J Biol Chem, Vol. 274, Issue 45, 31896-31902, November 5, 1999
The Kinetic Mechanism of EcoRI Endonuclease*
David J.
Wright
,
William E.
Jack§, and
Paul
Modrich¶
From the Department of Biochemistry and ¶ Howard Hughes
Medical Institute, Duke University Medical Center,
Durham, North Carolina 27710
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ABSTRACT |
Steady-state parameters governing cleavage of
pBR322 DNA by EcoRI endonuclease are highly sensitive to
ionic environment, with Km and
kcat increasing 1,000-fold and 15-fold,
respectively, when ionic strength is increased from 0.059 to 0.23 M. 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 spaced
EcoRI sites is governed by the same turnover number as
hydrolysis of parental pBR322, which contains only a single
EcoRI 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 to
EcoRI sequence recognition and subsequent to DNA cleavage.
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INTRODUCTION |
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 (1-4).
The enzyme functions as a homodimer of a 31-kDa polypeptide (5-9), and
in presence of Mg2+ introduces two staggered, single-strand
scissions into the symmetric recognition sequence, 5'-GAATTC-3' (10).
In the absence of a divalent cation, the endonuclease binds
specifically and with high affinity to its recognition sequence (7,
11-13).
Work from several laboratories has indicated that DNA cleavage by
EcoRI 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 (5, 14, 15).
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 (5, 14, 16-18).
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 (5, 15), and facilitated diffusion has
been implicated in the paths by which the endonuclease locates an
EcoRI sequence and departs from a cleaved site (19-22).
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.
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Fig. 1.
Kinetic mechanism for EcoRI
endonuclease. S, 1, and 2 represent DNA where the EcoRI site is intact (S), cleaved in
one strand (1), or cleaved in both strands (2).
Panel A, facilitated diffusion has been
implicated in the kinetic paths by which the endonuclease locates an
intact EcoRI sequence and and leaves the cleaved site under
some reaction conditions (19, 20). This effect is indicated by
(E·N)n, which represents the population
of nonspecific complexes involved in this phenomenon. Rate constants
k5 and k 5 govern the
fate of the E·1 intermediate, which can dissociate from
the endonuclease surface in the case of some DNA substrates (5, 14,
16-18). Panel B, since the nonspecific complexes implicated in
EcoRI endonuclease catalysis cannot be directly detected
during analysis of cleavage kinetics, the mechanism has been simplified
for kinetic studies. Since dissociation of the E·1
intermediate is negligible with the pBR322 substrate used in this study
(16, 18, 29), this step can be neglected in the experiments described
here.
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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 on
Km 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.
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MATERIALS AND METHODS |
Enzymes and DNA--
Homogeneous EcoRI endonuclease
was prepared as described previously (23). Other restriction
endonucleases were purchased from New England Biolabs (Beverly, MA).
Covalently closed, circular pBR322 and pBR322(RI)2 (Fig. 2;
see Ref. 20) were isolated by published methods (5, 24).
PvuII-linearized pBR322 was prepared and
5'-32P-end-labeled as described previously (15, 19). Nick
translation was used to prepare
[32P]pBR322(RI)2. Plasmid DNA was treated
with 35 units of ClaI endonuclease at 16 °C in 0.02 M 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 two
ClaI 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 [
-32P]dTTP (25),
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 M NaCl, 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.
Steady-state Cleavage Initiated by Endonuclease Addition to
Solutions Containing DNA and Mg2+--
Reactions under
standard EcoRI cleavage conditions were performed at
37 °C in 0.1 M Tris-HCl (pH 7.6), 0.05 M
NaCl, 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 M
Tris-HCl (pH 7.6), 5 mM MgCl2, 0.2 mM EDTA, 0.05 mg/ml bovine serum albumin, 0.025-0.20
M NaCl, and 5'-32P-end-labeled DNA as
indicated. Cleavage was initiated by addition of 0.05 volume of diluent
(5) 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.
Steady-state Cleavage Initiated by MgCl2 Addition to
Previously Formed Endonuclease·DNA Complexes--
Reactions
containing 0.02 M Tris-HCl (pH 7.6), 0.2 mM
EDTA, 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 (7). 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 MgCl2
concentration of 5 mM. Reactions were terminated and
products quantified as described above.
Pre-steady-state Cleavage Initiated by Mixing MgCl2
with Specific EcoRI Endonuclease·DNA Complexes--
Pre-steady-state
chemical quench experiments with previously formed endonuclease·DNA
complexes were performed by a modification of the previously described
procedure (15) 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 M1 min1 (26)). The value
for kOH determined with the KinTek instrument was 538 ± 30 M1 min1 (one
standard deviation, n = 12).
Specific EcoRI·DNA complexes were formed by incubating
endonuclease and [3H]pBR322 at 37 °C in 0.02 M 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 0.01 M 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
(7) and ensured that >97% of the plasmid DNA was bound specifically
by the enzyme.
Reactions were quenched by mixing with 0.5 volumes of 0.075 M 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 0.01 M 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 (27) using the integrated rate equations for the
mechanism shown in Reaction 1 as described previously (15). Values for k2 were obtained from fitting data for
disappearance of substrate to an exponential decay mechanism. These
k2 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 Cleavage Initiated by Mixing DNA with Solutions
Containing Endonuclease and Mg2+--
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, 0.025-0.20
M 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 of
EcoRI 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 Cleavage of HindIII-linearized
pBR322(RI)2 by EcoRI Endonuclease--
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 M
Tris-HCl (pH 7.6), 0.05-0.15 M NaCl, 0.2 mM
EDTA, 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 of
HindIII-linearized pBR322(RI)2 at either or both
EcoRI 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 on
HindIII-linearized pBR322(RI)2
(kcat) were calculated according to the
relationship: kcat = Vmax/[Ea], where
Vmax was estimated as the initial rate of
product formation at substrate concentrations at least 10 times the
Km value (Fig. 4). The quantity of active
endonuclease ([Ea]) was determined as
described previously (7), 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 on
HindIII-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 both
EcoRI 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 (20). Values for fp
were calculated as described by Terry et al. (20), using the
following equation,
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(Eq. 1)
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where fp is the fraction of cleaved
molecules that have participated in processive events and
V51, V80, and
V29 are the initial rates of formation of the
51-, 80-, and 29-bp cleavage products.
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Fig. 2.
Plasmid pBR322(RI)2.
A, plasmid pBR322(RI)2 (4557 base pairs) is a
derivative of pBR322 with a duplication of a 192-bp BspI
fragment containing the EcoRI site of the parent plasmid
(20). C, ClaI site; E,
EcoRI site; H, HindIII site.
B, digestion of covalently closed circular
pBR322(RI)2 with HindIII endonuclease produces a
linear form of the plasmid with two EcoRI sites
(E) located 80 and 29 bp from one end. Cleavage of a single
EcoRI site produces either 4477- and 80-bp fragments or
4528- and 29-bp DNA fragments. Cleavage of both sites produces 4477-, 51-, and 29-bp DNA fragments.
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RESULTS |
Dependence of Steady-state Kinetic Parameters on Ionic
Strength--
The equilibrium affinity of EcoRI
endonuclease for its recognition sequence (7, 13), kinetic parameters
governing formation and dissociation of specific complexes (19, 28),
and the effective length of the DNA segment scanned by positionally
correlated facilitated diffusion (20, 29) 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 the
Km for pBR322 and an increase in
kcat 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 the
kcat/Km ratio, the rate of
cleavage will be highest at low salt concentration because
Km 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 because kcat increases with salt concentration. The
significance of these effects in terms of mechanism will be considered
below.
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Fig. 3.
Dependence of steady-state cleavage
parameters on NaCl concentration. EcoRI endonuclease
cleavage of linear pBR322 was performed at 37 °C in 0.02 M Tris-HCl (pH 7.6), 5 mM MgCl2,
0.2 mM EDTA, 0.05 mg/ml bovine serum albumin, and the
indicated concentrations of NaCl. Km and
kcat values were determined by a DNA saturation
curve at each NaCl concentration. Values for these parameters were
obtained from double-reciprocal plots of 1/vo
versus 1/So, which were fit by the
weighted regression procedure of Wilkinson (41).  ,
Km;  , kcat.
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Rate Constants for Sequence Recognition and Strand Cleavage Steps
Are Independent of Ionic Strength--
For purposes of
pre-steady-state analysis, the mechanism shown in Fig. 1 may be
simplified to that shown in Reaction 1.
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(Reaction 1)
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This simplification is valid for pBR322 because the
E·1 intermediate does not dissociate with this DNA (16,
18, 29). A previous chemical quench flow analysis has indicated that
this scheme is an adequate representation of the mechanism (15). Since
this previous study was restricted to analysis of the fate of preformed
E·S complexes at a single ionic strength, we have extended
this analysis to a range of NaCl concentrations using two mixing
protocols. In the first, preformed endonuclease·pBR322 complexes
were mixed with a MgCl2 solution, whereas in the alternate protocol DNA was mixed with a solution of endonuclease and
MgCl2. The former method bypasses steps occurring prior to
specific complex formation, whereas the rate of first strand cleavage
in the latter protocol can potentially be limited by enzyme-DNA
association, or by other slow steps on the path to specific complex formation.
Fig. 4 shows an example of
pre-steady-state cleavage of endonuclease·pBR322 complexes formed
prior to initiation of DNA cleavage by addition of MgCl2.
The fit of the data to a two-step mechanism is excellent, with the only
notable deviation occurring late in the reaction and reflecting
persistence of a small amount of the open circular intermediate
species.2 We conclude that
double-strand cleavage occurring within previously formed site-specific
complexes can be described by the two step mechanism described above.
Essentially identical results were obtained when enzyme was mixed with
DNA, provided that concentrations of DNA and enzyme were chosen so that
association of the endonuclease with the EcoRI site of the
plasmid was not rate-limiting. The latter requirement restricted
analysis under these mixing conditions to [NaCl] concentrations of
0.025-0.1 M (Table I). Over
this concentration range, rate constants for first and second strand cleavage events were found to be independent of mixing protocol (Table
I), ruling out a slow first order step necessary to produce a
cleavage-competent endonuclease·DNA complex once the enzyme has
located the EcoRI site. Furthermore, as summarized in Table I, rate constants governing the two strand cleavage steps are essentially insensitive to [NaCl] in the range of 0.025-0.2
M. Increasing ionic strength from 0.059 M to
0.23 M resulted in only a 25% decrease in the rate of
first strand cleavage, whereas the rate of second strand cleavage was
not significantly affected. In all cases strand cleavage rate constants
are much larger than kcat.
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Fig. 4.
Pre-steady-state cleavage of
EcoRI endonuclease·DNA complexes. Pre-steady
cleavage of preformed enzyme·pBR322 complexes was performed in the
presence of 0.05 M NaCl as described under "Materials and
Methods." The procedure described previously (15) was used to correct
for the presence of small amounts of contaminating, open circular DNA
(4-7%) in plasmid preparations, and were fit by non-linear
least-squares regression analysis (27) to integrated rate equations for
the mechanism (40) E·S 224 E·1 224 E·2. E·S represents a specific
endonuclease·DNA complex, and 1 and 2 correspond to DNA species that
have suffered one or two phosphodiester hydrolytic events within the
EcoRI sequence (Fig. 1). Solid lines
correspond to results of the fitting routine (pseudo-first order rate
constants for first and second strand events of 2.6 and 1.6 s1, respectively). , uncleaved, closed circular
pBR322;  , open circular intermediate cleaved in one strand of the
EcoRI site; , linear product resulting from double-strand
cleavage.
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Table I
Rate constants for first and second strand cleavage of pBR322 by EcoRI
Pre-steady-state DNA cleavage as a function of NaCl concentration was
performed and data analyzed as described under "Materials and
Methods." Reactions were initiated by mixing MgCl2 with
previously formed endonuclease·pBR322 complexes (E·S) or
by mixing pBR322 with an endonuclease MgCl2 solution
(E + S). Each value for k2 or
k3 is the average of four determinations ± two
standard deviations. ND, not determined. Values for
kcat are from Fig. 3.
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Pre-steady-state Burst of Product Formation--
Comparison of
pre-steady-state and steady-state results suggest that the
rate-limiting step for endonuclease turnover occurs subsequent to
sequence recognition and chemistry at all ionic strengths tested,
predicting occurrence of a pre-steady-state burst of double-strand
cleavage due to rapid formation of E·2 prior to the
rate-limiting step. This was confirmed (Table
II), although the yield was significantly
less than 1 mol of double-strand breaks per mol of endonuclease dimer
under all conditions tested despite that fact that DNA concentrations
used were at least 5 times the Km value in all
cases.
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Table II
Pre-steady-state burst during EcoRI cleavage of pBR322
DNA cleavage was initiated by addition of EcoRI endonuclease
to reaction mixtures (see "Materials and Methods") pre-equilibrated
at 37 °C. Endonuclease and DNA concentrations were 1 and 10 nM, respectively, for all reactions except those containing
0.2 M NaCl where these concentrations were 10 and 100 nM due to the higher KM at this ionic
strength. These DNA concentrations are at least 5 times the
KM value at each ionic strength (Fig. 3).
Pre-steady-state bursts were determined by extrapolation to zero time
by linear least squares analysis and are expressed per mol of
endonuclease dimer.
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The less than stoichiometric burst amplitude was not due to significant
levels of inactive enzyme as judged by similar experiments in which
preformed endonuclease·[32P]pBR322 complexes (DNA
excess) were subsequently challenged with Mg2+ and an
excess of the unlabeled form of the DNA (Table
III). Under these conditions, product
yield was 0.8-1.0 mol of double-strand events per mol of endonuclease
dimer at ionic strengths of 0.059-0.13 M, decreasing
significantly only at an ionic strength of 0.23 M. We
attribute the higher product yield in this type of experiment, relative
to that observed during the pre-steady-state burst, to the involvement
of facilitated diffusion in the mechanism of EcoRI site
location. Since pBR322 has a single recognition sequence and 4360 nonspecific sites, initial endonuclease·DNA complexes are nonspecific
in nature, and these give rise to specific complexes by diffusion of
the protein within the domain of the polynucleotide (19, 20). The
enzyme thus kinetically partitions between nonspecific sites and the
EcoRI sequence during turnover (Fig. 1A). If a
subpopulation of nonspecifically bound enzyme fails to locate and
cleave the EcoRI site within a time period on the order of
that required for turnover, then the pre-steady-state burst will be
less than unity, as we have observed. The problem of kinetic
partitioning is circumvented in those experiments in which cleavage
preformed, specific complexes is initiated by addition of
Mg2+, conditions that result in near unity product yields
at low and moderate ionic strengths.
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Table III
Partitioning of EcoRI endonuclease·DNA complexes between cleavage and
dissociation
Cleavage of preformed complexes of endonuclease and
PvuII-linearized [32P]pBR322 was initiated by
addition of MgCl2 and a 17-fold molar excess of unlabeled
pBR322 DNA (see "Materials and Methods"). [32P]DNA
concentrations were 5 nM except at 0.2 M NaCl
where DNA was present at 25 nM due to reduced specific
affinity at high ionic strength (7). The EcoRI endonuclease
(dimer):DNA molar ratio was 1:10, and product yields were calculated as
described in Table I.
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The stoichiometry of preferential cleavage of preformed complexes at
ionic strengths of 0.059-0.13 M indicates that initial chemical step is fast relative to the competing dissociation reactions, i.e. k2 
(kS + kNS) (Fig.
1A). Under these conditions, formation of the
E·1 intermediate is therefore fast relative to conversion
of specific E·S complexes to kinetically nonproductive E·N complexes or to dissociation of the endonuclease from
the polynucleotide domain
(k2/(kS + kNS) > 10). Since these experiments
monitored double-strand cleavage events, these results also imply that
second strand cleavage is tightly coupled to first strand cleavage for
pBR322, as inferred previously (5, 16, 18).
The yield of preferential cleavage of preformed complexes was
significantly reduced at 0.23 M ionic strength, as compared with that observed at lower salt concentrations (Table
III).3 Because the reduced
yield of double-strand cleaved product is not due to dissociation of
the E·1 intermediate at the higher ionic strength (Table I
and Ref. 29), we interpret these findings in terms of partitioning of
specific complexes between cleavage and departure of the endonuclease
from an uncleaved site. This implies that k2 
kS + kNS (Fig. 1A) at
an ionic strength of 0.23 M.
Effects of Processive Action on EcoRI Endonuclease
Turnover--
The results described above indicate that the step that
limits endonuclease turnover occurs after double-strand cleavage. We
have considered three possibilities with respect to the nature of this
slow step. Since the endonuclease supports processive cleavage at low
to moderate ionic strengths (20), it is evident that the enzyme can
leave an EcoRI site after cleavage to diffuse within the
polynucleotide domain of a DNA product. Turnover may therefore be
limited by the effective lifetime of this population of nonspecific
complexes. A second possibility invokes slow release of the enzyme from
the cleaved EcoRI site, whereas the third attributes the
slow step to a rate-limiting conformational change that must occur
before the endonuclease can support another round of DNA cleavage.
To address potential involvement of slow release from a cleaved site or
a slow, post-cleavage conformational transition, we have exploited the
previous finding that EcoRI endonuclease can act
processively on DNA molecules bearing two EcoRI sites (20, 22). Because processive cleavage requires that the endonuclease leave a
cleaved site, locate the second EcoRI site on the same DNA
molecule, and cleave that site prior to dissociation into solution,
evaluation of the kcat for processive events
will reveal the presence of slow steps that occur between cleavage of
the first and second sites. However, if turnover is limited by the lifetime of nonspecific complexes formed after the two chemical steps,
multiple cleavage events within a single DNA could conceivably occur at
the same steady-state rate as nonprocessive events.
To assess this possibility, we examined EcoRI cleavage of a
linear form of pBR322(RI)2, a plasmid bearing two
EcoRI sites, which are separated by 51 base pairs and
embedded within identical flanking sequences (20). Cleavage of
HindIII-linearized pBR322(RI)2 can give rise to
three small DNA products: a 29-bp fragment produced by cleavage at the
site proximal to the DNA terminus or at both sites, an 80-bp fragment
produced by cleavage at the site more distal to the DNA terminus, and a
51-bp fragment produced by cleavage at both sites (Fig.
2). Rates of appearance of the 80-, 51-, and 29-bp products were used to calculate turnover numbers for both nonprocessive and processive cleavage events
(kcat and kproc, see
"Materials and Methods"). At ionic strengths of 0.086 and 0.13 M, where processive cleavage was observed, the two turnover numbers were identical (Table IV) and
similar to those determined using plasmid pBR322 (Table I). These
findings rule out endonuclease release from a cleaved EcoRI
site, as well as post-cleavage conformational changes as potential
rate-limiting steps in the reaction. Consequently, we infer that
endonuclease turnover is limited by the lifetime of the population of
nonspecific complexes that occur after DNA cleavage by departure of the
enzyme from the cleaved site onto flanking sequences and diffusion
within the domain of a DNA product prior to dissociation into
solution.
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Table IV
Processive cleavage of HindIII-linearized pBR322(RI)2
EcoRI endonuclease·[32P]pBR322(RI)2
complexes (0.2-0.5 nM, 0.1 mol of endonuclease dimer/mol
of plasmid) were formed at 37 °C (see "Materials and Methods")
and cleavage initiated by addition of an equal volume of a solution
containing 0.01 M MgCl2 and a 10-fold molar excess
of unlabeled pBR322(RI)2 DNA. Pre-steady-state product yields
were estimated from ordinate intercepts obtained by linear
extrapolation of cleavage data after the pre-steady-state reaction
phase to zero time (40). Turnover numbers governing substrate decay
(kcat) and generation of the 51-bp DNA product by
processive action (kproc) were calculated as
described under "Materials and Methods" using initial portions of
progress curves. The fraction of cleaved DNA molecules undergoing
processive hydrolysis (fp) was calculated as
described by Terry et al. (Ref. 20; see "Materials and
Methods"). UD, processive cleavage below detection limit. The
fraction of linear substrate that is subject to processive cleavage is
limited to 50% due to the fact the enzyme can associate with either of
the two fragments resulting from cleavage of the first site (20).
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DISCUSSION |
Despite their significant affinity for nonspecific sequences,
restriction endonucleases maintain high cleavage specificity for their
canonical recognition sites and are able to locate these sequences by
kinetically efficient pathways. The extensively studied EcoRI and EcoRV endonucleases address the first
problem via recognition site-dependent activation of the
DNA cleavage center (9, 31). Facilitated diffusion has been implicated
in the pathways by which both EcoRI (19-22) and
EcoRV (32, 33) endonucleases locate their recognition sites.
In this type of mechanism, diffusional search occurs within the domain
DNA chain after the initial protein-DNA collision, which favors
nonspecific complex formation by a large statistical factor (34).
Based on the experiments described here, we have concluded that
facilitated diffusion processes dominate the steady-state kinetic
behavior of the EcoRI endonuclease with natural DNA
substrates. Despite the large effects of ionic strength on
Km and kcat (Fig.
3), we have been unable to detect any
significant effects of this reaction parameter on the kinetics of
recognition and cleavage events that occur once the enzyme has arrived
at the EcoRI sequence (Table I). We therefore attribute the
increases in Km and kcat that
are associated with increased salt concentration to nonspecific
endonuclease·DNA interactions that occur prior to EcoRI
site location and subsequent to double-strand cleavage.
The differential effects of increases in ionic strength on
Km and kcat can be understood
in terms of the importance of nonspecific endonuclease·DNA complexes
in the reaction. At low to moderate ionic strengths, positionally
correlated facilitated diffusion, which is topologically equivalent to
"sliding" (34), plays an important role in the mechanism by which
EcoRI endonuclease locates its recognition sequence
(19-21). At an ionic strength of 0.08 M, the endonuclease
has been estimated to scan about 1,300 base pairs per DNA binding event
by a sliding type mechanism, with the effective chain length scanned in
this manner decreasing rapidly with increasing salt concentration (19).
At low ionic strengths, which favor a low Km, the
endonuclease reaction thus approaches the diffusion limit where each
protein·DNA collision event is productive with respect to DNA
cleavage (see also below). The efficiency of this search process
requires extensive sampling of the hexanucleotide sequence sets present
within the substrate. The effect of salt concentration on
kcat can be understood in similar terms.
Inasmuch as the slow step in the reaction occurs subsequent to cleavage
at all ionic strengths tested and since analysis of processive turnover
of the enzyme has ruled out a slow conformational change or slow
release from the cleaved site as rate determining, we ascribe the slow
step to the lifetime of the population of nonspecific complexes that
result from diffusion of the protein within the domain of the DNA
product prior to its dissociation into solution. This implies that DNA
products are also subject to extensive sequence sampling at low ionic
strength prior to rate-limiting dissociation, resulting in a low
kcat. Conversely, high ionic strength, which
favors reduced nonspecific affinity and less efficient positionally
correlated search (19, 20), results in elevation of both
kcat and Km.
Previous work has shown that the endonuclease supports processive
cleavage of EcoRI sites separated by 50-330 base pairs, with the efficiency of processive action decreasing from about 80% at
an ionic strength of 0.06 M to about 30% at 0.13 M (20), and we have confirmed these findings. However,
processive action has not been detected at ionic strengths of 0.18 M or higher (Table IV and Ref. 20). Nevertheless, the
results described here indicate that product release is rate-limiting
for turnover at ionic strengths of 0.05-0.18 M, and
largely rate determining at 0.23 M (Table I). Despite the
reduction in processive action observed with increases in salt
concentration, the latter findings indicate that the enzyme rarely
dissociates from the cleaved EcoRI site directly into
solution, but rather diffuses within the domain of a polynucleotide
product prior to release, even at high ionic strengths
(kPN kP, Fig.
1A). The reduced processivity at the higher salt
concentrations can be explained in several ways. In order for
processive action to occur, the DNA segment spanning the
EcoRI sites in question must be subject to efficient
sequence sampling, e.g. by positionally correlated
diffusion, and this must be coupled with productive recognition of an
EcoRI sequence upon encounter by the enzyme. A reduction in
the efficiency of EcoRI site recognition at the higher ionic
strengths may account for the failure to detect processive action of
the enzyme under these conditions. A second explanation for reduced
processivity under conditions where the endonuclease remains associated
with the DNA product is based on the potential involvement of a
distinct mode of sequence sampling at high ionic strength. In contrast to positionally correlated microscopic dissociation-reassociation (sliding), the intersegment transfer (macroscopic
dissociation-reassociation) mode of facilitated diffusion invokes
ligand transfer between distal DNA sites that happen to be in proximity
due to the flexibility of large DNA chains (34). Thus, reduction in the
DNA segment size that is subject to positionally correlated search
coupled with intersegment transfer of the endonuclease within the
domain of a DNA product could account for the reduction in processive behavior observed at high ionic strength under conditions where the
endonuclease nevertheless remains associated with a cleavage product.
Equations relating the steady-state kinetic parameters
Km and kcat to the rate
constants shown in the reduced mechanism of Fig. 1B can be
calculated as shown by Equations 2 and 3.
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(Eq. 2)
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(Eq. 3)
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The ratio kcat/Km is
given by Equation 4.
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(Eq. 4)
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We have used Equation 3 to compare measured values of
kcat to the first order rate constant governing
product release, k4, at several ionic strengths
using experimental values for kcat, k2, and k3 (Fig. 3 and
Table I). Measured values of kcat are 1.0-1.1
times k4 at ionic strengths of 0.18 M or less, and 1.27 times k4 at an
ionic strength of 0.23 M, consistent with the conclusion above that dissociation of the endonuclease from the DNA product is
rate-limiting for turnover. These values for k4,
experimental values for k2 and
k3, (Table I), and published values for
k1 and k1 (7, 19, 28)
allow calculation of Km according to Equation 2. For
these calculations, k1 and
k1 were corrected for the effects of 5 mM MgCl2 (k1/10 and
k1 × 3) based on the effects of the divalent
cation on the kinetics of association and dissociation of a mutant form
of the endonuclease that retains recognition function but is defective
in strand cleavage (15). Table V shows
that these calculated values (designated Km') are
within a factor of 4 of the measured Km values over
the ionic strength range of 0.059 to 0.18 M. Table V also
compares kcat/Km values
calculated in a similar manner according to Equation 4. These values
(kcat/Km') are within factors
of 2-5 of measured values. The close agreement of calculated and
experimental values for Km and
kcat/Km strongly suggests
that the kinetic scheme depicted in Fig. 1B is a valid
description of the EcoRI endonuclease kinetic mechanism.
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Table V
Measured and calculated parameters governing steady-state cleavage by
EcoRI endonuclease
Measured kcat/KM and
KM values are from Fig. 3. Values for
KM' and
kcat/KM' were calculated
according to Equations 2 and 4. Values of k1 and
k1 used in these calculations were from Refs. 7,
19, and 28 after correction for effects of 5 mM
MgCl2 (15). Values for KM' and
kcat/KM' were not calculated for
[NaCl] of 0.20 M since k1 and
k1 have not been determined at this salt
concentration.
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TOP
ABSTRACT
INTRODUCTION
Present address: Becton Dickinson Technologies, 21 Davis Dr.,
Research Triangle Park, NC 27709.
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed. Tel.: 919-684-2775; Fax: 919-681-7874; E-mail: modrich@biochem.duke.edu.
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