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Substrate-Assisted Catalysis in the Cleavage of DNA by the Ecori and Ecorv Restriction Enzymes

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Substrate-Assisted Catalysis in the Cleavage of DNA by the Ecori and Ecorv Restriction Enzymes Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8499-8503, September 1993 Biochemistry Substrate-assisted catalysis in the cleavage of DNA by the EcoRI and EcoRV restriction enzymes (acid base catalysis/mechanism of deavage/nuclease/chemicafly modiffed oligodeoxynucleotide/H-phosphonate) ALBERT JELTSCH, JURGEN ALVES, HEINER WOLFES, GUNTER MAASS, AND ALFRED PINGOUD* Zentrum Biochemie, Medizinische Hochschule Hannover, Konstanty-Gutschow-Strasse 8, 30623 Hannover, Germany Communicated by Thomas R. Cech, June 14, 1993 ABSTRACT The crystal structure analyses of the EcoRI- stereochemistry of their catalysis make it likely that these DNA and EcoRV-DNA complexes do not provide clear sug- enzymes cleave DNA by a similar mechanism that involves gestions as to which amino acid residues are responsible for the an attack of an activated water molecule in-line with the activation of water to carry out the DNA cleavage. Based on leaving 03' group. Recently, we published (11) a detailed molecular modeling, we have proposed recently that the at- proposal for the mechanism of DNA cleavage by EcoRI and tacking water molecule is activated by the negatively charged EcoRV based on structural data (Brookhaven data bank pro-Rp phosphoryl oxygen of the phosphate group 3' to the entries: 1RlE, 3RVE), molecular modeling, and results of scissile phosphodiester bond. We now present experimental site-directed mutagenesis experiments (ref. 12 and references evidence to support this proposal. (0) Oligodeoxynucleotide therein): Asp-91 and Glu-111 in EcoRI, Asp-74 and Asp-90 in substrates lacking this phosphate group in one strand are EcoRV, and the phosphate at which cleavage occurs were cleaved only in the other strand. (it) Oligodeoxynucleotide proposed to bind the essential cofactor Mg2+. The divalent substrates carrying an H-phosphonate substitution at this metal ion probably has several functions: it might help to bind position in both strands and, therefore, lacking a negatively and to position the DNA, it could also partially neutralize the charged oxygen at this position are cleaved at least four orders extra negative charge at phosphorus in the transition state ofmagitude more slowly than the unmodified substrate. These and supply a water molecule in its coordination sphere to results are supported by other modification studies: oligode- protonate the leaving group after cleavage. Lys-113 in EcoRI oxynucleotide substrates with a phosphorothioate substitution and Lys-92 in EcoRV might help to neutralize the extra at this position in both strands are cleaved only ifthe negatively negative charge in the transition state. In the proposed charged sulfur is in the Rp configwuration as shown for EcoRI mechanism, the attacking water molecule is activated by the [Koziolkiewicz, M. & Stec, W. J. (1992) Biochemistry 31, pro-Rp phosphoryl oxygen of the phosphate group on the 3' 9460-9466] and EcoRV (B. A. Connoily, personal communi- side ofthe scissile bond (Fig. 1). This assignment of a part of cation). As the phosphate residue 3' to the scissile phosphodi- the substrate to play an active role in the catalytic process ester bond is not needed for strong DNA binding by both was suggested by the fact that, in the structures ofthe specific enzymes, these fings strongly suggest that this phosphate EcoRI-DNA and EcoRV-DNA complexes, no amino acid group plays an active role during catalysis. This proposal, side chain was found in a position where it could function as furthermore, gives a straightforward explanation ofwhy in the a general base to activate water. A water molecule modeled EcoRI-DNA and EcoRV-DNA complexes the DNA is distorted into the structures of the EcoRI-DNA and EcoRV-DNA differently, but in each case the 3' phosphate group closely complexes, such that it takes up the appropriate position approaches the phosphate group that is attacked. Finally, an in-line with the bond to be cleaved, is in hydrogen-bonding alternative mechanism for DNA cleavage involving two metal distance to the pro-Rp phosphoryl oxygen of the phosphate ions is unlikely in the light of our finding that both EcoRI and group 3' adjacent to the scissile phosphodiester bond (11). EcoRV need only one Mg2+ per active site for cleavage. To analyze the role ofthe phosphate group 3' to the scissile phosphodiester bond and its negatively charged phosphoryl Type II restriction endonucleases bind and cleave double- oxygen in the mechanism of EcoRI- and EcoRV-catalyzed stranded DNA with high specificity at sites 4-6 bp long (for DNA cleavage, we have employed chemically modified oli- reviews, see refs. 1-5). All these endonucleases need Mg2+ godeoxynucleotide substrates lacking this phosphate group as a cofactor for catalysis, some also need it for specific or its negatively charged phosphoryl oxygen in "missing- binding, and produce fragments with 5'-phosphorylated contact" experiments (for a discussion of the principal prob- ends. For two restriction enzymes, EcoRI (for reviews, see lems associated with the use of modified oligodeoxynucle- refs. 2 and 3) and EcoRV (for review, see ref. 4), the otides in the study ofprotein-DNA interactions, see ref. 13). stereochemical course ofthe cleavage has been determined to Such experiments have proven to be very useful in identify- proceed with an inversion of configuration at phosphorus (6, ing the contribution of a base (14) or a nucleoside (15) to 7). As x-ray structures have been solved for EcoRI and specific binding of DNA by proteins. Missing-contact exper- EcoRV in complex with the specific DNA substrate (8, 9), the iments can also contribute to understanding catalysis. catalytic centers of these enzymes could be identified. In- triguingly, they turned out to be remarkably similar in struc- MATERIALS AND METHODS ture (10). This finding came as a surprise, considering the fact that EcoRI and EcoRV are not significantly homologous, EcoRI and EcoRV. EcoRI, EcoRV, and the EcoRV mutant recognize different DNA sequences, and cleave DNA to D90A (10) were homogeneous preparations obtained from produce sticky and blunt ends, respectively. The similarity of overproducing Escherichia coli strains by procedures as the catalytic centers of EcoRI and EcoRV and the identical described (16, 17). Oligodeoxynucleotide Substrates. Oligodeoxynucleotides were on solid support with a Milligen Cyclone The publication costs ofthis article were defrayed in part by page charge synthesized payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 8499 Downloaded by guest on September 30, 2021 8500 Biochemistry: Jeltsch et al. Proc. Natl. Acad. Sci. USA 90 (1993) EcoRV T A '\ G scissile <attacking I Lysll3 T bond H20 sssile H2O L92 bond 2+ Mg 2+ Mg ) ~~~~~~~~~~~protonating 4 protonating H20 H20 Ap9O Glulli Asp9l sp74 FIG. 1. Molecular model of the catalytic centers of EcoRI and EcoRV based on the coordinates of the EcoRI-DNA and EcoRV-DNA complexes (Brookhaven data bank entries, lRlE and 3RVE). The water molecules and the Mg2+ ion have been modeled into the structure, in the EcoRI-DNA complex also the amino acid side chains (for details, see ref. 11). For clarity only those amino acids directly involved in the catalytic mechanism and the sugar phosphate backbone of one strand ofthe DNA are shown. The attacking water molecule is hydrogen-bonded to the phosphate group 3' to the scissile bond. A second water molecule in the hydration sphere ofthe Mg2+ ion is hydrogen-bonded to the leaving group. DNA synthesizer using ,B-cyanoethylphosphoramidites ob- melting experiments carried out as described (19). Melting tained from MWG-Biotech (Ebersberg, Germany) and puri- temperature values are as follows: RI/RVum, 70°C; RIum, fied by HPLC (18). 400(; RVum, 45°C; RImp, 50°C; RVmp, 53°C; RIhp, 37°C; (i) Three unmodified (um) substrates serving as reference RVhp, 40°C. The melting curves of the missing-phosphate substrates for the modified substrates [recognition sequences substrates, RImp and RVmp, show a broader transition than are in boldface type]. the reference substrate, RI/RVum, indicating dissociation of the shorter oligodeoxynucleotide from the discontinuous RI/RVum: strand at around 350( (data not shown). EcoRI EcoRV DNA Cleavage Experiments. Oligodeoxynucleotides were GpGpGpCpTpApGpipkpTpTpCpGpIpTp&pTpCpGpCpTpApCpTpGpC labeled with T4 polynucleotide kinase (AGS, Heidelberg, CpCpCpGpApTpCpTpTpApkpGpCpTpIpTpApGpCpGpApTpGpApCpG Germany) or terminal deoxynucleotidyltransferase (Pharma- RIum: TpApTpApGpipipTpTpCpTpApT cia) by using [y-32P]ATP or dideoxyadenosine 5'-[a- TpApTpCpTpTpApipGpApTpApT 32P]triphosphate (Amersham-Buchler, Braunschweig, Ger- many). The cleavage reactions with the missing-phosphate RVum: CpGpCpGpGpIpTp&pTpCpCpGpC substrates were carried out at 4°C in 20 mM Tris-HCl, pH CpGpCpCpTpApTpApGpGpCpGpC 7.5/50 mM NaCl/10 mM Mg922, as described (19). All other (ii) Missing-phosphate (mp) substrates (note that these cleavage reactions were carried out at 210C. Products of double-stranded substrates have one continuous and one cleavage in one strand ("nicking") were separated by homo- discontinuous strand). chromatography or gel electrophoresis. Substrate concentra- tions were 0.1 or 1 ,uM. Enzyme concentrations were varied RImp: between S nM and 1 ,uM depending on whether multiple- GpGpGpCpTpApGpk &pTpTpCpGpApTpApTpCpGpCpTpApCpTpGpC turnover (RIum, RVum) or single-turnover experiments CpCpCpGpApTpCpTpTpkpIpGpCpTpApTpApGpCpGpApTpGpApCpG (modified
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