The Kinetic Mechanism of EcoRI Endonuclease

Steady-state parameters governing cleavage of pBR322 DNA by EcoRI endonuclease are highly sensitive to ionic environment, with Km andkcat increasing 1,000-fold and 15-fold, respectively, when ionic strength is increased from 0.059 to 0.23m. By contrast, pre-steady-state analysis has shown that recognition, as well as first and second strand cleavage events that occur once the enzyme has arrived at the EcoRI site, are essentially insensitive to ionic strength, and has demonstrated that the rate-limiting step for endonuclease turnover occurs after double-strand cleavage under all conditions tested. Furthermore, processive cleavage of a pBR322 variant bearing two closely spacedEcoRI sites is governed by the same turnover number as hydrolysis of parental pBR322, which contains only a singleEcoRI sequence, ruling out slow release of the enzyme from the cleaved site or a slow conformational change subsequent to double-strand cleavage. We attribute the effects of ionic strength on steady-state parameters to nonspecific endonuclease·DNA interactions, reflecting facilitated diffusion processes, that occur prior toEcoRI sequence recognition and subsequent to DNA cleavage. Steady-state parameters governing cleavage of pBR322 DNA by EcoRI endonuclease are highly sensitive to ionic environment, with Km andkcat increasing 1,000-fold and 15-fold, respectively, when ionic strength is increased from 0.059 to 0.23m. By contrast, pre-steady-state analysis has shown that recognition, as well as first and second strand cleavage events that occur once the enzyme has arrived at the EcoRI site, are essentially insensitive to ionic strength, and has demonstrated that the rate-limiting step for endonuclease turnover occurs after double-strand cleavage under all conditions tested. Furthermore, processive cleavage of a pBR322 variant bearing two closely spacedEcoRI sites is governed by the same turnover number as hydrolysis of parental pBR322, which contains only a singleEcoRI sequence, ruling out slow release of the enzyme from the cleaved site or a slow conformational change subsequent to double-strand cleavage. We attribute the effects of ionic strength on steady-state parameters to nonspecific endonuclease·DNA interactions, reflecting facilitated diffusion processes, that occur prior toEcoRI sequence recognition and subsequent to DNA cleavage. base pair(s) EcoRI endonuclease is among the simplest of the site-specific DNA enzymes and has proven useful for study of the mechanisms governing the interaction of such proteins with DNA (1Modrich P. Roberts R.J. Linn S.M. Roberts R.J. Nucleases. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 109-154Google Scholar, 2Heitman J. Model P. Proteins. 1990; 7: 185-197Crossref PubMed Scopus (36) Google Scholar, 3Rosenberg J. Curr. Opin. Struct. Biol. 1991; 1: 104-113Crossref Scopus (156) Google Scholar, 4Roberts R.J. Halford S.E. Linn S.M. Lloyd R.S. Roberts R.J. Nucleases. 2nd Ed. Cold Spring Harbor Laboratory Press, Plainview, NY1993: 35-88Google Scholar). The enzyme functions as a homodimer of a 31-kDa polypeptide (5Modrich P. Zabel D. J. Biol. Chem. 1976; 251: 5866-5874Abstract Full Text PDF PubMed Google Scholar, 6Lu A.-L. Jack W.E. Modrich P. J. Biol. Chem. 1981; 256: 13200-13206Abstract Full Text PDF PubMed Google Scholar, 7Terry B.J. Jack W.E. Rubin R.A. Modrich P. J. Biol. Chem. 1983; 258: 9820-9825Abstract Full Text PDF PubMed Google Scholar, 8McClarin J.A. Frederick C.A. Wang B.-C. Greene P. Boyer H.W. Grable J. Rosenberg J. Science. 1986; 234: 1526-1541Crossref PubMed Scopus (419) Google Scholar, 9Kim Y. Grable J.C. Love R. Greene P.J. Rosenberg J.M. Science. 1990; 249: 1307-1309Crossref PubMed Scopus (312) Google Scholar), and in presence of Mg2+ introduces two staggered, single-strand scissions into the symmetric recognition sequence, 5′-GAATTC-3′ (10Hedgpeth J. Goodman H.M. Boyer H.W. Proc. Natl. Acad. Sci. U. S. A. 1972; 80: 31-35Google Scholar). In the absence of a divalent cation, the endonuclease binds specifically and with high affinity to its recognition sequence (7Terry B.J. Jack W.E. Rubin R.A. Modrich P. J. Biol. Chem. 1983; 258: 9820-9825Abstract Full Text PDF PubMed Google Scholar,11Modrich P. Q. Rev. Biophys. 1979; 12: 315-369Crossref PubMed Scopus (78) Google Scholar, 12Halford S.E. Johnson N.P. Biochem. J. 1980; 191: 593-604Crossref PubMed Scopus (60) Google Scholar, 13Jen-Jacobson L. Kurpiewski M. Lesser D. Grable J. Boyer H. Rosenberg J.M. Greene P.J. J. Biol. Chem. 1983; 258: 14638-14646Abstract Full Text PDF PubMed Google Scholar). Work from several laboratories has indicated that DNA cleavage byEcoRI endonuclease proceeds by the mechanism shown in Fig.1, with double-strand cleavage proceeding via an intermediate species (E·1) containing one single-strand break within the EcoRI sequence (5Modrich P. Zabel D. J. Biol. Chem. 1976; 251: 5866-5874Abstract Full Text PDF PubMed Google Scholar, 14Halford S.E. Johnson N.P. Biochem. J. 1983; 211: 405-415Crossref PubMed Scopus (28) Google Scholar, 15King K. Benkovic S.J. Modrich P. J. Biol. Chem. 1989; 264: 11807-11815Abstract Full Text PDF PubMed Google Scholar). The fate of E·1 is determined by reaction conditions and the nature of the substrate: the intermediate dissociates from the enzyme in the case of some DNAs but not others (5Modrich P. Zabel D. J. Biol. Chem. 1976; 251: 5866-5874Abstract Full Text PDF PubMed Google Scholar, 14Halford S.E. Johnson N.P. Biochem. J. 1983; 211: 405-415Crossref PubMed Scopus (28) Google Scholar, 16Rubin R.A. Modrich P. Nucleic Acids Res. 1978; 5: 2991-2997Crossref PubMed Scopus (45) Google Scholar, 17Ruben G. Spielman P. Tu C.D. Jay E. Siegel B. Wu R. Nucleic Acids Res. 1977; 4: 1803-1813Crossref PubMed Scopus (25) Google Scholar, 18Langowski J. Urbanke C. Pingoud A. Maass G. Nucleic Acids Res. 1981; 9: 3483-3490Crossref PubMed Scopus (27) Google Scholar). Chemical quench experiments have indicated that product release is rate-limiting for turnover on ColE1 and pBR322 plasmid DNAs under one set of experimental conditions (5Modrich P. Zabel D. J. Biol. Chem. 1976; 251: 5866-5874Abstract Full Text PDF PubMed Google Scholar, 15King K. Benkovic S.J. Modrich P. J. Biol. Chem. 1989; 264: 11807-11815Abstract Full Text PDF PubMed Google Scholar), and facilitated diffusion has been implicated in the paths by which the endonuclease locates anEcoRI sequence and departs from a cleaved site (19Jack W.E. Terry B.J. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4010-4014Crossref PubMed Scopus (144) Google Scholar, 20Terry B.J. Jack W.E. Modrich P. J. Biol. Chem. 1985; 260: 13130-13137Abstract Full Text PDF PubMed Google Scholar, 21Ehbrecht H. Pingoud A. Urbanke C. Maass G. Gualerzi C. J. Biol. Chem. 1985; 260: 6160-6166Abstract Full Text PDF PubMed Google Scholar, 22Jeltsch A. Alves J. Wolfes H. Maass G. Pingoud A. Biochemistry. 1994; 33: 10215-10219Crossref PubMed Scopus (83) Google Scholar). However, the effects of reaction parameters that are expected to affect the efficiency of facilitated diffusion have not been examined with respect to catalytic behavior of the enzyme. We have therefore systematically analyzed the kinetics of the endonuclease using steady-state and pre-steady-state methods. This work demonstrates that product release is rate-limiting for turnover at ionic strengths of 0.06–0.23 m and show that, although variation of ionic strength has dramatic effects onKm and kcat, the kinetics of events that occur at the recognition sequence are essentially insensitive to variation of this parameter. We also show that the protein remains associated with the polynucleotide product after cleavage, undergoing facilitated transfer between DNA sites so rapidly that the kcat for processive cleavage of a two-site substrate is the same as that for cleavage of an otherwise identical DNA containing a single EcoRI site. These observations indicate that the steady-state behavior of the enzyme on natural substrates is dominated by nonspecific interactions, reflecting the significance of facilitated diffusion processes in the reaction mechanism. Homogeneous EcoRI endonuclease was prepared as described previously (23Cheng S.-C. Kim R. King K. Kim S.-H. Modrich P. J. Biol. Chem. 1984; 259: 11571-11575Abstract Full Text PDF PubMed Google Scholar). Other restriction endonucleases were purchased from New England Biolabs (Beverly, MA). Covalently closed, circular pBR322 and pBR322(RI)2 (Fig. 2; see Ref. 20Terry B.J. Jack W.E. Modrich P. J. Biol. Chem. 1985; 260: 13130-13137Abstract Full Text PDF PubMed Google Scholar) were isolated by published methods (5Modrich P. Zabel D. J. Biol. Chem. 1976; 251: 5866-5874Abstract Full Text PDF PubMed Google Scholar, 24Birnboim H.C. Doly J. Nucleic Acids Res. 1979; 7: 1513-1523Crossref PubMed Scopus (9908) Google Scholar). PvuII-linearized pBR322 was prepared and 5′-32P-end-labeled as described previously (15King K. Benkovic S.J. Modrich P. J. Biol. Chem. 1989; 264: 11807-11815Abstract Full Text PDF PubMed Google Scholar, 19Jack W.E. Terry B.J. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4010-4014Crossref PubMed Scopus (144) Google Scholar). Nick translation was used to prepare [32P]pBR322(RI)2. Plasmid DNA was treated with 35 units of ClaI endonuclease at 16 °C in 0.02m Tris-HCl (pH 7.6), 0.02 m NaCl, 6 mm MgCl2, 1 mm dithiothreitol, 0.10 mg/ml bovine serum albumin, and 75 μg/ml ethidium bromide for 30 min to produce molecules bearing a single-strand break at either of the twoClaI sites present on the plasmid. Final DNA preparations contained 75–80% open circles, 20–23% covalently closed circles, and 2–4% linear molecules. These preparations were radiolabeled by nick translation in the presence of [α-32P]dTTP (25Rigby P.W.J. Dieckmann M. Rhodes C. Berg P. J. Mol. Biol. 1977; 113: 237-251Crossref PubMed Scopus (6965) Google Scholar), followed by closure of the single-strand break with T4 DNA ligase. Nucleic acid pellets recovered after phenol extraction and ethanol precipitation were dried briefly in vacuo and resuspended in 0.1 ml of 0.02 m Tris-HCl (pH 7.6), 0.05 mNaCl, 1 mm EDTA. DNA was separated from unincorporated radiolabel by gel filtration through Sephacryl S-300 equilibrated in 0.02 m Tris-HCl (pH 7.6), 0.05 m NaCl, 1 mm EDTA. Two to 10 mol of radiolabeled dTMP were incorporated per mol of plasmid DNA, and ligation efficiencies were 88–92%. To prepare radiolabeled linear pBR322(RI)2 with both EcoRI sites located near one end (Fig. 2), nick-translated [32P]pBR322(RI)2 DNA was digested with HindIII endonuclease according to the manufacturer's instructions. Linear plasmid DNA was recovered after phenol extraction and ethanol precipitation as described above. Reactions under standard EcoRI cleavage conditions were performed at 37 °C in 0.1 m Tris-HCl (pH 7.6), 0.05 mNaCl, 5 mm MgCl2, 0.2 mm EDTA, 0.05 mg/ml bovine serum albumin, and DNA as indicated. Effects of NaCl on cleavage were determined in reactions containing 0.02 mTris-HCl (pH 7.6), 5 mm MgCl2, 0.2 mm EDTA, 0.05 mg/ml bovine serum albumin, 0.025–0.20m NaCl, and 5′-32P-end-labeled DNA as indicated. Cleavage was initiated by addition of 0.05 volume of diluent (5Modrich P. Zabel D. J. Biol. Chem. 1976; 251: 5866-5874Abstract Full Text PDF PubMed Google Scholar) containing an appropriate amount of EcoRI endonuclease. Reactions were terminated by addition of 0.2 vol 50% (w/v) glycerol, 1% sodium dodecyl sulfate, 0.05 m EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol, and products were resolved by electrophoresis through 1% agarose gels in a Tris borate buffer system. Bands were visualized by ethidium fluorescence or by autoradiography, excised, and DNA products quantified by liquid scintillation counting. Reactions containing 0.02 m Tris-HCl (pH 7.6), 0.2 mmEDTA, 0.05 mg/ml bovine serum albumin, 5′-32P-end-labeled DNA, EcoRI endonuclease, and NaCl as indicated were incubated at 37 °C until equilibrium was attained (7Terry B.J. Jack W.E. Rubin R.A. Modrich P. J. Biol. Chem. 1983; 258: 9820-9825Abstract Full Text PDF PubMed Google Scholar). Cleavage was initiated by adding reaction buffer containing MgCl2 and a 17-fold molar excess of unlabeled pBR322 DNA (relative to [32P]DNA) to yield a final MgCl2concentration of 5 mm. Reactions were terminated and products quantified as described above. Pre-steady-state chemical quench experiments with previously formed endonuclease·DNA complexes were performed by a modification of the previously described procedure (15King K. Benkovic S.J. Modrich P. J. Biol. Chem. 1989; 264: 11807-11815Abstract Full Text PDF PubMed Google Scholar) using a RQF-3 quench flow apparatus (KinTek Instruments). Reactant loops and mixing block were maintained at 37 °C by circulating water. Calibration was verified weekly by performing KOH-catalyzed hydrolysis of p-nitrophenyl acetate at 25 °C (second order rate constant kOH = 570 m−1 min−1 (26Kirsch J.F. Jencks W.P. J. Am. Chem. Soc. 1964; 86: 837-846Crossref Scopus (180) Google Scholar)). The value for kOH determined with the KinTek instrument was 538 ± 30 m−1 min−1 (one standard deviation, n = 12). Specific EcoRI·DNA complexes were formed by incubating endonuclease and [3H]pBR322 at 37 °C in 0.02m Tris-HCl (pH 7.6), 0.025–0.2 m NaCl, 0.2 mm EDTA, and 0.05 mg/ml bovine serum albumin. DNA cleavage was initiated by mixing samples (40 μl) with an equal volume of 0.01m MgCl2 in the same buffer. The final concentration of [3H]pBR322 was 1–10 nm, and final endonuclease concentrations varied between 20 and 200 nm, with higher concentrations of the two components being required to drive specific complex formation at higher ionic strengths. The component concentrations used in each experiment were based on equilibrium affinity constants determined under identical conditions (7Terry B.J. Jack W.E. Rubin R.A. Modrich P. J. Biol. Chem. 1983; 258: 9820-9825Abstract Full Text PDF PubMed Google Scholar) and ensured that >97% of the plasmid DNA was bound specifically by the enzyme. Reactions were quenched by mixing with 0.5 volumes of 0.075m EDTA. Collected samples also contained an additional 140 μl of 0.075 m EDTA delivered by the quench flow apparatus during sample ejection. Quenched samples were collected in 1.5-ml microcentrifuge tubes containing 20 μl of 10% sodium dodecyl sulfate and were stored on ice until all time points had been collected. Samples were dried in vacuo, resuspended in 75 μl of 0.01m Tris-HCl (pH 7.6), 0.05% bromphenol blue, 5% glycerol (v/v), and reaction products were quantified after agarose gel electrophoresis as described above. Rate constants governing first (k2) and second (k3) strand cleavage events were estimated by nonlinear least squares regression analysis (27Marquardt D.W. J. Soc. Ind. Appl. Math. 1963; 11: 431-441Crossref Google Scholar) using the integrated rate equations for the mechanism shown in Reaction 1 as described previously (15King K. Benkovic S.J. Modrich P. J. Biol. Chem. 1989; 264: 11807-11815Abstract Full Text PDF PubMed Google Scholar). Values fork2 were obtained from fitting data for disappearance of substrate to an exponential decay mechanism. Thesek2 values were used as initial estimates in fitting data for the open circular intermediate and the linear DNA product. The reported values for k3 were obtained from analysis of the appearance of the double-strand cleaved product and did not deviate from those obtained by analysis of the formation and decay of the open circular intermediate by more than 10%. Pre-steady-state experiments were also performed by mixing an endonuclease-MgCl2 solution with [3H]pBR322. Solutions containing 0.02 m Tris-HCl (pH 7.6), 0.2 mm EDTA, 0.05 mg/ml bovine serum albumin, 0.025–0.20m NaCl, and 2–20 nm [3H]pBR322 were prewarmed to 37 °C, after which DNA cleavage was initiated by mixing samples (40 μl) with an equal volume of a solution containing 0.01 m MgCl2 and EcoRI endonuclease in the same buffer. Enzyme samples were prepared for each time point by diluting endonuclease to the appropriate concentration using prewarmed reaction buffer immediately prior to the mixing experiment. To ensure that second order effects were not limiting the rate of cleavage, the endonuclease concentration was increased until no further change in reaction rate was observed. Zero time points were obtained by mixing quenching reagent with the reaction mixture before addition ofEcoRI endonuclease, while end points were determined by allowing the reactions to proceed for 30 s, by which time cleavage was complete. Reaction products were resolved on 1% agarose gels and quantified as described above. Steady-state DNA cleavage was assayed at 37 °C. EcoRI endonuclease (0.1 mol of enzyme dimer/mol of plasmid) was added to prewarmed solutions containing 2–5 nm HindIII-linearized [32P]pBR322(RI)2 DNA in 0.02 mTris-HCl (pH 7.6), 0.05–0.15 m NaCl, 0.2 mmEDTA, and 0.05 mg/ml bovine serum albumin. After incubation at 37 °C for 30 min to allow specific complex formation, cleavage was initiated by adding an equal volume of prewarmed buffer containing 10 mm MgCl2 and 20–50 nm unlabeled pBR322(RI)2 DNA (10 mol of unlabeled DNA/mol of radiolabeled DNA). Samples (20 μl) were removed as a function of time and mixed with 5 μl of a reaction quench solution as described above. Reaction products were separated by electrophoresis on 10% polyacrylamide gels in a Tris borate buffer system. Radiolabeled DNA bands were visualized by autoradiography, excised, and quantified by liquid scintillation counting as described above. Specific radioactivities of the reaction products were determined by allowing cleavage to proceed for 1 h at 37 °C, by which time all the substrate was cleaved. The large DNA fragments (4477 and 4528 bp)1 produced by cleavage ofHindIII-linearized pBR322(RI)2 at either or bothEcoRI sites were not resolved from one another or from unreacted substrate under these conditions. However, the amount of substrate consumed during cleavage reactions is equal to the sum of the amounts of 80- and 29-bp fragments produced. Steady-state turnover numbers governing endonucleolytic attack onHindIII-linearized pBR322(RI)2(kcat) were calculated according to the relationship: kcat =Vmax/[Ea], whereVmax was estimated as the initial rate of product formation at substrate concentrations at least 10 times theKm value (Fig. 4). The quantity of active endonuclease ([Ea]) was determined as described previously (7Terry B.J. Jack W.E. Rubin R.A. Modrich P. J. Biol. Chem. 1983; 258: 9820-9825Abstract Full Text PDF PubMed Google Scholar), and was typically 92%. Initial rates of product formation (Vmax) were calculated as the sum of initial rates of appearance of the 80-bp fragment produced by single site cleavage at the more central EcoRI site (V80) and the 29-bp fragment produced either by single site cleavage at the more terminal EcoRI site or by processive cleavage at both sites (V29; see Fig.2). Vmax values calculated in this manner thus include contributions from both nonprocessive and processive turnovers (see "Results"). Rate constants for generation of the 51-bp DNA fragment by processive cleavage at both EcoRI sites onHindIII-linearized pBR322(RI)2(kproc, see Fig.2) were calculated according to the relationship: kproc =V51/fp[Ea], where kproc is the rate constant for processive turnover, V51 is the initial rate of formation of the 51-bp fragment at concentrations of substrate that were approximately saturating, and fp is the fraction of cleaved DNA molecules undergoing processive cleavage at bothEcoRI sites. The factor fp is an experimentally determined value for the fraction of enzyme molecules that participate in processive cleavage, which is limited to a maximum of 0.5 on linear substrates (20Terry B.J. Jack W.E. Modrich P. J. Biol. Chem. 1985; 260: 13130-13137Abstract Full Text PDF PubMed Google Scholar). Values for fpwere calculated as described by Terry et al. (20Terry B.J. Jack W.E. Modrich P. J. Biol. Chem. 1985; 260: 13130-13137Abstract Full Text PDF PubMed Google Scholar), using the following equation,fp=V51/(V80+V29),(Eq. 1) where fp is the fraction of cleaved molecules that have participated in processive events andV51, V80, andV29 are the initial rates of formation of the 51-, 80-, and 29-bp cleavage products. The equilibrium affinity of EcoRI endonuclease for its recognition sequence (7Terry B.J. Jack W.E. Rubin R.A. Modrich P. J. Biol. Chem. 1983; 258: 9820-9825Abstract Full Text PDF PubMed Google Scholar, 13Jen-Jacobson L. Kurpiewski M. Lesser D. Grable J. Boyer H. Rosenberg J.M. Greene P.J. J. Biol. Chem. 1983; 258: 14638-14646Abstract Full Text PDF PubMed Google Scholar), kinetic parameters governing formation and dissociation of specific complexes (19Jack W.E. Terry B.J. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4010-4014Crossref PubMed Scopus (144) Google Scholar, 28Wright D.J. King K. Modrich P. J. Biol. Chem. 1989; 264: 11816-11821Abstract Full Text PDF PubMed Google Scholar), and the effective length of the DNA segment scanned by positionally correlated facilitated diffusion (20Terry B.J. Jack W.E. Modrich P. J. Biol. Chem. 1985; 260: 13130-13137Abstract Full Text PDF PubMed Google Scholar, 29Jack W.E. Participation of Outside DNA Sequences in the EcoRI Endonuclease Reaction Pathway Ph.D. thesis. Duke University, Durham, NC1983Google Scholar) are highly dependent on ionic strength. Fig. 3 demonstrates that salt concentration also has a dramatic effect on the steady-state parameters that govern cleavage of pBR322 DNA. An increase in the NaCl concentration from 0.025 to 0.2 m (ionic strength of 0.059–0.23 m) results in a 1,000-fold increase in theKm for pBR322 and an increase inkcat of about 15-fold. The endonuclease is thus unusual in the sense that optimal ionic conditions for cleavage are dependent on substrate concentration. This can be seen by considering the effects of ionic strength at the extremes of substrate concentration, i.e. [DNA] → 0 and [DNA] → ∞. At dilute DNA concentrations, where velocity is proportional to thekcat/Km ratio, the rate of cleavage will be highest at low salt concentration becauseKm decreases more rapidly with decreasing ionic strength than does kcat. However, at high DNA concentration where rate is kcat-limited, the cleavage rate will be highest at high ionic strength becausekcat increases with salt concentration. The significance of these effects in terms of mechanism will be considered below. For of pre-steady-state the mechanism shown in Fig. 1 be to that shown in Reaction 1) This is for pBR322 intermediate does not with this DNA R.A. Modrich P. Nucleic Acids Res. 1978; 5: 2991-2997Crossref PubMed Scopus (45) Google J. Urbanke C. Pingoud A. Maass G. Nucleic Acids Res. 1981; 9: 3483-3490Crossref PubMed Scopus (27) Google Scholar, 29Jack W.E. Participation of Outside DNA Sequences in the EcoRI Endonuclease Reaction Pathway Ph.D. thesis. Duke University, Durham, NC1983Google Scholar). chemical quench flow analysis has indicated that this is an of the mechanism (15King K. Benkovic S.J. Modrich P. J. Biol. Chem. 1989; 264: 11807-11815Abstract Full Text PDF PubMed Google Scholar). this study was to analysis of the fate of complexes at a single ionic strength, have this analysis to a of NaCl concentrations using two mixing In the complexes were mixed with a MgCl2 in the DNA was mixed with a solution of endonuclease and The prior to specific complex formation, the rate of first strand cleavage in the can be limited by or by slow on the to specific complex Fig. an of pre-steady-state cleavage of complexes formed prior to of DNA cleavage by addition of The of the data to a mechanism is with the only in the reaction and reflecting of a amount of the open circular intermediate high of endonuclease to DNA, have the of DNA molecules bearing a single-strand break at the EcoRI site that are to further cleavage not of this species was in experiments performed by either mixing and the fraction of DNA to this species on the of enzyme to DNA, but not on the concentrations of either At an enzyme to DNA of this species for of all cleaved DNA. This low by of EcoRI endonuclease preparations has been shown that the endonuclease will low efficiency of a single-strand break into the strand of a site Kurpiewski L. Science. 1990; PubMed Scopus Google Scholar). the with EcoRI during enzyme (23Cheng S.-C. Kim R. King K. Kim S.-H. Modrich P. J. Biol. Chem. 1984; 259: 11571-11575Abstract Full Text PDF PubMed Google Scholar), is to transfer under the conditions for endonuclease is to the endonuclease high affinity complexes sites under the conditions used for the to an of the amount of We have this by performing all single turnover DNA cleavage experiments at an endonuclease to DNA of or conditions that in of DNA molecules to second strand cleavage at a of DNA. We that double-strand cleavage within previously formed site-specific complexes can be described by the two step mechanism described above. identical results were obtained when enzyme was mixed with DNA, that concentrations of DNA and enzyme were so that of the endonuclease with the EcoRI site of the plasmid was not The analysis under these mixing conditions to concentrations of m this concentration rate constants for first and second strand cleavage events were to be of mixing ruling out a slow first order step to produce a endonuclease·DNA complex once the enzyme has located the EcoRI site. Furthermore, as in rate constants governing the two strand cleavage are essentially insensitive to in the of ionic strength from 0.059 m to m in only a in the rate of first strand cleavage, the rate of second strand cleavage was not In all strand cleavage rate constants are than constants for first and second strand cleavage of pBR322 by ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± DNA cleavage as a function of NaCl concentration was performed and data analyzed as described under and Reactions were initiated by mixing MgCl2 with previously formed complexes or by mixing pBR322 with an endonuclease MgCl2 solution value for is the of ± two standard not Values are from Fig. in a Pre-steady-state DNA cleavage as a function of NaCl concentration was performed and data analyzed as described under and Reactions were initiated by mixing MgCl2 with previously formed complexes or by mixing pBR322 with an endonuclease MgCl2 solution value for is the of ± two standard not Values are from Fig. of pre-steady-state and steady-state results that the rate-limiting step for endonuclease turnover occurs subsequent to sequence recognition and at all ionic strengths of a pre-steady-state of double-strand cleavage to formation of prior to the rate-limiting This was although the yield was than 1 mol of double-strand per mol of endonuclease under all conditions that that DNA concentrations used were at least 5 times the Km value in all during EcoRI cleavage of EcoRI cleavage was initiated by addition of EcoRI endonuclease to reaction (see and at 37 °C. Endonuclease and DNA concentrations were 1 and 10 nm, respectively, for all reactions those containing 0.2 m NaCl where these concentrations were 10 and nm to the higher at this ionic strength. These DNA concentrations are at