SsoII
His6-tagged R.SsoII was expressed in Escherichia coli JM109 and purified as described by Sheflyan et al. (28).

Oligodeoxynucleotides
The following oligodeoxynucleotides were used for photocross-linking and cleavage experiments:

5'-GCCAACCTGGCTCT-3' (S)

3'-CGGTTGGACCGAGA-5',

5'-GCCAACCUGGCTCT-3' (I)

3'-CGGTTGGACCGAGA-5',

5'-GCCAACUTGGCTCT-3' (II)

3'-CGGTTGGACCGAGA-5',

5'-GCATATATATATATATUATATATATCGT-3' (III)

3'-CGTATATATATATATAATATATATAGCA-5';

the latter three were monosubstituted with 5-IdU at the position indicated (U). All individual oligodeoxynucleotides were purchased from INTERACTIVA and purified by HPLC. For photocross-linking experiments the substituted strands of oligodeoxynucleotides I–III were radioactively labeled at the 5'-terminus using T4 polynucleotide kinase and [{gamma}-32P]ATP, annealed to the respective complementary strand by heating to 50°C and cooling to room temperature.

Cleavage of oligodeoxynucleotide S by SsoII
Cleavage experiments were performed in 10 µl cleavage buffer (10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 10 mM MgCl2 and 100 µg/ml bovine serum albumin) at 37°C with the 32P-labeled oligodeoxynucleotide S that was labeled on both strands.

Photocross-linking of SsoII
Approximately 20 µM SsoII was preincubated with 20 µM double-stranded oligodeoxynucleotide I and II as well as a non-specific oligodeoxynucleotide III (Fig. 1) for 10 min on ice in a volume of 50 µl cross-linking buffer (10 mM HEPES, pH 6.5, 50 mM NaCl). Photocross-linking was carried out on ice with a 40 mW helium/cadmium laser emitting at 325 nm (Laser 2000). The total irradiation time was usually 20 min, in kinetic experiments 0–60 min. Samples of 2.5 µl were withdrawn before and after cross-linking and analyzed on a 15% SDS–polyacrylamide gel. Gels were silver stained and dried, radioactive bands were visualized by autoradiography with intensifying screens or by using an imager. For preparative isolation of the cross-linked SsoII/oligodeoxynucleotide complex, the analytical scale reaction was repeated 10 times and the reaction mixtures were pooled.



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  		Figure 1. Photocross-linking of SsoII to different 5-IdU-substituted oligodeoxynucleotides. (A) Double-stranded oligodeoxynucleotides comprising either the specific SsoII recognition sequence (I and II) or a non-specific sequence (III) were mono-substituted with 5-IdU at the position indicated with U. (B) Results of the cross-linking reaction, for which 20 µM SsoII was incubated with 20 µM 32P-labeled oligodeoxynucleotide I, II or III for 10 min on ice, and then irradiated with a 40 mW helium/cadmium laser emitting at 325 nm for 30 min. The reaction products were analyzed by SDS–PAGE and silver stained. The positions representing cross-linked SsoII (CL R.SsoII) and uncross-linked SsoII are indicated. (C) Autoradiogram of a gel run in parallel.

 
Protease digestions of the cross-linked SsoII–oligodeoxy-nucleotide complex
Aliquots of the radioactively labeled cross-linked complex of SsoII with the oligodeoxynucleotide were digested in freshly prepared 100 mM ammoniumbicarbonate buffer by various proteases in a reaction volume of 50 µl. Trypsin and chymo­trypsin were added to give a final concentration of 20, 100 and 500 µg/ml. Proteinase K was added to give final concentrations of 100 and 500 µg/ml. The digestions were performed for 2 h at 37°C. The reactions were terminated by the addition of 30 µl of 8 M urea, 0.025% (w/v) bromophenol blue and 0.025% (w/v) xylene cyanol and subsequent heating for 5 min at 95°C. The samples were subjected to electrophoresis on a 15% polyacryl­amide gel containing 0.5x TBE and 2 M urea after a pre-run for 30 min. The gel was dried, and radioactive bands were visualized by autoradiography.

Identification of the cross-link in the cross-linked SsoII–oligodeoxynucleotide complex
An aliquot of 10 nmol SsoII in the cross-link reaction mixture (500 µl) was digested after the addition of 500 µl of 100 mM ammoniumbicarbonate buffer in the presence of 60 µg/ml of chymotrypsin. The digestion was performed for 2 h at 37°C. To test the progress of the chymotryptic proteolysis, aliquots of the reaction mixture were analyzed by electrophoresis. After the digestion was complete, the reaction mixture was adjusted to 50 mM Tris–HCl, pH 7.5, and applied onto a Mono Q column (Amersham Pharmacia Biotech). For elution, the following buffers were used: buffer A, 50 mM Tris–HCl, pH 7.5, 1 mM EDTA; buffer B, 50 mM Tris–HCl, pH 7.5, 1 mM EDTA and 1 M NaCl. The gradient applied was 0–80% B in 40 min. The flow rate was 1 ml/min. Fractions of 1 ml were collected and desalted on NAP10 (Amersham Pharmacia Biotech) columns which had been equilibrated with 10 mM ammonium bicarbonate buffer prior to concentrating in a speed-vac. Aliquots of the fractions were analyzed by electrophoresis on a 15% polyacrylamide, 0.5x TBE, 2 M urea gel. For further purification of the cross-linked peptide–oligodeoxy­nucleotide complex, the peak fractions from the Mono Q column were combined and subjected to preparative electrophoresis on a 12% polyacrylamide gel containing 0.5x Tris-borate, pH 7.0, 6 M urea, after a pre-run for 30 min with a cathode buffer containing 1 mM thioglycolic acid. The cross-linked peptide–oligodeoxynucleotide complex was extracted from the gel and eluted into 500 µl of 10 mM ammonium bicarbonate buffer, by shaking for 2 h at 37°C. This solution was lyophilized, and the cross-linked complex was redissolved in 50 µl of 10 mM ammonium bicarbonate buffer and then applied to a 4 ml column (0.5 x 18 cm) of Sephadex G25 (Amersham Pharmacia Biotech). After elution with the same buffer, the cross-linked peptide–oligodeoxynucleotide complex was lyophilized. The recovery was 150 pmol. For sequencing, the cross-linked peptide–oligodeoxynucleotide complex was dissolved in 200 µl H2O and centrifuged using a ProSorb cartridge (Applied Biosystems). The membrane was cut out, washed with 5% (v/v) methanol for 5 min, and dried. The peptide was sequenced on a pulsed liquid phase sequenator Model 477A (Applied Biosystems) with a 120A on-line high performance liquid chromatography system according to Thole et al. (29). An aliquot of 75 pmol of the material was amenable to sequencing.

Site-directed mutagenesis of the W61A variant of SsoII
Site-directed mutagenesis of the SsoII gene was performed by a PCR-based technique (30). The mutant gene was sequenced and found to contain only the mutation desired. It must be mentioned that the wild-type enzyme we are working with contains a Gln to Pro exchange at position 161.

Circular dichroism spectroscopy
Circular dichroism spectra were recorded with 10 µM solutions of wild-type SsoII and the W61A variant in 10 mM Tris, 50 mM NaCl in a Jasco J-710 dichrograph at ambient temperature in a cylindrical cuvette of 0.05 cm path length.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To identify regions of the restriction endonuclease SsoII in close contact with its DNA substrate, we decided to carry out photo-crosslinking experiments with a 5-IdU monosubstituted oligodeoxynucleotide carrying the modification at the central position of the recognition sequence CCNGG. Photo-crosslinking techniques using nucleic acids substituted with halogenated pyrimidines have been successfully applied to identify contacts in DNA– and RNA–protein complexes (31–37). 5-IdU is a near-perfect analogue of thymidine (38) and a photoactivated zero-length cross-linker which has the advantage that irradiation with long wavelength UV light (325 nm) does not lead to the excitation of other chromophores. Efficient cross-linking of 5-IdU requires the close proximity of a reactive amino acid such as Phe, Tyr, Trp, His and Met in a suitable orientation (38,39).

Cleavage of a 5-IdU substituted oligodeoxynucleotide by SsoII
In order to be sure that the results of our photocross-linking study can be interpreted in a meaningful manner, it was important to clarify whether the presence of the 5-IdU substitution in the SsoII recognition sequence does not prevent DNA cleavage by SsoII. We therefore performed cleavage experiments with the unmodified oligodeoxynucleotide S and the modified oligo­deoxynucleotide I and found that they are cleaved by SsoII with similar kinetics (data not shown).

Photocross-linking of the SsoII–oligodeoxynucleotide I complex
Oligodeoxynucleotide I can be cross-linked in good yield to SsoII by irradiation with UV light (325 nm) emitted from a HeCd laser. The crosslink is specific as oligodeoxynucleotide II (related in sequence to oligodeoxynucleotide I, but not a substrate for SsoII) and oligodeoxynucleotide III are not effectively cross-linked (Fig. 1). With oligodeoxynucleotide II a faint band representing a cross-linked complex is seen, both on the silver stained gel and the autoradiogram, while with oligo­deoxynucleotide III only on the autoradiogram a very faint band corresponding to the cross-linked complex is seen. Omission of SsoII or incubation without irradiation (or irradiation in the presence of excess of unmodified DNA) failed to yield any cross-linked product. To find the optimum irradiation time for preparative cross-linking, the time course of the photocross-linking reaction was analyzed (Fig. 2A). The yield of the photocross-linked complex in this experiment was 30–40% after 20 min of irradiation and could not be significantly increased by longer irradiation.



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  		Figure 2. The time course of the photocross-linking reaction with SsoII and the 5-IdU-substituted oligodeoxynucleotide I. The SsoII–oligodeoxynucleotide I complex was formed by incubating 20 µM SsoII with 20 µM 32P-labeled oligodeoxynucleotide containing the 5-IdU residue in the central position of the upper strand (Fig. 1A) for 10 min on ice. The mixture was irradiated as in Figure1 for the indicated time periods. (A) Silver-stained gel of the irradiated samples analyzed by 15% SDS–PAGE. S represents the molecular mass standard. (B) Quantitative evaluation of the autoradiogram is shown.

 
To obtain preparative amounts of the cross-linked complex the cross-linking reaction was performed 10 times in 50 µl reaction volumes containing 1 nmol of SsoII and 1 nmol of oligodeoxynucleotide I which was radioactively labeled on the 5'-end of the modified strand. Each individual irradiation was performed for 20 min on ice. An aliquot before irradiation and an aliquot after irradiation of the combined photocross-linking reaction mixtures was analyzed by SDS–PAGE. Figure 3A shows that the cross-linked SsoII–oligodeoxynucleotide complex was obtained in ~50% yield and represents a homo­geneous species.



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  		Figure 3. Preparative photocross-linking of SsoII and the 5-IdU substituted oligodeoxynucleotide I and digestion of the photocross-linked SsoII–DNA complex with various proteases. Preparative photocross-linking was performed 10 times in 50 µl aliquots containing 20 µM SsoII and 20 µM oligodeoxynucleotide I. Aliquots of 2.5 µl of the reaction mixture before (0 min) and after (20 min) irradiation were analyzed by SDS–PAGE. (A) Silver-stained gel; (B) corresponding autoradiogram. S, molecular weight standard. (C) Aliquots of the cross-linked SsoII–DNA complex were digested in a volume of 50 µl of 100 mM ammonium bicarbonate either without or with increasing amounts of the indicated protease [trypsin (T), chymotrypsin (C), a mixture of both (C/T) or proteinase K (K)] for 2 h at 37°C. The digests were analyzed on a 15% polyacrylamide gel containing 0.5x TBE and 2 M urea, followed by autoradiography. The lane designated Oligo shows the position of the upper strand which was substituted with 5-IdU and radioactively labeled with 32P. The band representing the cross-linked complex of SsoII and the oligodeoxynucleotide is indicated as well as the digested cross-linked peptides and the free 14mer oligodeoxynucleotide.

 
Proteolytic digestion of the photocross-linked SsoII–oligodeoxynucleotide I complex
To find out which protease would be most suitable to obtain a small and defined fragment of the cross-linked complex, proteolytic digestions were performed with aliquots of the radioactively labeled cross-linked SsoII–oligodeoxynucleotide complex in the presence of increasing amounts of trypsin and chymotrypsin and a combination of both enzymes as well as with proteinase K. Digestions were performed with varying amounts of proteases for 2 h at 37°C. Reaction products were analyzed by polyacrylamide gel electrophoresis in the presence of urea and visualized by autoradiography (Fig. 3C). Treatment of the photocross-linked SsoII–oligodeoxynucleotide complex with specific and unspecific proteases converted most of the cross-linked SsoII–DNA complex to small fragments. Chymotrypsin generated a smaller fragment than trypsin and the combined trypsin/chymotrypsin digestion further reduced the size of the chymotryptic fragment. The main product of the proteinase K proteolysis was of the same size as the chymo­tryptic product but was accompanied by smaller fragments. The chymotryptic degradation was chosen for further analysis, because it produced an apparently homogeneous end product.

Identification of the cross-link region in the chymotryptic fragment
To identify the amino acid involved in the cross-link between SsoII and oligodeoxynucleotide I, the reaction mixture from the preparative cross-linking experiment was incubated with chymotrypsin. An aliquot of the digested material was analyzed by polyacrylamide gel electrophoresis in the presence of urea to confirm complete proteolysis of the cross-linked SsoII–oligodeoxynucleotide I complex. The cross-linked peptide was separated from other peptides by anion-exchange chromatography on a Mono Q column (Fig. 4A). Aliquots of the fractions 23–27 were precipitated and analyzed on a urea polyacrylamide gel (Fig. 4B). Fractions 25 and 26 contained the cross-linked peptide–oligodeoxynucleotide I complex. Fractions 25 and 26 were desalted, concentrated and loaded onto a preparative urea polyacrylamide gel. The cross-linked peptide–oligodeoxynucleotide complex was extracted from the gel, desalted, lyophilized and subjected to gel filtration in order to remove residual salt. An aliquot of the final preparation of the cross-linked peptide–oligodeoxynucleotide complex was analyzed by urea polyacrylamide gel electrophoresis and shown to be homogeneous (Fig. 4C). The recovery was 150 pmol as determined by UV spectrophotometry. The identity of the photocross-linked peptide was determined by peptide sequencing. Only 75 pmol of the cross-linked peptide were amenable to sequencing, possibly due to partial N-terminal modification by urea which was used in the purification procedure. The sequenced peptide covalently linked to the central position in the upper strand of the SsoII recognition sequence, RKNXXKEFEP (where X represent amino acid residues that do not give rise to a defined PTH amino acid derivative) corres­ponds to amino acid residues 57–66 in the SsoII protein sequence. The amino acid residue at position 60 in the SsoII sequence is a serine, which often is not well detected in the Edman degradation. At position 61 in the SsoII sequence is a tryptophan, a very good candidate for the site of cross-linking.



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  		Figure 4. Purification of the cross-linked complex after digestion with chymotrypsin and isolation of the SsoII peptide involved in the cross-link. (A) Anion-exchange chromatography on a Mono Q column was used to separate the free peptides after chymotryptic digestion from the cross-linked peptide-oligodeoxynucleotide complex. Fractions of 1 ml were collected and the radioactivity monitored. (B) The cross-linked peptide-oligodeoxynucleotide complex was identified by analyzing aliquots of the fractions by electrophoresis on a 12% polyacrylamide gel in the presence of 0.5x TBE and 2 M urea, followed by autoradiography. Lane S, oligodeoxynucleotide size marker ranging from 8 to 32 nucleotides; lane 1, oligodeoxynucleotide I; lanes 2–6, aliquots of fractions 23–27 after precipitation. The position of the cross-linked peptide–oligodeoxynucleotide complex (CL Peptide) is shown. It is present in fractions 25 and 26. (C) The cross-linked SsoII peptide was cut out from a preparative gel (compare to Fig. 5B) and subjected to desalting on a NAP5 column and subsequently to gel filtration on a Sephadex G25 column. S represents the oligodeoxynucleotide size marker, lane 1 oligodeoxynucleotide I and lane 2 the final preparation of the cross-linked peptide oligodeoxynucleotide complex (CL Peptide) which was subjected to sequencing.

 
Mutational analysis of the presumptive cross-link site, Trp61, in SsoII
In order to confirm that Trp61 is the site of cross-linking and to find out whether Trp61 is involved in DNA binding and/or cleavage we have generated the W61A mutant. The His6-tagged protein was overexpressed in Escherichia coli and purified to near homogeneity by affinity chromatography using Ni-NTA agarose. The protein was inactive in DNA cleavage experiments (data not shown) and exhibited a decreased affinity towards DNA, as demonstrated in competition experiments in which increasing amounts of W61A were added to a mixture of wild-type SsoII and the oligodeoxynucleotide substrate (S), and the initial rates of DNA cleavage were measured (Fig. 5). As an ~50% inhibition of the wild-type SsoII cleavage rate was observed at a 20-fold excess of the variant over the wild-type protein, we conclude that W61A has an ~20-fold lower affinity for this oligodeoxynucleotide substrate than wild-type SsoII. To exclude that enzymatic inactivity and decrease in affinity is possibly due to improper folding, circular dichroism spectra of the wild-type and the mutant protein were recorded (Fig. 6). They were found to be identical within the limits of error, demonstrating that the W61A variant has the same secondary structure (and, therefore, most likely the same tertiary structure) as the wild-type protein. The spectra obtained were analyzed in terms of secondary structure composition (40). According to this analysis SsoII contains ~40% {alpha}-helix and 20% ß-sheet.



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  		Figure 5. Cleavage of a 14mer substrate by SsoII alone and in competition with its variant W61A. Cleavage of the 32P-labeled 14mer oligodeoxynucleotide S (100 nM) was performed with 100 nM SsoII after preincubation in the absence or presence of 2 µM W61A SsoII. Aliquots were withdrawn at the time points indicated and analyzed on a 20% polyacrylamide gel containing 1x TBE and 7 M urea. (A) Autoradiogram of the gel; (B) quantitative evaluation by an instant imager. Circles, cleavage in the absence of W61A SsoII; squares, cleavage in the presence of W61A SsoII.

 


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  		Figure 6. Circular dichroism spectra of wild-type SsoII and the mutant W61A. Circular dichroism spectra of 10 µM wild-type SsoII (black) and of 10 µM W61A variant (grey) are shown.

 
We have performed photocross-linking experiments with the W61A variant under the same conditions as with the wild-type enzyme, i.e. at concentrations sufficient for complex formation. As shown in Figure 7, the W61A variant cannot be cross-linked to oligodeoxynucleotide I, confirming that Trp61 is the site of cross-linking.



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  		Figure 7. Photocross-linking of W61A to 5-IdU substituted oligodeoxynucleotide I. Aliquots of 20 µM wild-type SsoII (lanes 1 and 2) or the W61A mutant (lanes 3 and 4) were incubated with 20 µM oligodeoxynucleotide I for 10 min on ice. The mixtures were irradiated for 20 min. Aliquots before (lanes 1 and 3) and after irradiation (lanes 2 and 4) were analyzed by electrophoresis on a 15% SDS–polyacrylamide gel that was silver stained. Lane S shows size markers.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have carried out photocross-linking experiments with the restriction endonuclease SsoII and an oligodeoxynucleotide carrying in the central position of the recognition sequence CCNGG a 5-IdU substitution in one strand. A region in SsoII was identified to be located in the protein–DNA interface of the SsoII–DNA recognition complex by a specific cross-link between Trp61 and the central base pair of the recognition sequence. As 5-iodopyrimidines are considered to be zero-length cross-linkers and the cross-linking yield was high (~50%), it can be assumed that during the recognition process Trp61 approaches the DNA closely, presumably by forming a van der Waals contact with the non-specified central base pair in the major groove of the DNA. This assumption is supported by the finding that the W61A variant of SsoII which must have a similar structure as the wild-type protein (as it has the same circular dichroism spectrum), is inactive in DNA cleavage and shows a decrease in affinity towards its substrate. The inactivity of the W61A variant is probably due to the fact that the small side chain of alanine allows for a conformational freedom at the protein–DNA interface that is not tolerated by the large side chain of tryptophan which therefore seems to be required for a precise fit of the DNA into the SsoII binding site.

The amino acid sequence of the peptide identified to be cross-linked to the oligodeoxynucleotide, RKNSWKEFEP (Trp61 underlined), does not show any pronounced homology to any known protein sequence, according to a BLAST search (41). Although SsoII shows significant sequence homology with the restriction enzymes EcoRII and PspGI (42) with 36 identical out of 122 amino acid residues and 51 out of 198, respectively, the homology does not extend to the N-terminal region in which Trp61 is located. In this context it is noteworthy that the available co-crystal structures of restriction endo­nuclease–DNA complexes suggest that the major DNA recognition elements are located in the C-terminal half of the respective enzymes, for example the extended chain motif of EcoRI: Met137–Asn142 (12), the R-loop of EcoRV: Gly182–Thr187 (13), i.e. distal from the catalytic PD/E...D/EXK motif which is located in the N-terminal half. It might be, therefore, that SsoII is an exception compared to these enzymes, or that we have only identified a minor recognition element, which is present for example in PvuII (Asp34) and EcoRV (Lys38), where they are responsible for base contacts in the minor groove (13,14). The involvement of other protein regions in DNA binding will be investigated in further photocross-linking studies with SsoII and for comparison also with EcoRII, for which a DNA binding site was identified in the C-terminal half, with homology to other restriction endonucleases, among them SsoII (23).


   ACKNOWLEDGEMENTS
 
We thank Frauke Christ for many valuable discussions and Dr Uwe Pieper for recording and analyzing the circular dichroism spectra as well as performing the BLAST search. This work has been supported by grants from the DFG and the RFBR.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +49 641 99 35402; Fax: +49 641 99 35409; Email: vera.pingoud@chemie.bio.uni-giessen.de This paper is dedicated to the memory of Professor Dr Z. Shabarova ({dagger} 19 September 1999) and Professor Dr D. Nathans ({dagger} 16 November 1999), pioneers in the study of the biochemistry of restriction enzymes Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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