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Effect of Tetrahydropyrimidine Derivatives on Protein-Nucleic Acids Interaction

1999; Elsevier BV; Volume: 274; Issue: 11 Linguagem: Inglês

10.1074/jbc.274.11.6920

1083-351X

Gennady Malin, Robert Iakobashvili, Aviva Lapidot,

RNA Interference and Gene Delivery

2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyri- midine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B) or ectoine) are now recognized as ubiquitous bacterial osmoprotectants. To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases. THP(A) completely arrested the cleavage of plasmid and bacteriophage λ DNA by EcoRI endonuclease at 0.4 mm and the oligonucleotide (d(CGCGAATTCGCG))2 at about 4.0 mm. THP(B) was 10-fold less effective than THP(A), whereas for betaine and proline, a notable inhibition was observed only at 100 mm. Similar effects of THP(A) were observed for all tested restriction endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mm THP(A). No effect of THP(A) on the activity of DNase I, RNase A, and Taq DNA polymerase was noticed. Gel-shift assays showed that THP(A) inhibited the EcoRI-(d(CGCGAATTCGCG))2complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carboxy group significantly decreased the activity of THPs, suggesting that their zwitterionic character is essential for the inhibition effect. Possible mechanisms of inhibition, the role of THPs in the modulation of the protein-DNA interaction, and the in vivo relevance of the observed phenomena are discussed. 2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyri- midine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B) or ectoine) are now recognized as ubiquitous bacterial osmoprotectants. To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases. THP(A) completely arrested the cleavage of plasmid and bacteriophage λ DNA by EcoRI endonuclease at 0.4 mm and the oligonucleotide (d(CGCGAATTCGCG))2 at about 4.0 mm. THP(B) was 10-fold less effective than THP(A), whereas for betaine and proline, a notable inhibition was observed only at 100 mm. Similar effects of THP(A) were observed for all tested restriction endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mm THP(A). No effect of THP(A) on the activity of DNase I, RNase A, and Taq DNA polymerase was noticed. Gel-shift assays showed that THP(A) inhibited the EcoRI-(d(CGCGAATTCGCG))2complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carboxy group significantly decreased the activity of THPs, suggesting that their zwitterionic character is essential for the inhibition effect. Possible mechanisms of inhibition, the role of THPs in the modulation of the protein-DNA interaction, and the in vivo relevance of the observed phenomena are discussed. Two tetrahydropyrimidine derivatives identified inStreptomyces bacteria (1Inbar L. Lapidot A. J. Bacteriol. 1988; 170: 4055-4064Crossref PubMed Google Scholar, 2Inbar L. Lapidot A. J. Biol. Chem. 1988; 263: 16014-16022Abstract Full Text PDF PubMed Google Scholar, 3Inbar L. Lapidot A. J. Bacteriol. 1991; 173: 7790-7801Crossref PubMed Google Scholar), one a previously unknown metabolite, THP(A), 1The abbreviations used are: THP(A), 2-methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyrimidine; THP(B), 2-methyl,4-carboxy-3,4,5,6-tetrahydropyrimidine; THP, tetrahydropyrimidine derivative; bp, base pair(s) and the other previously identified (as ectoine) in halophilic bacteria (4Galinski E.A. Pfeiffer H.P Truper H.G. Eur. J. Biochem. 1985; 149: 135-139Crossref PubMed Scopus (314) Google Scholar), THP(B), are now recognized as widely spread osmoprotectants within the bacterial world (5Csonka L.N. Epstein W. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. ASM Press, Washington, D. C.1996: 1210-1223Google Scholar). The role and activities of THPs are of special interest as they represent a limited group of osmoprotectants that are synthesized de novo, in the bacterial cell, in contrast to those transported from the medium (6Csonka L.N. Hanson A.D. Annu. Rev. Microbiol. 1991; 45: 569-606Crossref PubMed Scopus (654) Google Scholar). Their synthesis in a number ofStreptomyces strains as a response to increased salinity and elevated temperature was recently described (7Malin G. Lapidot A. J. Bacteriol. 1996; 178: 385-395Crossref PubMed Google Scholar). THPs are small molecules, highly soluble in water and neutral at physiological pH. NMR and x-ray crystallography data show that THPs are zwitterionic molecules with a delocalized π charge in the NCN group (Fig. 1) and form the half-chair conformation (8Inbar L. Frolow F. Lapidot A. Eur. J. Biochem. 1993; 214: 897-906Crossref PubMed Scopus (32) Google Scholar). More information has been accumulated lately on THPs activity in living cells. It was found that exogenously provided ectoine (THP(B)) could reverse growth inhibition, caused by osmotic stress, inEscherichia coli (9Jebbar M. Talibart R. Gloux K. Bernard T. Blanco C. J. Bacteriol. 1992; 174: 5027-5035Crossref PubMed Scopus (138) Google Scholar), Corynebacterium glutamicum(10Farwick M. Siewe R.M. Kramer R. J. Bacteriol. 1995; 177: 4690-4695Crossref PubMed Google Scholar), and the soil bacterium Rhizobium meliloti (11Talibart R. Jebbar M. Gousbet G. Himdi-Kabbab S. Wroblewski H. Blanco C. Bernard T. J. Bacteriol. 1994; 176: 5210-5217Crossref PubMed Google Scholar). We demonstrated that exogenously provided THP(A), like THP(B), reversed inhibition of E. coli growth by osmotic stress, and moreover, both THP(A) and THP(B) could stimulate growth of E. coli at an elevated temperature (43 °C) (7Malin G. Lapidot A. J. Bacteriol. 1996; 178: 385-395Crossref PubMed Google Scholar). Recently cloned genes for ectoine synthesis from Halomonas elongata (12Canovas D. Vargas C. Iglesias-Guerra F. Csonka L.N. Rhodes D. Ventosa A. Nieto J.J. J. Biol. Chem. 1997; 272: 25794-25801Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and from Marinococcus halophilus (13Lois P. Galinski E.A. Microbiology. 1997; 143: 1141-1149Crossref PubMed Scopus (160) Google Scholar) were demonstrated to be both necessary for halotolerance of ectoine-producing bacteria and osmotically regulated. The ability of osmoprotectants (e.g. proline and betaine) to overcome the inhibitory effect of osmotic stress in bacteria was traditionally explained in two ways. One hypothesis states that proline and betaine have special interactions with proteins that protect them from denaturation in the presence of high concentrations of electrolytes (14Csonka L.N. Microbiol. Rev. 1989; 53: 121-147Crossref PubMed Google Scholar, 15Schobert B. Tschesche H. Biochim. Biophys. Acta. 1978; 541: 270-277Crossref PubMed Scopus (240) Google Scholar). In support of this mechanism, it was recently shown that THPs stabilize proteins upon freezing and at elevated temperatures (16Galinski E.A. Experientia. 1993; 49: 487-495Crossref Scopus (268) Google Scholar, 17.Lapidot, A., Malin, G., and Iakobashvili, R. (1998) Israel Patent Application 123256, 10.02.1998.Google Scholar). According to another hypothesis, proline and betaine are merely aimed at maintaining cell turgor in media of high osmolarity, being compatible with normal cellular functions at high intracellular concentration. Intracellular concentrations of THPs attain up to 158 mm in nonhalophylicStreptomyces bacteria (7Malin G. Lapidot A. J. Bacteriol. 1996; 178: 385-395Crossref PubMed Google Scholar), and as high as 2.25 min the halophilic bacterium H. elongata (18Wohlfarth A. Severin J. Galinski E.A. J. Gen. Microbiol. 1990; 136: 705-712Crossref Scopus (121) Google Scholar), whereas betaine can be synthesized to 0.6 m in the Methanohalophilus strain Z7401 (19Lai M. Sowers K.R. Robertson D.E. Roberts M.F. Gunsalus R.P. J. Bacteriol. 1992; 173: 5352-5358Crossref Google Scholar) and proline to 0.7m in Bacillus subtilis (20Whatmore A.M. Chudek J.A. Reed R.H. J. Gen. Milcrobiol. 1990; 136: 2527-2535Crossref PubMed Scopus (214) Google Scholar). At these high concentrations, proline, betaine, and THP(B) demonstrate a pronounced destabilizing effect on DNA in vitro(22Rees W.A. Yager T.D. Korte J. von-Hippel P.H. Biochemistry. 1993; 32: 137-144Crossref PubMed Scopus (245) Google Scholar, 23Buche A. Colson P. Houssier C. J. Biomol. Struct. Dyn. 1993; 11: 95-119Crossref PubMed Scopus (28) Google Scholar, 24Rajendrakumar C.S.V. Suryanarayana T. Reddy A.R. FEBS Lett. 1997; 410: 201-205Crossref PubMed Scopus (114) Google Scholar). 2R. S. Iakobashvili, G. Malin, and A. Lapidot, submitted for publication. Moreover, THP(B) at certain concentrations, can, like betaine (22Rees W.A. Yager T.D. Korte J. von-Hippel P.H. Biochemistry. 1993; 32: 137-144Crossref PubMed Scopus (245) Google Scholar), eliminate base pair composition dependence of DNA melting.2 In addition, THP molecules share similarity in structure and geometry with pyrimidine bases and have the zwitterionic character, which was recently shown to be responsible for modification of DNA electrostatic interaction with counterions by a number of osmolytes (25Flock S. Labarbe R. Houssier C. Biophys. J. 1996; 71: 1519-1529Abstract Full Text PDF PubMed Scopus (25) Google Scholar). These notions led us to suggest that the impact of osmoprotectants, and THPs in particular, on protein-DNA interaction is, likely, underestimated. The destabilization of the DNA duplex could play a dual role in the interaction of DNA with proteins; it may either enhance binding, as it was shown for proline and SSB protein, or diminish it, as it was shown for DNaseI, for which double-stranded DNA serves as a substrate (24Rajendrakumar C.S.V. Suryanarayana T. Reddy A.R. FEBS Lett. 1997; 410: 201-205Crossref PubMed Scopus (114) Google Scholar). Yet the impact of osmolytes on the sequence-specific interaction of proteins with DNA (e.g. restriction endonucleases and transcription factors) could be even more complex and remains to be elucidated. Type II restriction enzymes are recognized as an ideal model system for evaluating site-specific protein-DNA interaction, as any interference with the precise alignment of the enzyme and substrate will affect the cleavage. It has been demonstrated that the binding of ligands, such as antibiotics and dyes, reduces the activity and specificity of restriction endonucleases (26Wells R.D. Klein R.D. Singleton C.K. Boyer P.D. The Enzymes. XIV. Academic Press, New York1981: 157-191Google Scholar). It has also been shown that the constituents of bacterial cells, such as polyamines and basic proteins (27Pingoud A. Urbanke C. Alves J. Ehbrecht H.J. Zabeau M. Gualerzi C. Biochemistry. 1984; 23: 5697-5703Crossref PubMed Scopus (44) Google Scholar) or polyphosphate (28Rodriguez R.J. Anal. Biochem. 1993; 209: 291-297Crossref PubMed Scopus (45) Google Scholar), are also capable of inhibiting the DNA cleavage by restriction endonucleases in vitro. Hence, the primary function of the restriction-modification system, aimed at protecting bacteria from phage infection or from other sources of foreign DNA, might be dependent on the composition of intracellular metabolites. In the present study, we have investigated the effect of bacterial osmolytes, such as THP(A), THP(B), proline, and glycine betaine, on DNA cleavage by several type II restriction endonucleases, taken as a model system for specific protein-DNA interaction. The results were compared with the effect on proteins that bind nucleic acid nonspecifically, such as DNaseI, RNase A, and Taq DNA polymerase. We report here that THP(A) and, to a lesser extent, THP(B) are capable of completely arresting the DNA cleavage by a number of restriction endonucleases, whereas proline and glycine betaine are almost ineffective. The effect of THPs on EcoRI binding to the oligonucleotide, containing its recognition site, and on linear diffusion was also examined. A possible role of THPs in the modulation of the restriction-modification system and in gene expression under stress is suggested. Plasmids pGEM1 and pBR322 were isolated from E. colistrain DH5 by alkaline lysis and purified by equilibrium centrifugation in a gradient of cesium chloride (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1.42-1.43Google Scholar). Restriction endonucleasesEcoRI, PvuII, AvaI, and DraI were purchased from New England Biolabs,SspI and SmaI were from MBI Fermentas, and SgrAI and EcoRV were from Boehringer Mannheim. Pancreatic bovine deoxyribonuclease I, ribonuclease A, yeast RNA, betaine monohydrate (glycine betaine), and spermine tetrahydrochloride were from Sigma, l-proline (99.5%) was from Fluka, and Thermus aquaticus DNA polymerase (recombinant) was from MBI Fermentas. THPs were prepared and purified as described previously (7Malin G. Lapidot A. J. Bacteriol. 1996; 178: 385-395Crossref PubMed Google Scholar). The activity of THPs preparations in the restriction assay was checked before and after passing through a Chelex column, and no difference was observed. THPs preparations were further analyzed by atomic flame photometry, and no detectable amounts of Mg, Ca, and Fe ions and less then 0.1% sodium and potassium ions were found. The micromolar final concentration of Na+ or K+ upon addition of THPs to the reaction mixture is negligible in comparison to 50–100 mm potassium in the buffer. For the preparation of 2-methyl, 4-methylcarboxylate, 5-hydroxy-3,4,5,6-tetrahydropyrimidine (methyl-THP(A)) and 2-methyl, 4-methylcarboxylate-3,4,5,6-tetrahydropyrimidine (methyl-THP(B)) (Fig. 1), an excess of thionylchloride was gradually added to a 0.5 m solution of THP(A) or THP(B) in methanol at 0 °C under continuous stirring. Resulting solutions were evaporated under reduced pressure and the residue, containing CH3-THP(A)·HCl or CH3-THP(B)·HCl, was dissolved in water and neutralized by NaOH to pH 7.0. Water was evaporated under reduced pressure, and the solid material was resuspended in methanol, separated from NaCl by filtration, and dried. The purity of methyl-THP(A) and methyl-THP(B) was determined by1H and 13C NMR (data not shown). (d(CGCGAATTCGCG))2 was prepared by solid phase synthesis (Chemical Services, Weizmann Institute of Science), purified on a denaturing polyacrylamide gel containing 7m urea, and desalted on Sephadex G-25. The oligonucleotide was labeled with [γ-32P]ATP (6000 Ci/mmol, New England Biolabs) by T4 polynucleotide kinase (30Connolly B.A. Kneale G.G. Methods in Molecular Biology. 30. Humana Press Inc., Totowa, NJ1994: 371-383Google Scholar), purified by denaturing polyacrylamide gel, and desalted on Sephadex G-25. The 32P-labeled oligonucleotide was heated for 5 min at 90 °C in a buffer containing 20 mm Tris-HCl (pH 7.4), 100 mm NaCl, 10 mm MgCl2, 0.1 mm dithiothreitol and then slowly cooled to 4 °C, desalted on Sephadex G-25, purified by step elution on DEAE-Sephadex (10 mm bis-Tris propane, pH 7.4), with increasing concentrations of KCl (up to 0.6 m KCl) and desalted by dialysis against water. About 95% of the resulting oligonucleotide was cleavable by EcoRI endonuclease. Cleavage of DNA by each restriction enzyme was carried out in a total volume of 20 μl of the reaction buffer recommended by manufacturer. The cleavage reaction was initiated by addition of the enzyme to the incubation mixture containing THPs or other additives and terminated by addition of EDTA to a final concentration of 20 mm. Reaction products were analyzed by electrophoresis on a horizontal 1% agarose gel at 3 V/cm. Gels were stained with ethidium bromide and photographed, and pictures were quantified by densitometry. Cleavage of dodecadeoxynucleotide by EcoRI was carried out in 20 μl of buffer containing 50 mm NaCl, 20 mm Tris-HCl (pH 7.4), 0.1 mm EDTA, 0.1 mm dithiothreitol, 10 mm MgCl2, and 50 mg/ml bovine serum albumin. Products of the 32P-labeled dodecadeoxynucleotide cleavage were analyzed by electrophoresis on a 20% polyacrylamide gel containing 7.0 m urea, visualized by autoradiography and quantified by densitometry. The DNase I assay was performed at 37 °C in 100 μl of buffer containing 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, and 100 mm NaCl. A minimal amount of enzyme (0.3 ng/ml) needed for complete transformation of supercoiled plasmid DNA into nicked circular within 1 h (as determined in a separate experiment in the absence of THPs) was added to the reaction mixture containing THPs. Aliquots, taken after short intervals of incubation (every 5 or 10 min), were immediately transferred into liquid nitrogen and kept frozen until loaded on the agarose gel. RNase A assay was carried out at 37 °C in 20 μl of buffer containing 1 μg of yeast RNA, 20 mm Tris-HCl, pH 7.2, 50 mm NaCl, and 2 mm MgCl2. The minimal amount of enzyme needed for 50% digest of RNA within 30 min (0.2 μg/ml, as determined in a separate experiment in the absence of THPs) was added to the reaction mixture containing THPs. Reaction products were analyzed by electrophoresis on a 1% agarose gel. Taq DNA polymerase activity assay was performed at 72 °C in a 15-μl reaction mixture containing 67 mm Tris-HCl (pH 8.8); 6.7 mm MgCl2; 50 mm NaCl; 1 mm β-mercaptoethanol; 0.1 mg/ml bovine serum albumin; 10 nm [α-32P]dATP; 1.0 μm dATP; dCTP, dGTP, and dTTP (200 μm each); 0.6 mmactivated calf thymus DNA (Sigma); 40 units/ml of Taq DNA polymerase; and different concentrations of THPs. After 15 min incubation (corresponding to the region of linear reaction kinetics, as determined in a separate experiment). The reaction was stopped by addition of 10 μl of 50 mm EDTA and applied to strips of chromatographic paper (Whatman No. 3). Strips were washed three times with ice-cold trichloroacetic acid and dried, and the radioactivity was measured by a scintillation counter. Assays on EcoRI binding to the dodecadeoxynucleotide were carried out in 20 μl of binding buffer containing 50 mm NaCl, 20 mm Tris-HCl (pH 7.4), 0.1 mm EDTA, 0.1 mm dithiothreitol, and 50 mg/ml bovine serum albumin. THPs were added to the reaction mixture and incubated at room temperature for 15 min before or after the addition of EcoRI, 8 μl of the loading buffer (40% w/v sucrose and 100 μg/ml bromphenol blue) was added, and the samples were loaded on the 9% (29:1, acrylamide:bisacrylamide) polyacrylamide gel, 45 mm Tris borate (pH 8.0), and 2.0 mm EDTA. Gels were prerun for 2–3 h prior to loading of samples and after electrophoresis for 3–4 h at 10–12 V/cm and 4 °C, dried, and autoradiographed, and the resulting negatives were subjected to densitometry. We have basically followed the experiment developed by Ehbrecht et al. (31Ehbrecht H.J. Pingoud A. Urbanke C. Maass G. Gualerzi C. J. Biol. Chem. 1985; 260: 6160-6166Abstract Full Text PDF PubMed Google Scholar). To evaluate the contribution of linear diffusion to the rate of the EcoRI-catalyzed reaction, the difference in the cleavage rate of short (378 bp) and long (4361 bp) fragments of pBR322 plasmid DNA with a centrally located target site by EcoRI endonuclease (see Fig. 5) was measured. To prepare short and long DNA fragments, 300 μg of pBR322 plasmid DNA was cleaved overnight bySspI/EcoRV or PvuII endonucleases (60 units of each) in the buffers recommended by the manufacturers. The resulting digest mixture was used without further purification as a substrate for EcoRI restriction endonuclease. As was shown previously, the components of the primary reaction do not affect the secondary reaction (31Ehbrecht H.J. Pingoud A. Urbanke C. Maass G. Gualerzi C. J. Biol. Chem. 1985; 260: 6160-6166Abstract Full Text PDF PubMed Google Scholar). This experimental setup ensures that factors of the reaction other than the length of the substrate are invariant; namely, all fragments with the EcoRI site have identical flanking sequences, and due to the presence of fragments not containing the EcoRI site, nonspecific binding is identical in all experiments. The secondary reaction (with EcoRI endonuclease) was carried out in the optimized buffer (31Ehbrecht H.J. Pingoud A. Urbanke C. Maass G. Gualerzi C. J. Biol. Chem. 1985; 260: 6160-6166Abstract Full Text PDF PubMed Google Scholar) containing 20 mm Tris-HCl, pH 7.2, 0.05 mg/ml bovine serum albumin, 50 mm NaCl, and 1 mm MgCl2 in the presence of varying amounts of THPs. We have studied the effect of THP(A), THP(B), proline, and betaine on plasmid DNA cleavage by EcoRI endonuclease. The rate of cleavage of pBR322 DNA by EcoRI endonuclease was notably decreased, starting from 0.1 mmTHP(A). It is worth noting that no intermediate products such as open circular DNA were accumulated upon inhibition (Fig. 2A). To determine the type of inhibition and estimate the inhibition constant, the steady state kinetics of cleavage was measured at constant DNA concentrations and varying concentrations of THP(A). A Dixon plot (32Dixon M. Biochem. J. 1953; 55: 170-171Crossref PubMed Scopus (3332) Google Scholar) of these data (Fig. 2B) with Vmax constant within the range of error, combined with a set of parallel lines in a Cornish-Bowden plot (33Cornish-Bowden A. Biochem. J. 1974; 137: 143-144Crossref PubMed Scopus (807) Google Scholar) (Fig. 2C), indicates a competitive inhibition (34Cornish-Bowden A. Cornish-Bowden A. Fundamentals of Enzyme Kinetics. Butterworth & Co. Ltd., London1979: 78-82Google Scholar) with K i = 0.16 ± 0.04 mm. The inhibition effect of other osmolytes was much less pronounced; i.e. whereas THP(A) completely arrested plasmid DNA cleavage at around 1.0 mm, THP(B) was 10-fold less effective, betaine and proline at low concentrations showed stimulation of DNA cleavage, and a notable inhibition was observed only at 100 mm (Fig. 3). It is worth noting that at this high concentration, additional effects, such as a decrease of water activity, a significant increase of the medium dielectric constant, or changes in the pH of the reaction mixture, are possible. The inhibitory effect of THP(A) on the EcoRI endonuclease reaction was not unique for the pBR322 plasmid DNA as a substrate; the same effect was observed for pGEM1 plasmid DNA and for bacteriophage λ linear DNA.Figure 3Effect of osmolytes and methyl derivatives of THPs on the rate of pBR322 DNA cleavage by EcoRI.Every data point reflects the initial rate of cleavage (the region of linear reaction kinetics has been determined in a separate experiment) of pBR322 DNA (37 μg/ml) by EcoRI restriction endonuclease (37 units/ml) in the presence of the corresponding solute, normalized by the rate of cleavage in the absence of additives. ▪, THP(A); ●, THP(B); ■, methyl-THP(A); ○, methyl-THP(B); ▵,l-proline; ▴, glycine betaine. Data points are the average of at least two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The cleavage of dodecadeoxynucleotide duplex by EcoRI was tested in the presence of THP(A) with the minimal amount of enzyme needed for complete cleavage and with a 5-fold excess of the enzyme. Similar results were observed in both cases (Fig. 4, A and B). The complete (95–100%) inhibition of the reaction was reached at 5 mm THP(A). The higher concentration of THP(A) needed for complete inhibition of the oligonucleotide cleavage (3–5-fold) as compared with plasmid DNA could be attributed to the different reaction conditions employed in the two experiments, different flanking sequence of recognition sites, and known differences in the reaction kinetics of restriction enzymes on short duplexes of 8–12 bp compared with those on longer DNA (35Greene P.J. Poonian M.S. Nussbaum A.L. Tobias L. Garfin D.E. Boyer H.W. Goodman H.M J. Mol. Biol. 1975; 99: 237-261Crossref PubMed Scopus (123) Google Scholar). The inhibition of EcoRI endonuclease reaction by THP(A) was not compensated by increased concentration of MgCl2 (20 mm instead of 10 mm) in the reaction mixture, suggesting that sequestering of Mg2+ ions is not responsible for the observed effect. In both cases, the inhibition of DNA cleavage by THP(A) was similar to that depicted in Fig. 3. The cleavage of the plasmid DNA substrate by restriction endonucleases is preceded by a number of consecutive events, such as a nonspecific association with the DNA and linear diffusion of the nonspecifically bound protein along the DNA until the recognition site is reached and specific binding occurs. On exploring the possible targets for THP(A) action, we further investigated its effect on the linear diffusion of EcoRI along the DNA by using the experimental approach developed by Ehbrecht et al. (31Ehbrecht H.J. Pingoud A. Urbanke C. Maass G. Gualerzi C. J. Biol. Chem. 1985; 260: 6160-6166Abstract Full Text PDF PubMed Google Scholar). To ensure that the difference in the rate of cleavage in this experiment originates solely from the chain length of the substrate, the plasmid DNA (bearing a single EcoRI site) was cleaved beforehand by other restriction endonucleases, producing two sets of DNA fragments of different length. The resulting mixture was used as a substrate forEcoRI endonuclease, thus excluding possible effect of different flanking sequences and equalizing the nonspecific binding. Plasmid pBR322 was cleaved by PvuII endonuclease, to yield a linear fragment of 4361 bp. Alternatively, plasmid DNA was cleaved byEcoRV/SspI, resulting in a 3983-bp fragment without a recognition site and a 378-bp fragment with a centrally located EcoRI site (Figs. 5and 6A). Using optimized concentration of Mg2+ (31Ehbrecht H.J. Pingoud A. Urbanke C. Maass G. Gualerzi C. J. Biol. Chem. 1985; 260: 6160-6166Abstract Full Text PDF PubMed Google Scholar) we observed a distinct difference in the cleavage rate of the long and short DNA fragments byEcoRI endonuclease, ranging from 20 to 8% (Fig. 6,A and B). This enhancement of the reaction rate, proportional to the length of the DNA substrate, is usually explained in terms of sliding or intersegment transfer (36von Hippel P.H. Berg O.G. J. Biol. Chem. 1989; 264: 675-678Abstract Full Text PDF PubMed Google Scholar). The sliding process can be viewed as a one-dimensional diffusion of the nonspecifically bound protein (totally electrostatic binding mode) along the DNA, which is thought to occur by the displacement of bound (delocalized) positive counterions from the DNA (36von Hippel P.H. Berg O.G. J. Biol. Chem. 1989; 264: 675-678Abstract Full Text PDF PubMed Google Scholar). Thus, any influence decreasing the lifetime of the nonspecifically bound state (e.g. increase of salt concentration (37Jeltsch A. Wenz C. Stahl F. Pingoud A. EMBO J. 1996; 15: 101-108Crossref Scopus (84) Google Scholar)) or presenting a steric obstacle to linear movement (e.g. nonsaturating amounts of histone-like protein Hu (31Ehbrecht H.J. Pingoud A. Urbanke C. Maass G. Gualerzi C. J. Biol. Chem. 1985; 260: 6160-6166Abstract Full Text PDF PubMed Google Scholar)) will notably diminish the reaction rate dependence on the DNA length. This was not the case for the inhibition of the EcoRI cleavage reaction by THPs. On the contrary, we found that the difference in the cleavage rate of the long and short DNA fragments somewhat increased upon increasing THPs concentrations (Fig. 6), suggesting that the linear diffusion was not significantly affected by THPs in the concentration range employed. Higher concentrations of THP(A) led to very low levels of remaining endonuclease activity for short DNA fragments to be quantified from a gel and compared with the rate of cleavage of long DNA fragments. It is known thatEcoRI endonuclease first binds to its recognition site and then the enzyme-DNA complex binds Mg2+ (38Halford S.E. Johnson N.P. Biochem. J. 1983; 211: 405-415Crossref PubMed Scopus (29) Google Scholar). Thus, the DNA-protein complex formation in the absence of Mg2+ can be used to probe the effect of THP(A) on EcoRI binding to DNA. The experiments were conducted with a minimal amount ofEcoRI endonuclease, needed for complete binding of the d(CGCGAATTCGCG) duplex, and with a 5-fold excess of the enzyme, and they exhibited similar results. As shown in Fig. 7, A and B, THP(A) inhibited EcoRI-oligonucleotide complex formation, starting at 1–1.5 mm concentrations, with an inhibition constant,K i (50% inhibition of binding), of 2.0–2.5 mm. Higher THP(A) concentrations, 3.5–4.0 mm, provided complete (95–100%) inhibition. These data indicate that THP(A) arrested the DNA cleavage by EcoRI as soon as the first step of the reaction by preventing EcoRI binding to its target DNA sequence. In order to establish whether the zwitterionic nature of THPs is necessary for the observed inhibition effect, we prepared carboxymethyl derivatives of THPs (Fig. 1) and tested their effect on the reaction catalyzed byEcoRI endonuclease. The ability of these compounds to inhibit the endonuclease reaction was almost 100-fold lower, in comparison to the respective THPs, and at low concentrations, the methylated THP(B) derivative even enhanced the reaction rate as seen in Fig. 3 (note the logarithmic scale). It is possible that the slight inhibition effect was observed due to the minor amounts of unmethylated THPs remaining in the preparation. The inhibition of DNA cleavage (95–100%) by THP(A) was also demonstrated for other type II restriction endonucleases (Table I), in the range of 2–4 mm. Although the incubation buffers were different for most of the enzymes used, we have not noticed any correlation of the observed inhibition effect to the buffer composition. The finding that THP(A)