From the Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803
Received for publication, August 8, 2002, and in revised form, November 21, 2002
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ABSTRACT |
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DNA binding properties of the Type 1 DNA
polymerases from Thermus aquaticus (Taq,
Klentaq) and Escherichia coli (Klenow) have been examined
as a function of [KCl] and [MgCl2]. Full-length Taq and its Klentaq "large fragment" behave similarly
in all assays. The two different species of polymerases bind DNA with
sub-micromolar affinities in very different salt concentration ranges.
Consequently, at similar [KCl] the binding of Klenow is ~ 3 kcal/mol (150×) tighter than that of Taq/Klentaq to the
same DNA. Linkage analysis reveals a net release of 2-3 ions upon DNA
binding of Taq/Klentaq and 4-5 ions upon binding of
Klenow. DNA binding of Taq at a higher temperature
(60 °C) slightly decreases the ion release. Linkage analysis of
binding versus [MgCl2] reports the ultimate
release of ~1 Mg2+ ion upon complex formation. However,
the MgCl2 dependence for Klenow, but not Klentaq, shows two
distinct phases. In 10 mM EDTA, both polymerase species
still bind DNA, but their binding affinity is significantly diminished,
Klenow more than Klentaq. In summary, the two polymerase species, when
binding to identical DNA, differ substantially in their sensitivity to
the salt concentration range, bind with very different affinities when
compared under similar conditions, release different numbers of ions
upon binding, and differ in their interactions with divalent cations.
The large fragment domains of the Type 1 DNA polymerases from
Escherichia coli (Klenow) and Thermus aquaticus
(Klentaq) are remarkably similar in structure (see Fig.
1), despite the fact that Taq
polymerase functions at temperatures 40-60o higher than
E. coli Pol
I1/Klenow (1-5). A number of
laboratories have been exploring the basis for the structural stability
of extremophilic proteins (for a recent review, see Ref. 6). This study
focuses primarily on the functional characteristics of this homologous
mesophilic/thermophilic pair of polymerases in an effort to understand
the functional similarities and differences between the two
polymerases. Herein we focus on the initial functional step in DNA
replication; that is, the binding of the polymerase to DNA and the
dependence of DNA binding on salt.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
X-ray crystal structures of Klenow (Protein
Data Bank (PDB) code 1KLN (40)) and Klentaq (PDB code 4KTQ (41))
polymerases bound to DNA.
Taq polymerase is a single chain polypeptide with a
molecular mass of 94 kDa (1-4). Due to its use in PCR, Taq
polymerase is one of the most important biotechnological reagents in
use in the world today. The enzyme has both polymerase activity and 5'
nuclease activity and is a member of the Pol I polymerase family by
virtue of its similarity to E. coli Pol I DNA polymerase
(1-4). Like other thermophilic eubacterial DNA polymerases (5), but unlike E. coli Pol I, Taq polymerase lacks 3' 5' exonuclease activity (so called "proofreading activity") (1-4).
Removal of the 5' nuclease domain from full-length E. coli
Pol I (103 kDa) produces the 68-kDa Klenow fragment (7). Removal of the
same domain from Taq polymerase produces the 62.5-kDa
Klentaq fragment (8).
The non-covalent driving forces that lead to stable protein-DNA
complexes are strongly influenced by the solution environment (salt
concentration and type, temperature, pH, etc.). As a result of this
dependence on the solution conditions it is impossible to understand
the forces that drive these interactions based solely on structural
considerations. Rather, the functional properties (thermodynamics and
kinetics) of these interactions must also be investigated as a function
of solution conditions to understand the origins of the stability of
the complexes. Models and mechanistic explanations of function in the
Pol I polymerase family are often extrapolated from one family member
to another. These extrapolations are well justified given the
structural similarities among the family members as revealed by the
extensive series of recent co-crystal structures of different family
members (for review, see Refs. 9 and 10) as well as sequence
similarities among the active sites (9). Assays of function and
mutagenesis studies, however, often reveal subtle and not so subtle
differences among the family members (e.g. Refs. 11 and 12),
but directly comparative functional studies between different family
members are relatively scarce. In this study, we have characterized the
basic binding equilibria of full-length Taq, Klentaq, and
Klenow polymerases to DNA using a fluorescence anisotropy assay. We
have further characterized the KCl and MgCl2
dependencies of DNA binding by the different polymerases and
have quantitated the linked ion releases upon DNA binding for all three
polymerases. The results show that these two species of polymerase,
when binding to the same DNA, differ in their salt "sensitivities,"
their intrinsic affinities, their linked ion releases, and their need
for bound divalent cations.
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EXPERIMENTAL PROCEDURES |
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Proteins--
The proteins examined are full-length
Taq DNA polymerase, the Klentaq fragment of Taq
polymerase (8), and the D424A mutant of Klenow polymerase (13). The
Klenow D424A mutant lacks 3'-exonuclease activity and is commonly
referred to as "Klenow exo minus" (KF exo). It is the predominant
variant of Klenow polymerase used in the majority of functional studies
of Klenow over the past decade. The Taq DNA polymerase gene
was cloned, and the protein was expressed and purified in our
laboratory. The clone of Klentaq was constructed by Wayne Barnes at
Washington University (8) and was obtained from the American Type
Culture Collection (Manassas, VA). The clone for D424A Klenow fragment
was a gift from Catherine Joyce at Yale University.
The gene for Taq DNA polymerase was PCR-amplified using the strategy and primers described by Engelke (14). The amplified product was cloned into a pTrc99A expression plasmid (Amersham Biosciences) using EcoR1 and BamH1 restriction sites. The gene was then cut from this clone using NcoI and SalI restriction sites, re-ligated into a pET-15b expression plasmid (Novagen), and transformed into the BL21(DE3) strain of E. coli for expression and purification of the protein. The correct Taq coding sequence of the resulting plasmid (pKDTaq3) was verified by DNA sequencing. The protein purification procedure for Taq was based largely on the procedure described by Barnes (15) for Klentaq with a number of modifications, as follows. Cell pellets were solubilized in the lysis buffer (50 mM Tris-Cl, 10 mM MgCl2, 50 mM dextrose, 250 mM KCl, pH 7.9) with lysozyme and heated to 75 °C for 1 h. Contaminating nucleic acids were then removed by polyethyleneimine precipitation in the presence of 250 mM KCl. The supernatant containing the protein was loaded over a Bio-Rex 70 ion-exchange column, pre-equilibrated with KTA buffer (20 mM Tris, 22 mM (NH4)2SO4, 1 mM dithiothreitol, 0.1 mM EDTA, 10% glycerol, pH 7.9) to remove the excess polyethyleneimine. The flow-through was then loaded over a heparin-Sepharose column in the same buffer, and Taq polymerase was eluted with a 22-270 mM (NH4)2SO4 gradient. The Taq eluent was applied to a second Bio-Rex 70 column in KTA buffer, pH 8.8, and the flow-through contained the purified protein. Klentaq polymerase was purified as published (15) with the following modifications; no surfactant was used during purification, the ammonium sulfate precipitation step was omitted, and a second Bio-Rex 70 column, at pH 9.1, was added subsequent to the heparin column. The flow-through of the second Bio-Rex column contained the purified protein. Klenow exo minus (plasmid pXS106) was transformed into E. coli expression strain CJ376 and grown and induced as described previously (16). The protein was purified as previously described for full-length Pol I (17) with omission of the DEAE-cellulose column. No surfactant was used during purification or storage of any of the polymerases. Protein concentration was measured using the Bradford method (18).
DNA-- Stoichiometric and equilibrium DNA binding experiments were performed with the following primer-template sets: 13/20-mer, 5'-TCGCAGCCGTCCA-3' and 3'-AGCGTCGGCAGGTTCCCAAA-5', and 63/70-mer, 5'-TACGCAGCGTACATGCTCGTGACTGGGATAACCGTGCCGTTTGCCGACTTTCGCAGCCGTCCA-3' and 3'-ATGCGT- CGCATGTACGAGCACTGACCCTATTGGCACGGCAAACGGCTGAA- AGCGTCGGCAGGTTCCCAAA-5'.
The 13/20-mer primer template set used is the same as that used for kinetic studies of Klenow DNA binding by Benkovic and co-workers (19). The longer primer-template pair (63/70-mer) was designed for use at higher temperatures. The 63/70-mer uses the same putative binding region sequence as the 13/20-mer, with added "random" sequence selected from the same region of the M13 bacteriophage sequence. DNA oligonucleotides were purchased from Integrated DNA Technologies, Inc. Fluorescently labeled DNA was labeled at the 5' end of the primer strand with rhodamine-X (ROX) and was purchased directly from Integrated DNA Technologies.
Fluorescence Anisotropy Assay-- ROX-labeled DNA was titrated with increasing concentrations of protein, and binding was monitored using the anisotropy signal change as protein-DNA complex is formed (20, 21). Fluorescence anisotropy measurements were performed using a FluoroMax-2 fluorometer equipped with an automated polarizer and regulated at the indicated temperatures. The excitation and emission wavelengths were 583 and 605 nm respectively, with 8-nm band-pass and an integration time of 10 s. For all the equilibrium titrations the DNA concentration used was 1 nM. In all the experiments the protein was titrated into fluorescently labeled DNA, with the total [DNA] < Kd. After each addition the sample was equilibrated at the required temperature for 8 min, and anisotropy was measured. All titrations were performed in 10 mM Tris, pH 7.9, buffer at the indicated salt (KCl and MgCl2) concentrations and temperature. The pH was adjusted by mixing Tris base and Tris-HCl. KCl and MgCl2 concentrations were varied across the widest possible range for each polymerase. Limiting titrations at low KCl concentrations were those where the Kd approached the total [DNA]. Limiting values at high KCl concentrations were the point where well behaved, reproducible isotherms could no longer be obtained. For assays examining the effects of EDTA on binding, the titrations were carried out in 10 mM Tris, 10 mM EDTA, pH 7.9, with KCl concentrations of 50 mM for Klentaq and 300 mM for Klenow. Before measurement of the binding in the presence of EDTA the proteins were extensively dialyzed against this same buffer.
Data Analysis-- Stoichiometric binding curves were fit to the equation,
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(Eq. 1) |
Equilibrium binding curves were fit both to the equation above and to a
simplified standard single site isotherm usable when the [DNA]
Kd, and which thus assumes effective equality between the free polymerase concentration and the total polymerase concentration,
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(Eq. 2) |
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(Eq. 3) |
Thus, the slope of a plot of ln 1/Kd
versus ln [KCl] will be equivalent to the net number of
ions (nions) that are bound or released when
the protein-DNA complex is formed, where
nions is the net sum of the binding and
release of both the anions and cations (
nK+ +
nCl
) (24-26).
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RESULTS |
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To obtain adequate amounts of Taq polymerase to perform biophysical studies, we recloned the gene for Taq polymerase into a overexpressing pET vector (Novagen) using modification of the strategy and amplification primers originally described by Engelke et al. (14). There have been a number of previously published or patented descriptions of the purification of Taq DNA polymerase (1-4, 14, 15, 27, 28, 29-34). For routine use in PCR, full purification of the polymerase is not required, and the majority of the published or patented descriptions of Taq purification are aimed at producing Taq for use in PCR. Our procedures have been designed for producing Taq for use in biochemical and biophysical studies. As such, no additional tagged sequences (such as His tags) have been added to the polymerase. Also, no surfactants have been used in the purification, since their effects on the polymerase remain uncharacterized. In addition, we assayed for potential damaging effects of the ubiquitous 70 °C heat incubation step included in published and patented Taq purification procedures. Full purification of the polymerase without the heat step was performed; however, this "cold prep" polymerase behaved identically in all of our assays as the normal "heat-prepared" polymerase (data not shown), and so the heat incubation step was retained in the final purification protocol.
Binding Stoichiometry of the Polymerases--
Stoichiometric
titrations of each of the polymerases versus DNA were
performed using fluorescence anisotropy, and the results are shown in
Fig. 2 for Taq polymerase.
Polymerases were titrated into ROX-labeled DNA at high concentrations
of DNA ([DNA] Kd). The anisotropy of the DNA
increases as protein binds due to the decreasing rate of molecular
rotation of the DNA in the complex. The stoichiometry of binding was
determined by fitting the data to Equation 1 as described under
"Experimental Procedures." The ratio of bound protein to DNA at
saturation is 0.97 for Taq, 1.04 for Klentaq, and 0.82-0.95
for Klenow binding to the 13/20-mer primer-template pair. The binding
stoichiometry to the longer 63/70-mer primer-template pair was 1.1 for
Taq and 1.15 for Klentaq. As a control, it was determined
whether unlabeled DNA could effectively compete with fluorescently
labeled DNA for binding to the polymerase. The second curve in the
stoichiometric titration plots of Fig. 2 shows a stoichiometric
titration of protein versus double the amount of DNA, where
half the DNA is ROX-labeled, and half is not. Only the fluorescently
labeled DNA is anisotropically visible, so the displacement and
apparent doubling of the stoichiometric value for this second curve
indicates approximately equally effective binding of both the labeled
and unlabeled DNA to the polymerases. Unlabeled DNA effectively
competed with ROX-labeled DNA binding to all three polymerases. It
should be noted that DNA labeled with fluorescein, which was examined
first, is not effectively competed by unlabeled DNA for binding (data
not shown), underscoring the importance of the unlabeled competitive
control.
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KCl Dependence of DNA Binding--
Fig.
3 shows representative fluorescence
anisotropic titrations of the binding of Klenow and Klentaq polymerases
to identical pieces of DNA at several different KCl concentrations at
25 °C. Each titration curve fits well to a single site binding
isotherm, and it can be seen from the precision of the data that even
modest shifts in Kd can be readily quantitated. Fig.
4 shows the thermodynamic linkage plots
for DNA binding as a function of KCl concentration
(ln1/Kd versus
ln[salt]) for the polymerases in the presence and absence of 5 mM
MgCl2. The negative slopes of the linkage plots are
indicative of net ion release upon formation of the protein-DNA complex
(23-26). As observed for most DNA-binding proteins, DNA binding is
linked to ion release for all the polymerases. Full-length
Taq polymerase and the Klentaq fragment behave essentially
identically. The exact linked ion releases are reported in Table
I.
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Several points are notable about these data. The most striking
observation is the significant difference in salt ranges where sub-micromolar binding occurs. DNA binding across the ~10-300 nM Kd range occurs at KCl concentrations
about an order of magnitude higher for Klenow than for
Taq/Klentaq. This means that under similar solution
conditions the binding of Klenow to DNA is much tighter than that of
Taq/Klentaq. Both species of polymerase were titrated over
the widest possible KCl concentration ranges that produced acceptable
and analyzable titration curves. Although the two [KCl] ranges do not
overlap, by extrapolation the binding of Klenow is, on average, about 3 kcal/mol (~150×) tighter than the binding of Taq/Klentaq
to the same DNA at similar salt concentrations. This difference is also
reflected in the extrapolation of the linkage plots to 1 M
salt (ln[KCl] = 0), which provides an estimate of the
non-electrostatic components of the binding interaction (24-26). At 1 M salt the binding free energy for Klenow to DNA is ~2
kcal/mol tighter than the Gbinding of
Taq/Klentaq to the same DNA (Table I).
In addition to the intrinsic affinity difference between the E. coli and Taq polymerases, the slopes of the linkage plots with KCl indicate that the binding of Klenow to DNA results in the release of 1.5 more ions than the binding of Taq/Klentaq to the same DNA both in the presence and absence of MgCl2. This is an ~50% increase in the linked ion release for Klenow relative to Taq/Klentaq. For both polymerases, removal of MgCl2 from the buffer increases the reported linked ion release by about 0.6 ions, indicating that part of the ion release in the presence of MgCl2 is due to magnesium ion release.
MgCl2 Dependence of DNA Binding--
Binding
titrations were also performed as a function of MgCl2 at
fixed KCl concentrations for Klentaq and Klenow polymerases. Fig.
5 shows the individual titration curves
at different MgCl2 concentrations for the two polymerases
as well as the thermodynamic linkage plots for binding
versus [MgCl2]. It is clear that binding of
Klentaq is linked to the release of Mg2+ across the entire
[MgCl2] range, but the [MgCl2] dependence
of Klenow DNA binding is not monotonic. The MgCl2 linkage
plot for Klentaq (Fig. 5C) indicates a linked
Mg2+ release of 0.9 ions. For Klenow, on the other hand,
the MgCl2 linkage plot and inspection of the titration
curves themselves (Fig. 5B) both show the absence of a
linked Mg2+ release (rather, a small uptake) upon binding
of Klenow to DNA up until 10 mM MgCl2, whereas
above 10 mM MgCl2 there is a net release of 1.2 Mg2+ upon binding (Table
II).
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To further investigate the Mg2+ requirements for DNA
binding by the two polymerases, we EDTA-treated Klentaq and Klenow to
remove all excess and weakly bound Mg2+ and then assayed
DNA binding affinity in the presence of EDTA. In both cases, EDTA
significantly decreases the affinity of the polymerases for DNA, as
shown in Fig. 6. This effect is slightly more dramatic for Klenow than for Klentaq (13× reduction of affinity for Klenow versus a 5.5-fold reduction of affinity for
Klentaq). Surprisingly, however, removal of EDTA almost completely
restores the original binding affinity of the polymerases (data not
shown). These data indicate that free Mg2+ is not required
for DNA binding by either polymerase but suggest that some
protein-bound divalent cations are required for highest affinity
binding by both species of polymerase (see "Discussion").
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KCl Dependence of Taq Binding at High Temperature--
T.
aquaticus lives at a temperature optimum of 70-75 °C (35).
Because it is possible that increased temperature may alter the way
Taq polymerase binds DNA, we also assayed for salt
dependence of binding at higher temperatures. Binding at 60 °C
required design and use of a longer DNA primer-template pair
(63/70-mer), since the shorter 13/20-mer primer-template had a
Tm of ~55 °C. Fig.
7 shows that the salt dependence of
binding of Taq polymerase to DNA at the 2 temperatures is
similar but slightly decreased at 60 °C relative to 25 °C.
Kd values are reported in Table
3. Fig. 7 also shows the KCl dependence
for the longer 63/70-mer DNA at 25 °C to account for any possible
changes due to the differences between the two lengths of DNA. At
25 °C, the binding affinity of Taq for the 63/70-mer is
somewhat tighter (~3-4 × or ~1 kcal/mol) than the binding
affinity for the 13/20-mer, but the ln1/Kd versus
ln[KCl] dependencies are the same. It is also
interesting to note that Taq binds the 63/70-mer DNA with
somewhat higher affinity at 25 °C than at 60 °C. Thermophilic
proteins are generally almost catalytically inactive at room
temperature, and this is also the case for Taq, which has
been shown to have little or no polymerization activity below about
40 °C (4). Taq and Klentaq bind DNA quite happily at
25 °C, however, and even lower. A complete characterization of the
temperature dependencies of binding for these polymerases is currently
in progress in our laboratory.
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DISCUSSION |
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KCl Dependence of DNA Binding by the Polymerases--
The linked
ion releases for binding of the two different species of polymerases to
DNA are both relatively small compared with the range of ion releases
generally reported for DNA-binding proteins (24-26, 36, 37). Some
similar small ion releases that have been reported include the release
of 3 ions upon binding of Sso7d to DNA (38) and a 1-2 ion release upon
binding of the -subunit of E. coli RNA polymerase to DNA
(39). Although the ion release is small for both species of polymerase,
the binding of Klenow polymerase results in a larger release of ions
than the binding of Klentaq or Taq polymerase. On the scale
of the overall ion release for the two polymerases, this difference
between Taq/Klentaq and Klenow is somewhat significant and
is indicative of differences in the way the two polymerases interface
with the DNA. Even though both species of polymerase bind to the same
DNA with ~1:1 stoichiometry, their different linked ion releases
suggest that they bind with different footprints. Below, we discuss
this in light of the available crystal structures of the binary
complexes of the polymerases bound to primer-template DNA (40, 41).
The ion release for Taq polymerase at 60 °C is slightly lower than the ion release at 25 °C, further distancing the salt-dependent behaviors of the two species of polymerase when they are compared at temperatures near their respective optimal growth temperatures. It may be possible that the temperature dependence of the ion release for Taq is more complex than is revealed by looking at only these two temperatures and that we have fortuitously chosen two temperatures for Taq where the ion releases are similar. There may exist specific separate solution conditions under which Klentaq and Klenow polymerases release the same number of ions upon DNA binding, but under the identical solution conditions examined herein, they do not.
Table I also shows that the ln1/Kd
versus
ln[KCl] linkage plots for Taq and
Klentaq have identical slopes. The Taq and Klentaq
ln1/Kd versus
ln[KCl]
dependencies, shown in Figs. 4 and 7, are nearly co-linear if plotted
on the same plot. This finding suggests that in both cases only the
polymerase domain of the protein is binding DNA, i.e. that
the 5'-nuclease domain of full-length Taq does not bind DNA
under these conditions. If both the 5'-nuclease domain and the
polymerase domain of the full-length enzyme bound the DNA, one would
expect the full-length protein to interact with a larger amount of the
DNA and, hence, exhibit a different ion release.
Intrinsic DNA Binding Affinity and Salt Tolerance Differences between the Polymerases-- One of the most striking differences between the DNA binding properties of Klenow versus Taq/Klentaq polymerases is the shift in salt ranges for sub-micromolar binding. A consequence of this difference is that at any particular salt concentration, the relative affinity of Klenow polymerase for DNA is significantly tighter than that of Taq/Klentaq. This is further suggested by the estimated difference in non-electrostatic binding free energies for the polymerases, estimated from the extrapolation of the KCl dependence data to 1 M salt. Both of these lines of evidence indicate that Klenow binds DNA about 3 kcal/mol tighter than Taq/Klentaq DNA. The data at high temperatures for Taq polymerase show that this large affinity difference is not due to the fact that we are assaying the binding of Taq/Klentaq to DNA at 25 °C. This large difference in relative binding affinities for the two different species of polymerase, like the difference in linked ion release, further suggests differences in their initial DNA binding modes.
It should be noted that the expression clones for Taq versus Klentaq polymerases in this study are from completely different sources, with no overlap of materials or design. The clone for full-length Taq was produced in our laboratory, whereas that for Klentaq was produced by Wayne Barnes (8, 15). The fact that the clones from the two independent sources produce Taq and Klentaq proteins that behave almost identically indicates that the decreased DNA binding affinity of Taq/Klentaq is an intrinsic property of the polymerase and is not the result of some sort of unintentionally introduced mutation. Furthermore, we examined the possibility that the heating step, included in almost all published Taq/Klentaq purifications, might somehow have partially damaged the protein. However, Taq polymerase purified without the heating step behaves identically to the protein purified with the heating step.
It is currently not clear why the E. coli Klenow polymerase binds DNA with so much higher affinity than the equivalent polymerase from T. aquaticus. Another way to look at the data of Fig. 4 is to note that the two species of polymerase bind DNA with similar affinities in very different salt concentration ranges. However, neither E. coli nor T. aquaticus are halophilic. In fact neither will grow well in media containing >1% NaCl (42, 43). How sensitive their growth and viability are in the 0-1% salt range has not been characterized, however, so it is possible that there are subtle differences in the salt sensitivities of the two bacteria that might correlate with the salt sensitivity of their Pol I-type polymerases. Several other potential explanations for the observed differences may be postulated. 1) There may be other factors in the intracellular environments of the two bacteria that either lower the affinity of the E. coli enzyme or increase the affinity of the Taq enzyme in vivo or both (i.e. allosteric regulators, specific anions, other proteins, etc.). Specific anions have been shown to regulate DNA binding affinity in several systems (25, 26, 44). 2) High temperature stability of Taq/Klentaq may be achieved at the cost of the loss of high affinity DNA binding. 3) The two polymerases might show significant differences in sequence specificity, e.g. Taq might bind GC-rich sequences (more common in thermostable bacteria) tighter than mixed DNA sequences. 4) The operational or tolerable functional affinity range for Pol I-type polymerases may be quite wide in vivo, such that any affinity in the nanomolar to picomolar range is adequate for appropriate function or easily compensated for in vivo by increased/decreased expression of the particular polymerase. 5) These Pol I type polymerases from E. coli and T. aquaticus, although widely considered homologues, may not be completely functionally equivalent in the two bacteria, and therefore, might not be expected to have similar DNA affinities. 6) DNA may simply bind to the two polymerases in different ways. This point requires expansion. It has long been known that Taq DNA polymerase is devoid of 3'-exonuclease (or proofreading) activity. The proofreading activity of Klenow polymerase, on the other hand, is so efficient that it interferes with direct binding and kinetics studies, such that Klenow mutants with reduced or eliminated exonuclease activity have been used in almost all such studies for more than a decade. Removal of the exonuclease catalytic activity does not, however, necessarily abolish DNA binding to the exonuclease site. In fact the co-crystal of Klenow bound to primer-template DNA utilizes one of these exonuclease deficient mutants, yet unexpectedly shows the DNA bound in a so called "editing mode," which includes contacts in the exonuclease domain, the edge of the polymerase domain cleft, and a second cleft that is formed upon DNA binding and runs roughly between the two active sites and roughly perpendicular to the polymerase domain cleft (40). Co-crystal structures of DNA bound to Klentaq show the DNA bound further up into the polymerase active site cleft (although it does not pass all the way through the cleft) (41). The position of the DNA in the crystal structure of the binary complex of full-length Taq and DNA (45) is similar to that for Klentaq. The binary complexes for Klenow and Klentaq are the structures that are shown in Fig. 1. It may be that as 3' exonuclease activity evolved, the strength of the interaction of the DNA with the exonuclease site shifted the location of the initial binding site for DNA. There are a billion years of evolutionary distance between the two bacterial families (46). This hypothesis of two different initial binding sites would be consistent with the available Klenow co-crystal structure (and the two-site shuttling model proposed for movement of the DNA between sites) (40), consistent with electrostatic calculations that were performed on the Klenow structural data (47) and consistent with the significantly higher binding affinity of Klenow for DNA versus Klentaq and the larger linked ion release upon binding by Klenow relative to Klentaq. Further studies are required for elucidation of which, if any, of these possibilities can explain the binding and salt sensitivity differences between the two species of polymerases.
MgCl2 Dependence of DNA Binding by the Polymerases-- The MgCl2 concentration ranges showing linked ion releases for Klentaq and Klenow are, like the KCl concentration ranges, separated by about an order of magnitude. Klentaq DNA binding releases 0.9 net Mg2+ ions in the 0-10 mM [MgCl2] range, whereas Klenow DNA binding releases 1.2 net Mg2+ ions in the 10-50 mM [MgCl2] range. As with the majority of DNA binding proteins assayed to date, the Mg2+ linkages are lower than the monovalent ion linkages. For many DNA-binding proteins, the linked ion release in MgCl2 is half that in monovalent salts, indicative of cation-specific effects (24-26). Deviations from this ratio can be an indication of specific ion effects. The fact that the net Mg2+ releases for Klentaq and Klenow are lower than half the K+ release is likely due to the fact that the MgCl2 dependencies were (by necessity) determined in the presence of KCl (24-26). Because the net ion release is much lower for Mg2+, the statistical significance of the difference between the values for the two polymerases is less reliable. It is interesting to note, however, that similar to the findings with KCl, Klenow DNA binding is linked to the release of more Mg2+ than Klentaq DNA binding (about 30% more). Thus, like the results with KCl, the linked ion releases of Mg2+ upon DNA binding and the significant shift in the salt ranges for similar affinity binding of the two polymerases both suggest different binding conformations or binding footprints for the two polymerases in their respective protein-DNA complexes.
Binding of both species of polymerase to DNA is linked to the release
of Mg2+ at higher MgCl2 concentrations, but
both species of polymerase appear to require protein-bound divalent
ions to achieve their highest DNA binding affinity. Treatment of the
polymerases with EDTA lowers the DNA binding affinity of Klentaq and
Klenow by ~6× (G = 1.0 kcal/mol) and ~13×
(
G = 1.53 kcal/mol), respectively. Removal of
EDTA, however, almost completely restores the binding affinity
originally observed in the absence of EDTA. This suggests that EDTA
interacts with metals on the protein surface and either competitively
(in the binding site) or allosterically diminishes DNA binding affinity
but does not remove the required metals from the protein at least at
the concentration used in this experiment. Because EDTA also chelates
other divalent cations, from this experiment alone it is not conclusive
that the required ion is Mg2+. Bound zinc has been reported
for both species of polymerase (48, 49). Both species of polymerase are
known to require Mg2+ or Mn2+ for catalytic
activity (1, 50, 51). E. coli Pol I has been reported to
bind up to 21 magnesium and/or manganese ions using electron
paramagnetic resonance (52).
Conclusion--
We have examined the DNA binding properties of the
Type 1 DNA polymerases from E. coli and T. aquaticus as a function of KCl and MgCl2. The results
of this study show that these two species of polymerase, when binding
to the same DNA, 1) bind with very different affinities under similar
solution and salt concentration conditions, 2) differ substantially in
their sensitivity to the salt concentration range for binding, 3)
release different numbers of K+ and Mg2+ ions
upon formation of the protein-DNA complex, and 4) differ in their
requirements for protein bound divalent cations. It is not yet
determined which, if any, of these functional differences are
correlated with the ability of Taq/Klentaq polymerase to
function at such dramatically higher temperatures than Klenow.
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ACKNOWLEDGEMENTS |
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We thank Carmen Ruiz for excellent technical support. We thank Catherine Joyce for the gift of the expression clone and host strain for Klenow. We thank Joe Beechem for the suggestion to use ROX, and we thank Tomasz Heyduk for technical advice on the anisotropy measurements.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant MCB 9904680.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 225-578-5233;
Fax: 225-578-2597; Email: licata@lsu.edu.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M208133200
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ABBREVIATIONS |
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The abbreviations used are: Pol I, polymerase I; ROX, rhodamine X.
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