Restriction Endonuclease SsoII with Photoregulated Activity—a

ARTICLE pubs.acs.org/bc

Restriction Endonuclease SsoII with Photoregulated Activity—a “Molecular Gate” Approach Le Thi Hien,†,|| Timofei S. Zatsepin,†,§ Benno Schierling,# Eugene M. Volkov,† Wolfgang Wende,# Alfred Pingoud,# Elena A. Kubareva,‡ and Tatiana S. Oretskaya*,†,‡ †

Department of Chemistry Belozersky Institute of Physico-Chemical Biology Lomonosov Moscow State University, Leninskie gory, Moscow 119991, Russia § Central Research Institute of Epidemiology, Novogireevskaya street 3a, Moscow 111123, Russia # Justus-Liebig-Universit€at, Institut f€ur Biochemie, FB 08, Heinrich-Buff-Ring, Giessen, D-35392 Germany ‡

bS Supporting Information ABSTRACT: A novel method for regulating the activity of homodimeric proteins—“molecular gate” approach—was proposed and its usefulness illustrated for the type II restriction endonuclease SsoII (R.SsoII) as a model. The “molecular gate” approach is based on the modification of R.SsoII with azobenzene derivatives, which allows regulating DNA binding and cleavage via illumination with light. R. SsoII variants with single cysteine residues introduced at selected positions were obtained and modified with maleimidoazobenzene derivatives. A twofold change in the enzymatic activity after illumination with light of wavelengths of 365 and 470 nm, respectively, was demonstrated when one or two molecules of azobenzene derivatives were attached to the R.SsoII at the entrance of or within the DNA-binding site.

’ INTRODUCTION In “genome surgery”, a number of enzymes are used as molecular tools for cleaving defective genes—restriction and homing endonucleases, fusion of DNA cleavage domains (e.g., the nonspecific DNA cleavage domain of FokI) with DNA binding modules (e.g., “zinc fingers”).1,2 Specific DNA cleavage at the desired position and at the appropriate time is a key requirement for precise gene targeting. In the case of the human genome, only so-called meganucleases (sequence specific endonucleases with extended recognition sites) could be used to cleave a single unique site.3 However, even such enzymes can show undesirable activity in the cell. There are numerous methods to regulate the protein activity on the level of gene expression4,5 or through the reversible or irreversible interaction with different types of molecules including oligonucleotides.6
9 Usually, attaching low molecular mass ligands to proteins allows the synthesis of bioconjugates with special properties— i.e., modified or novel activity.10
12 One of the methods used is based on the photoregulation of the activity of a protein via an azobenzene “photoswitch”.13 Azobenzene can reversibly isomerize from the extended trans-configuration to the more compact cis-configuration by illumination with UV light (trans f cis) or vice versa by blue light (cis f trans). Thermal relaxation is also possible (cis f trans).14 Rapid and effective cis
trans isomerization of azobenzene and its derivatives allows their application for in vivo studies.15 The regulation of the activity of restriction endonucleases (RE) or homing endonucleases (HE) by light r 2011 American Chemical Society

opens up new prospects for controlling DNA duplex cleavage and creating safer molecular tools for gene manipulations. Thus, the modified nuclease can be delivered to the cell nucleus in the less active state for the subsequent remote activation by an external signal (UV light) after binding to the DNA at a specific site. Undesirable DNA cleavage can be reduced by lowering the nuclease activity after hydrolysis of the target area by illumination with blue light. The primary requirement for production of a photoregulated protein is the selective attachment of appropriate light-sensitive compounds. So far, there are only reports about two RE for which activity can be regulated by light, namely, for BamHI (R.BamHI)16
18 and PvuII (R.PvuII).19 R.BamHI specifically hydrolyzes doublestranded DNA only after formation of the correct homodimeric form of the enzyme. Nakayama et al. introduced phenylazophenylalanine (AzoAla) into the dimer interface to regulate the activity of this enzyme by affecting the dimerization process.20 The activity of the enzyme in the dark (trans-AzoAla-BamHI) was suppressed, but after illumination with UV light, the activity of the enzyme (cis-AzoAla-BamHI) was restored. However, this method is time-consuming and tedious. Previously, we have demonstrated an efficient method, which one might call the “molecular spring” approach for regulating the Received: January 31, 2011 Revised: May 4, 2011 Published: June 20, 2011 1366

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Bioconjugate Chemistry Scheme 1. Photoregulation of Modified RE SsoII by “Molecular Gate” Approach

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X-ray structure analysis of its homologue—restriction endonuclease Ecl18kI (R.Ecl18kI)22 in order to introduce Cys residues in appropriate positions; (3) production of mutant forms of R.SsoII, containing Cys residue(s) in the selected positions; (4) modification of R.SsoII variants by azobenzene derivatives and analysis of the DNA cleavage activity of modified R.SsoII when illuminated by UV and blue light, respectively.

’ EXPERIMENTAL SECTION

activity of R.PvuII.19 A symmetric azobenzene derivative—bis(maleimido)azobenzene—was introduced as a cross-linker to connect two neighboring cysteine residues next to the active site of the protein. When the exciting light changes from UV to blue and vice versa, the isomerization of azobenzene works like a spring, altering the local conformation of the protein, which can lead to a change in the activity of the enzyme. The “molecular spring” method is better applied to proteins of small molecular mass. In this case, the isomerization of bis(maleimidoazobenzene) changes the local structure of the protein. In addition, linkage of the two subunits of the homodimer protein by a peptide spacer proved to be advantageous for achieving a high photoswitching effect. Here, the N- and C-termini of the two subunits of the wild-type R.PvuII are brought close to each other with the help of a four-amino-acid linker. In this study, we propose a “molecular gate” approach to regulate the activity of a restriction endonuclease. This approach is based on the fact that most type II restriction endonucleases in the active state are homodimers with the DNA-binding center located in the interface between the two subunits. Modification of the protein with azobenzene derivatives (A) (Scheme 1) at the entrance of the DNA binding site might make the DNA binding site inaccessible due to steric hindrance in the case of the extended configuration of trans-azobenzene (B). When illuminated by UV, the extended trans-form of azobenzene isomerizes into the compact cis-form (C) that might increase the distance R between the ends of the two molecules of azobenzene (Scheme 1). Therefore, in the cis-form of azobenzene the DNA substrate could possibly reach the DNA-binding center of the protein. The subject of our investigation was the homodimeric restriction endonuclease SsoII (R.SsoII). It recognizes and cleaves the double-stranded pentanucleotide sequence: 50 3 3 3 V CCNGG 3 3 3 30 /30 3 3 3 GGNCCv 3 3 3 50 (N = A, G, C, T). A thiol group of a cysteine residue can be introduced into the protein by site-directed mutagenesis and this is the most convenient way for the specific modification of the protein by various reagents. Previously, we synthesized symmetrical bis(maleimido)azobenzene and asymmetrical azobenzene derivatives bearing a maleimide group in one of the benzene rings for binding to a cysteine of the protein and a hydroxyl or carboxyl group in the other ring. The optical properties of the azobenzene derivatives in free form and after its attachment to the R.SsoII variants were studied.21 The aim of the present investigation was to modify variants of R.SsoII by azobenzene derivatives, for the purpose of regulating the activity of the enzyme by light. The tasks of the work were as follows: (1) synthesis of azobenzene derivatives, confirmation of their structures, investigation of their chemical and optical properties; (2) selection of amino acid residues in R.SsoII on the basis of the

General. Materials and reagents were obtained from commercial suppliers and used without further purification unless otherwise noted. The following reagents were used in this study: monomaleimide derivative of 2.3 kDa poly(ethylene glycol) (Pierce, USA) and 5 kDa poly(ethylene glycol) (PEG-Mal) (Sigma, USA), Ni2+-NTA-agarose (Qiagen, Germany), protein ladder PageRuler, DNA-marker pUC mix (Fermentas, Lithuania). € kta FPLC (GE Heathcare, USA) was used with HiTrap G-25 A Superfine Sephadex desalting 5 mL column (GE Healthcare, USA). Mass spectra were recorded on a system that consisted of an ACQUITY UPLC and a TQD tandem quadrupole massspectrometric detector (Waters, USA). NMR spectra were recorded on the Avance 400 spectrometer (Bruker, Germany) at 25 °C. CHCl3 served as an internal standard. 4-Maleimido-40 -[2-(2-hydroxyethylthio)succinimido]azobenzene (MAB
OH) (1). MAB-OH was synthesized as described previously21 with minor modifications (Scheme 2). A solution of 2-mercaptoethanol (0.4 mmol) in dioxane (2 mL) was added to a solution of bis(maleimido)azobenzene (150 mg, 0.4 mmol) in dioxane (20 mL) within 30 min while stirring solution. The target product (Rf 0.23) was isolated by chromatography on a silica gel column with an ethanol gradient in chloroform (0
5%) with a yield of 45 mg (25%). 1H NMR (CDCl3, π, ppm): 8.0 (4 H, d), 7.7 (4 H, d), 7.3 (2 H, s), 4.2 (1 H, s), 3.7 (2 H, m), 3.0 (2 H, m), 2.7 (2 H, m); MS: C22H18N4O5S (MH)+ calcd/found: 450.10/451.24. 4-Maleimido-40 -(2-(2-amino-2-carboxyethylthio)succinimido)azobenzene (MAB-Cys) (2). An aqueous solution of L-cysteine hydrochloride (10 mL, 63 mg, 0.4 mmol) was added to a solution of bis(maleimido)azobenzene (200 mg, 0.5 mmol) in dioxane (30 mL) within 30 min while stirring solution. The mixture obtained was extracted twice with 30 mL chloroform. The precipitate, containing products, was filtered and dried in air. The yield of a mixture of products with one 2 and two cysteine residues 2.1 (Scheme 2) was 111 mg (50%). MS: C23H19N5O6S (MH)+ calcd/found: 493.49/494.31. Mutagenesis, Protein Expression, and Purification. Sitedirected mutagenesis of the R.SsoII gene was performed using a PCR method.23 The plasmids pQESso9 carrying the gene coding for the restriction endonuclease SsoII and pACMS7 containing the gene coding for the methyltransferase SsoII were a generous gift from Dr. V. Pingoud (Institute of Biochemistry, Justus-Liebig University, Giessen, Germany). The plasmid pQESso9 or plasmids carrying mutant forms of the R.SsoII gene were used to transform the E. coli strain JM109 which had previously been transformed with the plasmid pACMS7. All genetic constructs were confirmed by DNA sequencing over the entire coding region. Cultures were grown at 37 °C to OD600 ≈ 0.7 and protein expression was induced by the addition of 1 mM IPTG. After 3 h of induction, cells were harvested by centrifugation for 15 min, 4000 g at 4 °C. The cell pellet was resuspended in 20 mM Tris-HCl buffer (pH 7.9), containing 500 mM NaCl, 20 mM imidazole, 1367

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Bioconjugate Chemistry

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Scheme 2. Synthesis of Monosubstituted Azobenzene Derivatives

1 mM phenylmethanesulfonylfluoride, pH 7.9, and lysed by sonication. Cell debris was removed by centrifugation (17000  g) for 30 min at 4 °C. The recombinant His-tagged proteins were purified by affinity chromatography over Ni-NTA agarose using 20 mM Tris-HCl (pH 7.9), 500 mM NaCl, 200 mM imidazole, 5 mM 1,4-dithiothreitol (DTT) for elution. Fractions containing pure protein were dialyzed overnight at 4 °C against 10 mM potassium phosphate buffer (pH 7.4), containing 200 mM KCl, 50% v/v glycerol, and stored at
20 °C. Protein purification was monitored by SDS-PAGE analysis, and protein concentration was determined by absorbance measurements at 280 nm. The molecular mass of the recombinant R. SsoII was determined as 37.2 kDa. Protein Labeling with Azobenzene Derivatives. To confirm reactivity of cysteine residues introduced into R.SsoII mutants, 5 μM R.SsoII in 20 mM K-phosphate buffer (pH 7.4), containing 150 mM NaCl, was incubated with a 50-fold molar excess of PEG-Mal (5 kDa) (freshly dissolved in DMSO) for 10 min at RT. The conjugates were analyzed by SDS-PAGE. To synthesize azobenzene
protein conjugates, 5 μM R.SsoII variant in 20 mM K-phosphate buffer (pH 7.4), containing 150 mM NaCl, was incubated with a 5-fold molar excess (over the number of cysteine residues in the protein) of the azobenzene derivative 1 or 2 (freshly dissolved in DMSO) for 10 min at RT. The yield of the conjugate was estimated in the following way. A 50-fold molar excess of PEG-Mal was added to an aliquot of the reaction mixture and analyzed by SDS-PAGE. The reaction € kta FPLC gel filtration and dialyzed mixture was purified by A overnight at 4 °C against 10 mM K-phosphate buffer (pH 7.4), containing 200 mM KCl, 50% v/v glycerol. Purified protein was stored at
20 °C. DNA Cleavage by Modified R.SsoII after Specific Illumination. Modified R.SsoII was pre-illuminated with light and then the cleavage reaction was started under continuous illumination. The “pre-illumination” of the R.SsoII variants was performed as follows: protein solution was illuminated with blue light (470 nm) for 2 min at a distance of 15 cm to transform azobenzene in the modified R.SsoII variants to the trans-state and was illuminated for 7 min with a UV lamp (365 nm) at a distance of 9 cm to transfer azobenzene in the modified R.SsoII

variants to the cis-state. The pre-illuminated enzyme was incubated with the DNA substrate under continuous specific illumination. After every 1, 2, 3, 5, 7, and 10 min, an aliquot of the reaction mixture was taken and analyzed by SDS-PAGE. DNA cleavage activity was analyzed by using an 873 base pair (bp) PCR fragment as substrate, which has a single R.SsoII site. The DNA substrate was incubated with R.SsoII for 15 min at 37 °C in 20 mL of 10 mM Tris-HCl buffer (pH 7.5), containing 10 mM MgCl2, 5 mM NaCl, 1 mM DTT, and 0.1 mg/mL bovine serum albumin (BSA). The concentration of DNA substrate was 5
10 nM (for R.SsoII variants with low activity) or 20 nM (for R.SsoII variants with high activity). Concentration of wt and mutant forms of R.SsoII ranged from 20 to 500 nM. Reaction products (DNA fragments of 530 bp and 338 bp in length) were analyzed by electrophoresis in a 4
6% polyacrylamide gel under nondenaturing conditions. Gels were stained with ethidium bromide, and the fluorescence was measured with a BioDocAnalyze system (Biometra) and quantitated. Intensities of DNAcontaining bands were determined by processing the gel image using the ImageQuant program (GE Healthcare). The amount of cleavage (%) was calculated as the ratio between the intensities of the product bands and the total intensities of all the bands (initial DNA substrate and products). WtR.SsoII was used as a control. When R.SsoII variants, modified with MAB-Cys (2) were used, the cleavage reaction was carried out in 10 mM Tris-HCl buffer (pH 7.5) without DTT, containing 10 mM MgCl2, 5 mM NaCl, 0,1 mg/mL BSA and, in addition, 1 μM NiCl2 or 10 μM CoSO4 (for forming a complex between the Cys residue in MAB-Cys and metal ions) was used.

’ RESULTS AND DISCUSSION Synthesis of Azobenzene Derivatives. Asymmetric azobenzene derivatives MAB-OH (1) and MAB-Cys (2) were obtained by the reaction of two different thiols with bis(maleimido)azobenzene (Scheme 2). However, compound 2 was quite hydrophilic and could not be isolated by silica gel column chromatography. Therefore, the reaction mixture was extracted with chloroform to remove bis(maleimido)azobenzene. Further, the mixture of 2 and 2.1 was 1368

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Bioconjugate Chemistry used for modification of protein, since only compound 2 is able to react with the cysteine residue of a protein. Design of R.SsoII Variants and Their Properties. There are two native cysteine residues at positions 33 and 60 of wtR.SsoII. The variant without cysteine residues R.SsoII(C33S/C60S), Cys-free R.SsoII (cfR.SsoII or 2CS), was produced by sitedirected mutagenesis. Other R.SsoII variants, in which some amino acids were changed for cysteine, were produced from the cfR.SsoII gene. Previously, the structure of R.Ecl18kI with a DNA substrate was determined.22 This RE is an isoschizomer of R. SsoII and differs from it by one amino acid only (Val232 in R. Ecl18kI corresponds to Ile232 in R.SsoII). The amino acid residues to be substituted by mutagenesis were chosen based on the R.Ecl18kI structure (Figure 1). The homodimeric protein contains internal and external clamps that surround the DNA substrate. Two amino acid residues S171 and R174 on the external clamp and three amino acids R198, I220, and A224 on the internal clamps were selected and changed for cysteine residues for subsequent modification by azobenzene derivatives (Figure 1). Thus, 5 variants each with one cysteine residue at the positions listed above and 6 variants with 2, 3, or 4 cysteine residues in various combinations were produced. It was expected that the larger the number of molecules of azobenzene located near the entrance of the DNA-binding site of a protein, the greater the steric effect and the greater should be the influence of illumination on the activity of the protein.

Figure 1. Structure model of the homodimer R.SsoII (based on the X-ray structure analysis for R.Ecl18kI22). Amino acid residues S171, R174, R198, I220, and A224 that were changed to cysteine are marked with arrows.

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The enzymatic activity of R.SsoII was assessed by its ability to hydrolyze a 873 bp DNA substrate, having only one recognition site for this enzyme. It was shown that the enzymatic activity of variants of R.SsoII depends on the position and the number of cysteine residues introduced into the enzymes (Figure 2). The closer the Cys residue is to the DNA-binding center of the enzyme and the greater the number of cysteine residues introduced is, the less active the mutant form of R.SsoII is likely to be. Thus, the cleavage of a DNA substrate by the 2CS/S171C variant is 70% of the activity of wtR.SsoII, of the 2CS/S171C/R174C variant is about 40%, and of the 2CS/S171C/R174C/I220C/ A224C variant is only 25% even with 3-fold higher concentration of this enzyme. This activity comparison is shown in Figure 2. For further studies, enzyme concentrations were selected so that the DNA cleavage ranged from 20% to 80%. In the variant 2CS/ R198C, the replaced R198 residue is located next to E195, which takes part in catalysis; this explains the low enzymatic activity of this variant among the other ones that have only a single cysteine residue modified per subunit. Variants with two cysteine residues modified per subunit, one of which at position 198, show the lowest activity (2CS/R198C/A224C) or almost no activity (2CS/R174C/R198C). Modification of Protein with Maleimide Derivatives. Poly(ethylene glycol) containing a maleimide group (PEG-Mal) was used to assess the reactivity of cysteine residues in R.SsoII variants. The high molecular mass of PEG-Mal (2.3 or 5 kDa) allows one to measure the formation of its conjugate with mutant

Figure 3. SDS-PAGE analysis of R.SsoII variants (5 μM) containing 1 (lane 5), 2 (lane 4), 3 (lane 3), or 4 (lane 2) cysteine residues per protein monomer modified with with PEG-Mal (500 μM). Lane 1
Protein molecular mass marker. Gel was stained with a Coomassie solution. a - unmodified protein; b - conjugate of 2CS/I220C with PEG-Mal; c - conjugate of 2CS/S171C/R174C with PEG-Mal; d - conjugate of 2CS/ S171C/R174C/I220C with PEG-Mal; e - conjugate of 2CS/S171C/ R174C/I220C/A224C with PEG-Mal.

Figure 2. Histogram showing the amount of cleavage of a DNA substrate by R.SsoII variants. The degree of DNA cleavage was determined as the arithmetic mean of three independent experiments. 1369

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Table 1. Comparison of the Enzymatic Activity of R.SsoII Variants Modified with the Azobenzene Derivative MAB-OH, under UV and Blue Light Illumination, Respectively enzyme

Figure 4. Analysis of R.SsoII(2CS/R174C) (5 μM) modified with PEG-Mal (2.3 kDa, 500 μM) and (or) MAB-OH (50 μM) by SDS gel electrophoresis. The gel was stained with a Coomassie solution. The protein was incubated with PEG-Mal (lane 2), with MAB-OH (lane 3), initially with MAB-OH, and then with PEG-Mal (lane 4). Lane M protein molecular mass marker, lane 1 - protein control. a - R.SsoII(2CS/R174C); b - conjugate of R.SsoII(2CS/R174C) with PEG-Mal.

forms of R.SsoII by SDS-PAGE. PEG-Mal reacted almost quantitatively with the majority of the R.SsoII variants with one and two cysteine residues per subunit, which indicated the high reactivity of cysteine residues toward the maleimide group (Figure 3). However, the yield of modification of the R.SsoII variants with three and four cystein residues per subunit was low due to steric hindrance, so we excluded these variants from further studies. A 5-fold excess of a solution of one of the azobenzene derivatives (1 or 2) (over the number of cysteine residues) was added to the protein solution at room temperature in order to modify Cys residues of R.SsoII variants. The molecular mass of azobenzene derivatives (2 times) by specific illumination was obtained for the modified 2CS/R174C/A224C-MAB-OH variant, in which one molecule of MAB-OH was attached per subunit to the Cys residue at the entrance of the DNA-binding site (C224) and the other one to the Cys residue on the outer surface of the protein (C174). We assume that hydrogen bonds could form between the hydroxyl groups of the azobenzene derivatives (MAB-OH) in the transform, which could interfere with DNA binding (Figure 5). In the present work, maleimidoazobenzene that contains a cysteine residue in one of the benzene rings, MAB-Cys (2), was 1370

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Table 3. Comparison of the Enzymatic Activity of R.SsoII and Its Variants, Modified with the Azobenzene Derivative MAB-Cys, under UV and Blue Light Illumination, Respectively enzyme

Figure 6. (A) DNA cleavage by the enzyme 2CS/R171C-MAB-Cys in the presence of Ni2+ ions (concentration 1 μM) under specific illumination. (B) Schematic detail of the 2CS/R171C-MAB-Cys variant with metal ions.

Table 2. Kinetic Parameters of 20 bp DNA Duplex Hydrolysis by wtR.SsoII and the 2CS/S171-MAB-Cys Variant enzyme wtR.SsoII 2CS/S171C-MAB-Cys

Vmax, nM/min

KM, nM

Vmax/Km

100 ( 13

4.07 ( 0.65

24.6

77 ( 5

17.03 ( 0.09

42.3

synthesized. A cysteine residue was selected as the substituent in one of the benzene rings of maleimidoazobenzene, because cysteine and its derivatives form complexes with many metal ions.24 In the modified protein, the Cys residue of MAB-Cys might coordinate the metal ion when the MAB-Cys is in the trans-form. Such a “barrier” (one metal ion complexed by two opposite MAB-Cys residues) should prevent the entrance of the substrate into the DNA-binding site. Analysis of the R.Ecl18kI structure showed that the most preferred variant for attachment of the MAB-Cys was the 2CS/S171C variant (Figures 1, 6). Kinetic parameters of DNA hydrolysis by wild-type R.SsoII and protein 2CS/S171C-MAB-Cys were determined without illumination and without adding any nickel or cobalt ions. Initial hydrolysis rates of a 20 bp DNA duplex, containing one recognition site of R.SsoII, by the enzymes were calculated. Different substrate concentrations were used. From the data, the values of the Michaelis constant (KM) and the maximum rate of the enzymatic reaction (Vmax) were determined (Table 2). It was demonstrated that the KM of 2CS/S171C-MAB-Cys was 4 times higher than that of wtR.SsoII, while the maximum hydrolysis rate Vmax of both enzymes differed less significantly. Thus, a 20 bp duplex has lower affinity for the 2CS/S171C-MABCys variant compared to the wild-type enzyme. This was consistent with our expectation, because azobenzene derivatives, which are next to the entrance to the DNA binding site, should impair substrate binding to the enzyme. It was shown previously that Ni2+ and Co2+ ions form complexes with cysteine and its derivatives, and chelate stability constants of these complexes are higher in the case of Ni2+ ions.24 However, Ni2+ ions show a higher inhibitory effect on the activity of RE.25,26 Therefore, we determined the maximum concentration of the metal ions at which wtR.SsoII was still active. It was found that wtR.SsoII was able to hydrolyze the DNA substrate if the reaction mixture contains 1 to 10 mM CoSO4 or 1 mM NiCl2

photoswitch effect

1

wt

1.00 ( 0.01

2

wt +1 mM NiCl2

1.00 ( 0.02 1.1 ( 0.1

3

wt +1 mM CoSO4

4

2CS/S171C-MAB-Cys

1.1 ( 0.1

5

2CS/S171C-MAB-Cys +1 mM NiCl2

2.2 ( 0.2

6

2CS/S171C-MAB-Cys +1 mM CoSO4

1.2 ( 0.1

7

2CS/S171C-MAB-Cys +10 mM CoSO4

1.6 ( 0.1

in the presence of 10 mM MgCl2 (data not shown). The cleavage rates of the DNA substrate by wtR.SsoII under specific illumination in the absence and in the presence of Co2+ or Ni2+ are comparable (Table 3). The same result was obtained for the 2CS/R171C-MABCys variant in the absence of metal ions Co2+ or Ni2+ (Table 3), while in the presence of nickel ions (concentration 1 mM), the initial cleavage rate under UV light was 2-fold higher than that after irradiation with blue light (Figure 6). It can be concluded that the observed difference in the activity 2CS/R171C-MAB-Cys when illuminated by UV and blue light, respectively, was due to the formation of the complex with metal ions. In the presence of 10 mM CoSO4, the photoswitch effect was 1.6 (Table 3). It needs to be discussed whether DNA damage by UV illumination is more critical for the integrity of the genome than DNA cleavage by a nonspecific nuclease. We are fully convinced that a low-intensity UV-B illumination is much less toxic to the cell than uncontrolled nonspecific DNA cleavage. It is clear that UV-B light (290
320 nm) is much more harmful for the cell than UV-A light (320
400 nm). However, the dose (400 nm.29

’ CONCLUSIONS We developed a “molecular gate” approach for the regulation of the DNA cleavage activity of R.SsoII by light illumination. A number of R.SsoII mutants were produced that have cysteine residue(s) at selected positions, modified with maleimidoazobenzene derivatives and tested for DNA cleavage activity when illuminated by UV and blue light, respectively. A twofold change in the enzymatic activity of the modified R.SsoII by illumination with UV and blue light was demonstrated in the case of 2CS/ R174C/A224C-MAB-OH and 2CS/S171C-MAB-Cys in the presence of Ni2+ ions. The “molecular gate” approach developed in the present work might be most suitable for proteins with high molecular mass and a large surface area near the entrance of the substrate-binding center of the enzyme. The latter allows one to vary the position of modification by azobenzene derivatives in order to achieve the largest effect of “closure” and “opening” of the gate. To determine the optimal position for modification in the “molecular gate” approach is less tedious than in the “molecular spring” 1371

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Bioconjugate Chemistry method, because the site of modification should be next to the entrance of the DNA substrate into the DNA-binding center of the enzyme. A significant advantage of the “molecular gate” approach is that only one product is formed during attachment azobenzene derivatives to the protein. The method of regulation of enzyme activity by light can be applied not only to restriction endonucleases, but also to any homodimeric DNA- or RNA-binding protein, and other proteins. Regulation of enzymatic activity of a RE by light opens up new prospects for controlling DNA duplex cleavage and creating safer molecular tools for manipulating genes for gene targeting, including gene therapy.

’ ASSOCIATED CONTENT

bS

Supporting Information. Cartoon-style principle of the method, example of analysis of DNA cleavage activity of wtR. SsoII, and example of the absorption spectrum of the conjugate 2CS/S171C/R174C with the azobenzene derivative 1 and its changes during trans f cis and cis f trans isomerization. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Department of Chemistry, Lomonosov Moscow State University, Leninskie gory, Moscow 119991, Russia. Tel.: +7-495-939-5411, Fax: +7-495-939-3181, e-mail: [email protected]. )

Present Addresses

Department of Engineering Physics and Nanotechnology, University of Engineering and Technology,144 Xuan Thuy, Hanoi, Vietnam

’ ACKNOWLEDGMENT This work has been supported by the German Research Foundation and the Russian Foundation for Basic Research by the DFG-RFBR program “International Research Training Groups” (grants GRK 1384, 11-04-91338), Russian Foundation for Basic Research (grants 10-04-01578 and 09-04-93108), and grant for Leading Scientific Schools (3314.2010.4). ’ REFERENCES (1) Pingoud, A., and Silva, G. H. (2007) Precision genome surgery. Nat. Biotechnol. 25, 743–744. (2) Grizot, S., Smith, J., Daboussi, F., Prieto, J., Redondo, P., Merino, N., Villate, M., Thomas, S., Lemaire, L., Montoya, G., Blanco, F. J., P^aques, F., and Duchateau, P. (2009) Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease. Nucleic Acids Res. 37, 5405–5419. (3) Pingoud, A., Fuxreiter, M., Pingoud, V., and Wende, W. (2005) Type II restriction endonucleases: structure and mechanism. Cell. Mol. Life Sci. 62, 685–707. (4) Bumcrot, D., Manoharan, M., Koteliansky, V., and Sah, D. W. (2006) RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat. Chem. Biol. 2, 711–719. (5) Li, C., Feng, Y., Coukos, G., and Zhang, L. (2009) Therapeutic microRNA strategies in human cancer. AAPS J. 11, 747–757. (6) Sarkar, F. H., and Li, Y. (2008) NF-kappaB: a potential target for cancer chemoprevention and therapy. Front. Biosci. 13, 2950–2959. (7) Dolinnaya, N. G., Kubareva, E. A., Kazanova, E. V., Zigangirova, N. A., Naroditsky, B. S., Gintsburg, A. L., and Oretskaya, T. S. (2008)

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