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Heterogeneity in Molecular Recognition by Restriction

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Heterogeneity in Molecular Recognition by Restriction Proc. Natl. Acad. Sci. USA Vol. 92, pp. 3444-3448, April 1995 Biochemistry Heterogeneity in molecular recognition by restriction endonucleases: Osmotic and hydrostatic pressure effects on BamHI, Pvu II, and EcoRV specificity (protein-DNA recognition/star activity/bound water) CLIFFORD R. ROBINSONt AND STEPHEN G. SLIGARt School of Chemical Sciences and Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 Communicated by Jiri Jonas, University of Illinois, Urbana, IL, December 5, 1994 (received for review August 18, 1994) ABSTRACT The cleavage specificity of the Pvu II and and hydrostatic-pressure techniques to study the role of water BamHI restriction endonucleases is found to be dramatically in mediating site-specific interactions of the EcoRI endonu- reduced at elevated osmotic pressure. Relaxation in specificity clease with canonical and alternate DNA sequences (16, 18). of these otherwise highly accurate and specific enzymes, To investigate whether this nontraditional role for water that previously termed "star activity," is uniquely correlated with was discovered in EcoRI represents a common feature of osmotic pressure between 0 and 100 atmospheres. No other restriction enzyme systems, we have examined the Pvu II, colligative solvent property exhibits a uniform correlation BamHI, and EcoRV endonucleases. Along with EcoRI, they with star activity for all of the compounds tested. Application are the only members of the type II restriction endonuclease of hydrostatic pressure counteracts the effects of osmotic family to be characterized by high-resolution structural anal- pressure and restores the natural selectivity ofthe enzymes for ysis (4, 19-23). For these three enzymes, cleavage at alternate their canonical recognition sequences. These results indicate sites has been observed previously, either by addition of that water solvation plays an important role in the site- organic solvents to the reaction buffer or by changes in ionic specific recognition of DNA by many restriction enzymes. strength, pH, or divalent cation concentration or identity Osmotic pressure did not induce an analogous effect on the (24-28). Cleavage at alternate sites by these and other restric- specificity of the EcoRV endonuclease, implying that selective tion enzymes has been termed "star activity" by analogy with hydration effects do not participate in DNA recognition in this EcoRI star (EcoRI*) activity (29). system. Hydrostatic pressure was found to have little effect on Each subunit of the Pvu II endonuclease from Proteus the star activity induced by changes in ionic strength, pH, or vulgaris has 157 amino acids, and the homodimer recognizes divalent cation, suggesting that distinct mechanisms may exist symmetric CAGCTG duplex DNA sequences, cleaving both for these observed alterations in specificity. Recent evidence strands in the middle of the site to leave blunt ends (30). Pvu has indicated that BamHI and EcoRI share similar structural II* sites include all single base substitutions at the four internal motifs, while Pvu II and EcoRV belong to a different structural positions (CNGCTG, CANCTG, CAGNTG, and CAGCNG), family. Evidently, the use of hydration water to assist in as well as A or G at the first position and T or C at the sixth site-specific recognition is a motif neither limited to nor position (24). The dimeric BamHI endonuclease from Bacillus defined by structural families. amyloliquefaciens H consists of identical 213-amino acid resi- due subunits, and cleaves GGATCC duplex DNA sequences Type II restriction endonucleases, along with their companion between the two guanine bases on both strands, leaving methyltransferases, make up bacterial restriction/modification four-base 5' overhangs (31). Reported BamHI* sites include systems, which protect bacteria from foreign DNA. They GGAACC, GGCTCC, GAATCC, GGATCN, and comple- typically consist of symmetric homodimers, which recognize mentary sequences. The EcoRV endonuclease derives from symmetric DNA sequences of four to eight bases, and require the restriction/modification system in Escherichia coli. EcoRV only Mg2+ as a cofactor to cleave duplex DNA (1). Despite the is a dimer of identical subunits of 244 amino acids. It recog- vast number of type II enzymes identified to date (nearly 2400) nizes the symmetric GATATC duplex DNA sequences and no homology and little sequence similarity exist (2). Despite cleaves both strands in the middle of the site, leaving blunt ends this lack of homology, recent reports have identified significant (32). EcoRV has been reported to cut at alternate sites with structural similarities among these enzymes (3), and a new single base changes from the canonical site, including GT- classification of restriction enzymes has been proposed on the TATC, AATATC, GANATC, and complementary sequences basis of the location of the scissile bond (4). Recent structural (33, 34). evidence has led to appreciation of structural and functional In this study we test whether the participation of water is a roles for water in site-specific interactions in protein-DNA general phenomenon in restriction enzyme-DNA interactions, complexes (5-8). thereby seeking to advance a general understanding of themes The use of osmotic pressure represents a powerful method in protein-DNA recognition and to provide molecular expla- for identifying and studying the participation of hydration nations for the flawed molecular recognition events that are water in biomolecular systems (9). By examining the correla- manifested as restriction enzyme star activity. tion between functional or structural properties and osmotic pressure, solvent water has been shown to play key roles in biochemical processes, such as substrate binding (10), protein- MATERIALS AND METHODS protein interactions (11), allosteric effects and conformational For each enzyme, a standard buffer was defined in which changes (12), catalysis (13), protein stability (14), and ion cleavage was observed only at the canonical site. Sets of channel formation (15). We have previously utilized osmotic- Abbreviations: DMSO, dimethyl sulfoxide; atm, atmosphere. The publication costs of this article were defrayed in part by page charge tPresent address: Department of Biology, Massachusetts Institute of payment. This article must therefore be hereby marked "advertisement" in Technology, Cambridge, MA 02139 accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 3444 Downloaded by guest on September 28, 2021 Biochemistry: Robinson and Sligar Proc. Nati Acad Sci. USA 92 (1995) 3445 reactions were performed in the presence of increasing con- the intensity of the bands produced by cleavage at star sites centrations of the various compounds (osmolytes) added to the divided by the intensity of all bands (canonical site fragments standard buffer. Enzyme reactions were initiated by addition plus star site fragments). A fraction star activity of 0.5 means of enzyme to the solution. Order of addition had no observable that half of the plasmids cut at the canonical sites were also cut effect. Enzymes were incubated with DNA containing canon- at a star site. In all cases in which fraction star activities were ical and alternate ("star") sites in total volumes of 20 p.l at 37°C determined, restriction mapping confirmed that cleavage re- for 4 h and then quenched by addition of EDTA to a final actions proceeded to completion at the canonical sites before concentration of 20 mM. any star sites were cut. Osmotic pressures were determined as Restriction enzymes were purchased from GIBCO/BRL. described previously (16) by using a UIC 70 vapor-phase Purified pUC18 DNA was prepared by using Promega Magic osmometer or by calculation from tabulated data (35). Vis- Mega-Preps, from 1 liter cultures of E. coli grown in medium cosities and dielectric constants were taken from tabulated containing 200 mg of ampicillin. Reaction mixtures contained data (35, 36). 1 unit/,ul of enzyme and 0.5 ,g/,ul of pUC18 DNA, unless otherwise noted. For EcoRV, enzymatic activity at the canon- ical GATATC site was investigated by using purified A DNA RESULTS obtained from GIBCO/BRL. EcoRV* activity was tested by The pUC18 plasmid contains two canonical Pvu II sites using linear or circular pUC18 or A DNA. All three forms of (CAGCTG), at positions 306 and 628. Cleavage at these sites DNA gave equivalent results. yields fragments of 2368 and 318 bp. The most readily cleaved The standard buffers which allowed canonical site cleavages Pvu II* site in pUC18 was CAGCGG at position 112, gener- were: Pvu II, 50 mM Tris HCl, pH 7.4/6 mM MgCl2/50 mM ating fragments of 2174, 318, and 194 bp. The presence of KCl/50 mM NaCl; BamHI, 50 mM Tris HCl, pH 8.0/2 mM osmolytes in the reaction buffer promoted cleavage at alter- 2-mercaptoethanol/10 mM MgCl2/100 mM NaCl; EcoRV, 50 nate sites in pUC18 yielding additional fragments. DMSO, mM Tris HCl, pH 7.5/100 mM NaCl/10 mM MgCl2/10 mM ethylene glycol, glycerol, ethanol, and 2-propanol promoted 2-mercaptoethanol/100 ,tg of bovine serum albumin per ml. Pvu II* activity. Star activity is tightly correlated with osmotic Glycerol, ethylene glycol, dimethyl sulfoxide (DMSO), etha- pressure (Fig. 1A) but correlated less well with dielectric nol, 2-propanol, sucrose, and dextrose were added to the constant (Fig. 2A) or relative viscosity (Fig. 2B). standard buffers to final concentrations of 0-6 M to induce Cleavage at the unique canonical BamHI site in pUC18 osmotic pressures from 0 to 150 atmospheres (atm; 1 atm = (position 429) linearizes the plasmid. There are 11 potential 101.3 kPa). BamHI* sites in pUC18. Addition of DMSO, glycerol, ethylene Reactions at elevated hydrostatic pressures ranging from 1 glycol, sucrose, or dextrose promoted BamHI* site cleavage, to 1600 atm were performed as described (16) in 400-,lI yielding a distinctive band pattern consisting of eight new DNA polypropylene tubes (Cole-Palmer) filled with 0.2 g of zirco- fragments. The fraction of star activity was quantitated from nium oxide beads (Biospec Products, Bartlesville, OK).
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