From the Laboratory of Structural Biology, NIEHS,
National Institutes of Health, Research Triangle Park, North
Carolina 27709 and the ¶ Laboratory of Biophysical Chemistry,
NHLBI, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, August 19, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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DNA polymerase (pol) DNA polymerase (pol)1
Because of its small size, lack of accessory proteins, and excellent
expression properties, pol Comparison of the structure of pol DNA polymerases are more than nucleotidyl transferase catalytic
centers. Some DNA polymerases are processive and remain bound to the
DNA substrate as they move along the template strand synthesizing DNA.
DNA polymerase We remain cognizant that we are using dynamics measured in hundreds of
picoseconds to a few nanoseconds to study cycles requiring at least
hundreds of milliseconds; nevertheless, nanosecond flexibility is
clearly relevant to an understanding of the conformational events in
that cycle. Gangal et al. (22) have shown that the 1-3-ns
rotational correlation times of fluorescein conjugated to engineered
surface cysteines correlate strongly with the long-term structural
disorder reported by crystallographic B-factors for the adjacent
backbone elements. The measurement of segmental flexibility has a long
history (see Ref. 23); in particular, emission anisotropy has a long
history of revealing local motions (24, 25). In addition, time-resolved
methods have the advantage of quantifying both the angular extent and
the rate of these motions (24, 26, 27). In favorable cases, the fast
motion of a segment on a loose "hinge" can be interpreted
both in terms of the size of the independent segment and the angular
range of facile hinging. Even in flexible coiled polymers, the fast
term presents a measure of granularity and offers insight into the
persistence of rigid segments (28). In all, nanosecond flexibility
appears to be an important predictor of functional flexibility.
Wild-type pol Materials--
Wild-type and mutant derivatives of human
recombinant pol
A 24-mer DNA substrate was prepared by annealing three high pressure
liquid chromatography-purified oligonucleotides (Oligos Etc.,
Wilsonville, OR) to create a single-nucleotide gap at position 13. Each
oligonucleotide was resuspended in 50 mM Tris-HCl, pH 7.4, and 1 mM EDTA, and the concentration was determined from their UV absorbance at 260 nm. The annealing reactions were carried out
by incubating a solution of 10 µM primer with 11 µM of both the downstream and template oligonucleotides
at 90 °C for 2 min followed by slow cooling to room temperature. The
sequence of the gapped DNA substrate was as follows: upstream 12-mer
primer, 5'-CCGCTGATGCGC-3'; downstream 11-mer oligonucleotide,
5'-GTCGGTGGGCC-3'; 24-mer template, 5'-GGCCCACCGACAGCGCATCAGCGG-3'. The
3'-end of the upstream primer was synthesized without a 3'-hydroxyl so
it would not be extended with the addition of pol Absorption and Steady-State Fluorescence--
Absorption spectra
were recorded on a Hewlett Packard 8453 UV-VIS spectrophotometer (Palo
Alto, CA), and fluorescence emission spectra were obtained on an SLM
AB-2 (Thermo Spectronics, Rochester, NY) or SLM8000 (SLM Instruments,
Urbana, IL) spectrofluorometer. The protein concentration was between
2.5 and 8 µM, and the sample absorbance at 295 nm was
<0.1 with 3-mm path length. Excitation and emission monochromators
were set at 4-nm band pass. Emission spectra were collected with
excitation polarizer at 54.7° and emission polarizer at 0° to the
vertical, and background fluorescence from a solvent blank was
subtracted. Steady-state anisotropies were measured using the L-format
method with Glan-Thomson calcite prism polarizers. Anisotropy
(A) was calculated from the intensity of the vertical
(Iv) and horizontal (Ih)
measurements with a grating correction factor (G):
A = (Iv - GIh)/(Iv + 2GIh). G was calculated from the
ratio of intensities
(Iv/Ih) when the
excitation light was polarized horizontally. Stern-Volmer quenching
experiments were carried out as described previously (29). Temperature
was controlled at 20 °C with a circulating water bath.
Circular Dichroism--
Circular dichroism spectra were measured
using an Applied Photophysics PiStar-180 spectrometer using a 0.1-cm
cell thermostated at 20 °C. All measurements were corrected for
background signal.
Time-resolved Fluorescence Decay--
Measurements were
performed in 50 mM Tris-HCl, pH 7.4, and 0.1 M
KCl at 20 °C with a protein concentration of 4.7 to 9.4 µM. To avoid tyrosine absorbance, samples were excited at
295 nm, and the absorbance at 280 nm was 0.1-0.2. The fluorescence
intensity profiles were measured by the time-correlated single-photon
counting technique as described (28). Time per channel was 85 ps, and 512 channels were recorded. Samples were excited at 295 nm using a
synchronously pumped, cavity-dumped, frequency-doubled dye laser as
excitation source, with a repetition rate of 4 MHz, a pulse width of 5 ps, and an average UV power under 200 microwatts. The Hamamatsu R2809
MCP photomultiplier operated with a time spread (half-width) of about
90 ps, allowing one to resolve fluorescence lifetimes and/or
correlation times as short as 30 ps. Intensity decays were analyzed by
sum of two or more exponential functions, as shown in Equation 1.
For fluorescence anisotropy decay measurements, protein fluorescence at
340 nm was monitored through a film polarizer oriented parallel
IVV(t) and/or perpendicular
IVH(t) to the vertical excitation polarization and alternatively recorded. Data were collected up to
20,000 counts in the maximum channel at 20 °C. For each sample, sixteen fluorescence intensity decays were obtained contemporaneously and summed to generate the anisotropy decay,
r(t), as follows in Equation 2.
The fluorescence data were analyzed by the "sum and
difference" method described previously (34). The general
procedure was to fit the experimental anisotropy decays to the sum of
two exponential functions, shown in Equation 3, where
Site-specific Introduction of Tryptophan in pol
For study of the 8-kDa lyase domain, we substituted tryptophan for
Phe-25 (i.e. F25W/W325A; from this point on, referred to simply as F25W). The amino-terminal lyase domain possesses lyase, single-stranded DNA binding, and 5'-phosphate binding activities (1).
Several of the key residues for these activities have been
characterized (36-39). Trp-25 is located on the first helix of the
protein ( Characterization of Polymerase Conformation and Catalytic
Activity--
The purity of the recombinant proteins was accessed by
SDS-PAGE and found to be greater than 99% (Fig.
2, inset). To access the
effect of the mutations on the protein conformation, we measured circular dichroism spectra for wild-type, F25W, and L287W proteins (Fig. 2). The spectra indicate that the mutations had no effect on
overall protein folding. The catalytic activity of wild-type enzyme and
the mutant derivatives were assessed on a single-nucleotide gapped DNA
substrate utilizing Mg2+ as the divalent cation. The single
(W325A) or double mutants (F25W/W325A and L287W/W325A) exhibited
minimal effects on catalytic activity; kcat for
wild-type enzyme was 0.22 ± 0.05 compared with 0.20 ± 0.06, 0.24 ± 0.06, and 0.13 ± 0.02 for W325A, F25W, and L287W,
respectively. The DNA binding affinity of the mutant proteins for
gapped DNA substrates was also not affected by the mutation as assessed
by analytical ultracentrifugation (data not shown).
Steady-State Fluorescence of Wild-type and Mutant
Proteins--
The fluorescence emission spectra for F25W, L287W, and
wild-type proteins were measured in the absence and presence of
substrates. The emission spectra for F25W, L287W, and wild-type
proteins are shown in Fig. 3A.
The emission maximum for F25W was ~340 nm, whereas it was 348 nm for
the L287W and wild-type proteins indicating solvent-exposed tryptophan
microenvironments in these later instances. For F25W, the emission
maximum suggests that Trp-25 is less exposed to solvent as compared
with other tryptophans. The relative integrated intensities of F25W and
L287W were about 2- and 3-fold weaker than that of Trp-325 in wild-type
pol
The effects of Mg2+ and DNA binding on steady-state
fluorescence were also determined. The binding of 10 mM
Mg2+ resulted in a minimal fluorescence decrease for
wild-type (2.7%; see Fig. 3B) and L287W proteins (7.8%;
see Fig. 3D). In contrast, Mg2+ binding to F25W
resulted in a significant decrease of fluorescence intensity (24.8%;
see Fig. 3C). The fluorescence quenching detected with F25W
suggests metal-induced structural changes around Trp-25. Metal soaking
experiments with pol Stern-Volmer Quenching of Wild-type and Mutant
Proteins--
Effects of substrate binding on the microenvironment of
the tryptophan residues were probed by collisional quenching
experiments using acrylamide as a quencher (see Table
I and Fig.
4). In Fig. 4A, Stern-Volmer
plots for F25W, L287W, and wild-type proteins in the absence and
presence of Mg2+, single-nucleotide gapped DNA, and dTTP
are compared. The data, in the absence of substrates, were linear
yielding Stern-Volmer constants (Ksv) of
17.34 ± 0.24 M
For Trp-287, there was ~30% reduction in Ksv
without a change in the emission maximum. The smaller
Ksv is consistent with the structural location
of residue 287 near the template backbone in the closed liganded
ternary complex, whereas it is solvent-exposed in the absence of
substrates (open complex). A tryptophan modeled at this position
suggests it would be situated 5-6 Å from the template backbone. The
relatively small reduction of Ksv for wild-type enzyme is also consistent with the location of its lone tryptophan (Trp-325) on the outer surface of the N-subdomain near the carboxyl terminus. However, the 14.7% decrease of Ksv
suggests that substrate binding can influence the accessibility of
Trp-325, even though this residue is located on the side of the
N-subdomain away from the active site. In the presence of substrates,
the relative accessibility of the tryptophans probes is in the order of
Trp-25 < Trp-325 < Trp-287. Thus, the most prominent
protection from the quenching agent was observed for Trp-25 in the F25W
mutant. Even though substrates yielded a decrease in
Ksv, the wild-type (Trp-325) and L287W
tryptophan environments appear to still be highly accessible to the
quencher (Ksv of 13.91 and 14.74 M
Because of the sensitive location of the tryptophan introduced on
In Table I, the bimolecular quenching rate constants
(kq) were calculated from the mean fluorescence
lifetimes ( Time-resolved Fluorescence Decay--
The tryptophan fluorescence
lifetime decays of wild-type and mutant proteins were measured in the
presence and absence of substrates (Table II). The fluorescence
lifetimes of wild-type and L287W can be well represented by the sum of
two exponential decays (reduced
Binding of substrates to wild-type and mutant proteins had a minimal
effect on their decay parameters. For example, there were less than 7 and 5% decreases in the mean lifetimes of F25W and L287W,
respectively, in the presence of all substrates (Table II). This is in
contrast to the 15 and 36% substrate-induced quenching observed in the
steady state for F25W and L287W, respectively (Fig. 3). The wild-type
enzyme exhibited the smallest change (<2%) in the mean lifetime
induced by substrate binding (Table II). Hence, the steady-state
intensity losses are predominantly static. The decay-associated spectra
for L287W in the absence of substrates are shown in Fig.
5. The two lifetime components of L287W
are in solvent-exposed environments based on the emission maximum (350 nm). The longer lifetime component contributes about 59% to total
fluorescence. The decay-associated spectra for L287W in the presence of
substrates are similar to those without substrates (not shown)
suggesting a similar Trp-287 microenvironment when the N-subdomain is
open or closed.
Time-resolved Fluorescence Anisotropy Decay--
Fluorescence
anisotropy decays were collected for the F25W, L287W, and wild-type
proteins at 340 nm in the presence and absence of substrates. The
fitted parameters (
For wild-type protein, the fast component of Trp-325 has a rotational
correlation time of ~0.8 ns contributing 23% to total depolarization. Substrate binding did not alter the fast component (
For Trp-25, 30% of the depolarization originated from the fast
component (0.78-0.98 ns) in the absence of substrates. In contrast to
the significant effects that Mg2+ binding had on the
emission spectra (Fig. 3C), metal binding had little effect
on the nanosecond mobility of Trp-25 suggesting that this divalent
cation only produces static quenching without affecting the dynamics of
Trp-25. The longer rotational correlation times are much smaller than
those expected for rigid motion of a 39-kDa protein alone (10.76 ns) or
complexed with DNA (21.89 ns). It is possible to estimate the
rotational correlation time of a rigid hydrated sphere from the
Stokes-Einstein equation, shown in Equation 4, where
Mw is the molecular mass of F25W,
The anisotropy decay of unliganded Trp-287 can be best
described with two rotational correlation times of 0.93 and
21.57 ns (Table III). However, in contrast to tryptophan in wild-type
enzyme or F25W, the contribution from the fast component
(
In the presence of Mg2+ and DNA, the slower correlation
time increased to ~33 ns because of DNA binding. Most interesting,
further addition of the correct nucleotide, dTTP, resulted in a
dramatic decrease in the amplitude of the fast component (44 to 20%)
with a minor decrease in the correlation time (0.87 ns). If the short correlation time reflected local side chain rotation only, we would
expect the amplitude to remain stable. This is reasonable when we
consider that other indicators of the local tryptophan environment
indicate that dTTP binding has no effect on fluorescence quenching
(Table I), emission maximum (Fig. 3D), and lifetime parameters (Table II). Further, crystallographic structures suggest that Leu-287 would not be affected (solvent exposure) by nucleotide binding (6). The significant reduction of angular freedom induced by
correct nucleotide binding can only be observed on the tryptophan located on Fluorescence characterization of structure-based site-specific
introduction of tryptophan can be an instructive approach in investigating not only its microenvironment but also the segmental dynamics of its structural support. In this study, we have used the
wild-type protein with its lone tryptophan (Trp-325) and two engineered
tryptophan mutants (F25W and L287W) where Trp-325 had been altered to
alanine (W325A) (Fig. 1, A and B). These
tryptophan residues were used to probe the effects of substrate binding
on the lyase (Trp-25) and polymerase (Trp-287 and Trp-325) domains.
With regard to the amino-terminal lyase domain, Mg2+
significantly quenched Trp-25 (25%; see Fig. 3C). The other
tryptophan probes also sensed metal binding to a smaller extent
(<8%). Despite the metal-induced steady-state fluorescence quenching
observed for Trp-25, metal binding had almost no effect on Trp-25
flexibility (Table III). The 8-kDa lyase domain is connected to the
31-kDa polymerase domain through a protease-sensitive hinge (Fig.
1B). The anisotropy decay parameters for Trp-25 strongly
support the conclusion that the lyase domain is highly mobile in
solution as evidenced from the much faster rotational correlation time (11 ns) than expected for an ideal sphere (~16 ns). To define the
contribution of the lyase domain, we attempted to collect additional
data but failed to uncover a single segmental correlation time
corresponding to that anticipated for an isolated spherical lyase
domain motion (3 ns). We believe that To probe dynamics within the polymerase domain, we studied the
tryptophan in wild-type enzyme (Trp-325) and a tryptophan engineered into The flexibility of is a two-domain
DNA repair enzyme that undergoes structural transitions upon binding
substrates. Crystallographic structures indicate that these transitions
include movement of the amino-terminal 8-kDa lyase domain relative to
the 31-kDa polymerase domain. Additionally, a polymerase subdomain
moves toward the nucleotide-binding pocket after nucleotide binding,
resulting in critical contacts between
-helix N and the nascent base
pair. Kinetic and structural characterization of pol
has suggested that these conformational changes participate in stabilizing the ternary enzyme-substrate complex facilitating chemistry. To probe the
microenvironment and dynamics of both the lyase domain and
-helix N
in the polymerase domain, the single native tryptophan (Trp-325) of
wild-type enzyme was replaced with alanine, and tryptophan was
strategically substituted for residues in the lyase domain (F25W/W325A)
or near the end of
-helix N (L287W/W325A). Influences of substrate
on the fluorescence anisotropy decay of these single tryptophan forms
of pol
were determined. The results revealed that the segmental
motion of
-helix N was rapid (~1 ns) and far more rapid than the
step that limits chemistry. Binding of Mg2+ and/or
gapped DNA did not cause a noticeable change in the rotational correlation time or angular amplitude of tryptophan in
-helix N. More important, binding of a correct nucleotide significantly limited
the angular range of the nanosecond motion within
-helix N. In
contrast, the segmental motion of the 8-kDa domain was
"frozen" upon DNA binding alone, and this restriction did
not increase further upon nucleotide binding. The dynamics of
-helix
N are discussed from the perspective of the "open" to
"closed" conformational change of pol
deduced from
crystallography, and the results are more generally discussed in the
context of reaction cycle-regulated flexibility for proteins acting as
molecular motors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is a 39-kDa enzyme composed of two distinct domains of 8- and
31-kDa connected by a protease-hypersensitive hinge region. The
amino-terminal 8-kDa domain possesses the 5'-deoxyribose phosphate
lyase activity required to process the 5'-terminus in a DNA gap during
base excision repair. The polymerase active site is part of the
carboxyl-terminal 31-kDa domain (for a review see Ref. 1). The domain
and subdomain organization of pol
is illustrated in Fig. 1,
A and B. Although pol
appears to have evolved
separately from other families of polymerases of known structure (2),
it shares many general structural and mechanistic features with other
polymerases. The polymerase domain is composed of three functionally
distinguishable subdomains. The polymerase catalytic subdomain
coordinates two divalent metal cations that assist the nucleotidyl
transferase reaction. Two additional subdomains that have primary roles
in duplex DNA binding and nascent base pair (nucleoside 5'-triphosphate
and templating nucleotide) binding border the catalytic subdomain.
These subdomains will be referred to as C (catalytic)-,
D (duplex DNA binding)-, and N
(nascent base pair binding)-subdomains to discern their
intrinsic function. These would correspond to the palm, thumb, and
fingers subdomains, respectively, according to the nomenclature that
utilizes the architectural analogy to a right hand
(3).2
serves as an excellent model for study
of the nucleotidyl transferase reaction, DNA synthesis fidelity, and
protein-DNA interactions. Many revealing x-ray crystallographic structures of apoenzyme (4), binary DNA substrate complexes (substrate
and product) (5, 6), and ternary substrate complexes (DNA and
2'-deoxynucleoside 5'-triphosphate) (6-8) have been solved.
in different liganded states
indicates that there are numerous structural transitions that occur
upon substrate binding. These transitions include repositioning of the
amino-terminal lyase domain toward the carboxyl polymerase domain upon
binding DNA. Additionally, a large, ~30°, rotation of
-helix M
repositions
-helix N adjacent to the nascent base pair upon binding
a dNTP to form a ternary complex (9). The repositioning of
-helix N
upon binding a nucleotide results in an "open" (
dNTP) to
"closed" (+dNTP) conformational transition and forms an effective
active site that coordinates two Mg2+ ions (see Fig.
1C). When bound to single-nucleotide gapped DNA, the overall
architecture of pol
resembles a doughnut, which suggests that
nucleotide access may be restricted (9, 10). Upon binding a correct
dNTP and forming a closed complex, extensive contacts are observed
between the polymerase and nascent base pair (6, 7). The structure of
pol
bound to single-nucleotide gapped DNA indicates that the
template strand is bent ~90° as it enters the polymerase active
site, permitting polymerase stacking interactions with one face of the
nascent base pair and the DNA minor groove. This allows for a survey of
the geometry of the nascent base pair to probe whether it conforms to
Watson-Crick geometry. The structure of a "catalytic
intermediate" ternary complex of pol
indicates that
closure of the N-subdomain can be induced by metal-dNTP binding in the
absence of the catalytic metal (8). Kinetic approaches to study
nucleotide insertion and fidelity have indicated that rate-limiting
conformational changes occur before and after chemistry, suggesting an
induced-fit mechanism for nucleotide insertion and fidelity (see Ref.
11). The identity of the proposed conformational changes are unknown, but recent kinetic (8, 10, 12), structural (8), and modeling (11)
studies with pol
indicate that it is not the large subdomain
movements inferred from structural studies. However, the rate and
function of this large structural transition remains unknown.
is known to be processive on short gapped DNA but
distributive on non-gapped DNA (13). Because the isolated polymerase
domain (31-kDa domain) of pol
exhibits only distributive DNA
synthesis (14), the 8-kDa lyase domain confers processive DNA synthesis
to the intact enzyme. The dynamic description of processive DNA
polymerases should include their catalytic properties (e.g.
DNA and dNTP binding, conformational changes, and chemistry), as well
as their movement (i.e. translocation). The force and velocity of RNA polymerase has been studied as a function of load (15),
and it appears to function as a molecular motor. Recently, the
vectorial nature of these motors has been modeled in terms of a
"Brownian Ratchet" (16-18). The requirements for
rectification of Brownian motion seem to be as follows: 1) a cycle of
flexibility and/or hinging motion followed by one or more rigid states
in the biopolymer, 2) the coupling of nearly irreversible chemical events to selected portions of the cycle, and 3) a change in
conformation or binding affinity associated with those events. The
myosin-actin and dynein-tubulin systems are prototypes for this model
(17, 18). Regulation of flexibility also plays a role in recognition; in particular, some protein-protein associations are mediated by
"target-induced folding" of disordered domains (19-21). In
those systems, target-dependent structure seems to be a
compromise to achieve relatively tight binding without the stringent,
and selective, needs of a "lock and key" interface.
is comprised of 335 amino acids and includes a single
tryptophan near the carboxyl terminus (Trp-325; see Fig. 1,
A and B). The effects of metal binding on the
tryptophan fluorescence and the hydrodynamic shape of wild-type rat pol
were characterized previously (29) by time-resolved fluorescence and analytical ultracentrifugation. In this study, we have
strategically positioned tryptophan residues in the lyase domain
(F25W/W325A) or N-subdomain (L287W/W325A) by site-directed mutagenesis
to monitor changes in flexibility of these structural elements upon
binding substrates. We interpret changes in flexibility in the context of pol
function.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were overexpressed in Escherichia coli
and purified as described (30). Oligonucleotide site-directed
mutagenesis and activity measurements were performed as described
previously (31). The enzyme concentration was determined from the
absorbance at 280 nm using an extinction coefficient of 21,200 cm
1 M
1 (32). Acrylamide and
melatonin were obtained from Sigma.
and dNTP. The downstream primer was synthesized with a 5'-phosphate.
The relative amplitudes (
(Eq. 1)
i) and decay
constants (
i) were extracted from the fit.
The decay-associated spectra were obtained as described previously (29,
33).
The factor G was reduced to unity with a wedge
depolarizer (Optics for Research, Caldwell, NJ) by placing the
depolarizer at the entrance slit of the monochromator. The reference
lamp profile and color shift used for convolution analysis was tested with a monoexponential standard (aqueous solution of melatonin with a
fluorescence lifetime of 5.27 ns). The convolution was compared with
the experimental fluorescence intensity decay by a nonlinear
least-squares (reduced chi-squared;
(Eq. 2)
2)
minimization. The weighted residuals and autocorrelation function of
the weighted residuals were calculated to estimate the reliability of
each fit.
fast,
slow
fast, and
slow were the variable
parameters.
The pre-exponential
(Eq. 3)
terms directly relate to the
amplitude of rotational motion, i.e. are measures of
the extent to which the emission is depolarized by each rotational
component, thereby measuring the restriction upon diffusion of the
residue reporter (35).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
In
wild-type pol
, the carboxyl-terminal Trp-325 is located on the
surface of the enzyme just after the last helix (
O) (Fig. 1B). X-ray structures of rat
and human pol
indicate that one side of the indole ring is exposed
to solvent, and the opposite face makes van der Waals contact with
His-285 and Ile-323 (6, 7). Those observations were in excellent
agreement with our earlier characterization (29) of the fluorescence
properties of Trp-325 in rat pol
. To probe dynamic aspects of other
regions of pol
, Trp-325 was replaced with alanine, and tryptophan
residues were substituted at other strategic positions. In the
L287W/W325A double mutant (from this point on, referred to simply as
L287W), the tryptophan was introduced near the end of
helix N in
the N-subdomain (Fig. 1). Residue 287 was selected for tryptophan substitution because of its solvent-exposed environment and minimal contact with substrates as deduced from crystal structures with and
without substrates (6). Crystallographic characterization of pol
in
various liganded states indicates that numerous structural transitions
occur upon binding substrates. The most prominent conformational event
occurs upon binding of the correct nucleotide resulting in an
open to closed transition where
-helix N tilts toward the
active site intimately contacting the nascent base pair. The
characteristics of L287W thus enabled us to use it as a probe for the
effects of substrate binding on the dynamics of
-helix N.
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Fig. 1.
DNA polymerase and DNA substrate organization
and structure. A, functional organization and
domain/subdomain nomenclature of pol . DNA polymerase
contributes two enzymatic activities essential during base excision
repair. The active sites for these activities (an 8-kDa amino-terminal
lyase domain (purple) and the carboxyl-terminal polymerase
domain) are found on distinct domains. The polymerase domain is
composed of three functionally distinct subdomains referred to as D-,
C-, and N-subdomains in reference to their intrinsic function (see
text). These subdomains correspond to the thumb, palm, and fingers
subdomains for DNA polymerases that utilize the architectural analogy
to a right hand (3). The location of the intrinsic tryptophan (Trp-325)
and site-specifically engineered tryptophan residues (Trp-25 and
Trp-287) are indicated. B, structure of the ternary complex
of pol
complexed with ddCTP and DNA (blue) (6). The
subdomains are colored according to the scheme used in A. C, a comparison of the position of
-helixes M and N in
the open (
dNTP) and closed (+dNTP) forms of pol
. Rotation around
an axis (
-helix M) positions
-helix N adjacent to the nascent
base pair (blue) to probe whether correct Watson-Crick
geometry occurs. A modeled tryptophan at residue 287 is illustrated,
and the distance between C
of Leu-287 in the open and closed
forms is indicated.
A; see Fig. 1, A and B) and is
predicted to be partially solvent-exposed. The crystallographic
structure of the pol
ternary complex indicates that there are
no direct substrate contacts with this residue, and residue
25 is located about 10 Å from the lyase active site (i.e.
Lys-72) (6). The structure of the apoenzyme form of rat pol
(4), or
in complex with non-gapped DNA (7), indicates that the lyase domain is
distant from the carboxyl-terminal subdomain (i.e.
N-subdomain), in contrast to its position when bound to gapped DNA (6).
The open and closed conformations of the amino-terminal lyase domain
suggest that this domain is flexible in solution. The dynamic
properties of the lyase domain in full-length enzyme are unknown even
though the NMR structure of the 8-kDa lyase domain has been solved
(37).
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Fig. 2.
Circular dichroism spectra for wild-type and
mutant pol . Spectra were collected at 20 °C in a reaction
mixture with 50 mM Tris-HCl, pH 7.4, and 0.1 M
KCl and a protein concentration of 10.6 µM. The spectra
for wild-type enzyme and the F25W and L287W mutants of pol
were
nearly identical. The inset shows a photograph of a 12%
SDS-PAGE gel. Molecular mass standards are in the left
lane (M), and 6 µg of wild-type (lane 1), F25W
(lane 2), and L287W (lane 3) pol
were loaded
on the gel.
, respectively. The steady-state anisotropy values for F25W,
L287W, and wild-type pol
were 0.13 ± 0.01, 0.14 ± 0.003, and 0.16 ± 0.005, respectively, indicating a more flexible
tryptophan in F25W and L287W than the wild-type protein. The
steady-state parameters for wild-type human pol
were similar to
those of rat enzyme determined previously (29).
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Fig. 3.
Effect of substrates on the fluorescence
emission spectra of wild-type and mutant pol . A, the
relative fluorescence of wild-type enzyme compared with the F25W and
L287W mutants of pol
. The protein concentration was 2.0 µM. The emission spectra of wild-type (B),
F25W (C), and L287W (D) were collected in the
presence (dotted lines) of various substrates in 50 mM Tris-HCl, pH 7.4, and 0.1 M KCl. The
concentrations of ligands were as follows:
+Mg2+, 10 mM; +DNA, 10 mM Mg2+ and a stoichiometric concentration
(relative to protein) of gapped DNA; +2× DNA, 10 mM Mg2+ and 2-fold molar excess of gapped DNA;
+dTTP, same as +DNA with 50 µM dTTP. The
solid line in each panel represents the spectrum
in the absence of ligands.
crystals indicated that Mg2+ could
bind to the hairpin of the first Helix-hairpin-Helix motif (40). This
metal binding motif is also found in the D-subdomain and is proposed to
facilitate DNA binding (5). Our data suggest that the Trp-25 probe in
the lyase domain senses metal binding, even though it is not proximate
to these hairpin residues. Further binding of gapped DNA and dTTP
resulted in 25.8% (F25W), 32.3% (L287W), and 29.3% (wild-type)
fluorescence decreases as compared with proteins without substrates.
Notably, DNA binding resulted in ~7 nm of blue shift in the emission
maximum of F25W (Fig. 3C), further indicating that the
environment around Trp-25 is altered with DNA binding. Although Phe-25
of wild-type protein is not implicated in DNA binding as deduced from
crystallographic structures, its location adjacent to a positively
charged groove may indicate a role in DNA binding. The longer DNA
molecule used here (24 bp) may interact with a larger binding surface
than that observed in crystallographic structures utilizing shorter (16 bp) DNA molecules (6).
1, 20.97 ± 0.57 M
1, and 16.31 ± 0.12 M
1 for F25W, L287W, and wild-type enzymes,
respectively. From these results, the relative accessibility of the
tryptophans to the quencher was determined to be as follows:
wild-type < F25W < L287W. In the presence of substrates
(Mg2+, single-nucleotide gapped DNA, and dTTP),
Ksv of the respective tryptophans decreased
50.2% (8.64 ± 0.06 M
1), 29.7%
(14.74 ± 0.26 M
1), and 14.7%
(13.91 ± 0.27 M
1) with F25W, L287W, and
wild-type proteins, respectively. The large decrease in
Ksv for Trp-25 and the 7-nm blue shift in the emission maximum (Fig. 3C) are consistent with the role of
this residue in DNA binding.
Acrylamide quenching and fluorescence parameters for wild-type and
single tryptophan mutants of pol
View larger version (16K):
[in a new window]
Fig. 4.
Acrylamide quenching of tryptophan in pol
. Measurements were performed at 20 °C in a reaction mixture
with 2.0 µM enzyme in 50 mM Tris-HCl, pH 7.4, and 0.1 M KCl. Samples were excited at 296 nm, and emission
was recorded at 350 nm. Ligand concentrations were as follows: 10 mM Mg2+, 4 µM gapped DNA, and 50 µM dTTP. A, Stern-Volmer acrylamide quenching
of wild-type (
and solid lines), F25W (
and
dotted lines), and L287W (
and dashed lines)
enzymes in the absence (open) and presence
(filled) of ligands. B, Stern-Volmer acrylamide
quenching of L287W in the presence (dotted lines) and
absence (
; solid line) of various substrates as
follows:
, +Mg2+;
, +Mg2+/dTTP;
,
+Mg2+/gapped DNA; and
, +Mg2+/gapped
DNA/dTTP.
1, respectively).
-helix N of the N-subdomain (Trp-287) near the templating base, the
microenvironment of Trp-287 was studied in the presence of different
substrate combinations (see Fig. 4B and Table I). Based on
these results, Mg2+ alone, or Mg2+ and dTTP,
did not afford significant protection of this tryptophan from
acrylamide quenching (Table I). In contrast, binding of the gapped DNA
alone resulted in ~21% decrease in accessibility (Ksv = 16.66 M
1)
suggesting that most of the protection was coming from DNA itself. The
binding of the correct nucleotide, dTTP, had no effect on tryptophan
accessibility. This is consistent with ternary complex structures of
pol
indicating that Leu-287 is exposed to solvent (6, 7). The
linearity of the Stern-Volmer plots (Fig. 4), without a hint of
curvature, suggests that the quenching mechanism for these proteins is
dynamic and that the tryptophan microenvironments are homogeneously accessible.
m; Table II)
and Ksv. The quenching rate constants for the
all the proteins appear to be near diffusion-controlled
(109-1010
M
1s
1). The relative decreases
in kq in the presence of substrates (last
column in Table I) are very close to the reductions observed in
Ksv indicating that the observed decreases in
tryptophan accessibility are primarily because of a decrease in dynamic
encounters. The similar quenching parameters for L287W
(Ksv and kq) in the
presence and absence of the correct nucleotide suggest that the closure of the N-subdomain upon correct nucleotide binding has no effect on
acrylamide accessibility for Trp-287 on
-helix N.
Fluorescence lifetime decay parameters for wild-type and single
tryptophan mutants of pol
2 = 1.04-1.30). In
the absence of substrates, the lifetimes of Trp-325 (wild-type) were
0.95 and 8.14 ns, and those of Trp-287 were 1.42 and 5.02 ns. These
lifetime parameters for the human wild-type enzyme are similar to those
of rat pol
(1.27 and 8.44 ns) measured previously (29). Thus,
despite the 14-amino acid difference between the human and rat enzymes,
the microenvironments of Trp-325 from these two sources appear to be
similar, and the unusually long lifetime component characteristic of
rat pol
is also observed with the human enzyme. For the Trp-25
mutant, fits with a two-exponential model showed systematic deviations and high
2 values. Three-exponential models fit the F25W
decay data adequately (reduced
2 = 0.99-1.24).
View larger version (18K):
[in a new window]
Fig. 5.
Decay-associated spectra for L287W pol .
Spectra were determined in 50 mM Tris-HCl, pH 7.4, and
0.1 M KCl at 20 °C in the absence of substrates with
excitation at 295 nm.
fast,
fast,
slow,
slow) and
2 values
are presented together with initial anisotropy
(ro) in Table III.
In all cases, the anisotropy decays could be fit best by a sum of two
exponentials. Single exponential fits gave more than 5-fold increases
in
2 for all cases, and addition of a third exponential
term led to no significant improvement in
2.
Time-resolved fluorescence anisotropy decay parameters for wild-type
and single tryptophan mutants of pol
fast and
fast) but increased the slower
correlation time (27 and 37 ns in the absence and presence of
substrates, respectively; see Table III). This increase in the longer
correlation time is consistent with the binding of the
single-nucleotide gapped DNA (14.45-kDa). Nucleotide (dTTP) binding did
not cause changes in the rotational parameters.
is the
calculated partial specific volume (0.739 cm3/g for human
pol
at 20 °C), h is the degree of hydration (assuming 0.3 cm3/g of protein),
is the solution viscosity
(~1.002 centipoise at 20 °C), R is the gas
constant (8.314 × J M
1
K
1), and T is the absolute temperature (293.15 K).
From Equation 4, the predicted rotational correlation time for pol
(Eq. 4)
is ~16.3 ns. Further, the asymmetric nature of pol
can only
yield correlation times that exceed those for a sphere of the same size
(29). Hence, the lower
slow in F25W (11.34 ns) indicates
segmental motion of the amino-terminal lyase domain. Most importantly,
DNA binding increased
slow and decreased the depolarization amplitude of the fast component (
fast)
suggesting that both the fast (local) movement of Trp-25 and the 8-kDa
lyase domain are restricted in the presence of DNA and are not
restricted further upon nucleotide binding.
fast) is noticeably higher (44%), indicating a
significant angular contribution from the fast component to the total
depolarization. The fast correlation time for L287W could be attributed
to segmental motion involving several residues in
-helix N. Alternatively, it may represent an unusually slow local motion of
Trp-287; i.e. the internal rotation of the indole ring about
the C
-C
and
C
-C
bonds corresponding to the
1 and
2 dihedral angles of the
tryptophan side chain (28, 41). The rotational motion of tryptophan
side chains in intact proteins typically yield correlation times of 50-400 ps (42-47). Because some of these determinations probably include averaging with slower segmental contributions, we consider 400 ps an upper estimate for this process. Thus, indole side-chain motions
are too fast to correspond to a correlation time of 0.93 ns. This
correlation time is more reasonably assigned to
-helix N tilting in
the solitary enzyme.
-helix N (L287W) that interacts with the nascent base pair in the closed conformation. Because the conformational transitions of pol
are benchmarked with crystallographic structures (6), our
data suggest that the reduced motion of Trp-287 represents rigidity of
-helix N in the vicinity of the nascent base pair subsequent to
closing of the N-subdomain. In contrast to the results observed for
binding the correct nucleotide, the binding of the incorrect
nucleotide, dCTP, did not result in a restrained Trp-287 mobility
(Table III). This may primarily be because of the fact that DNA
polymerases generally bind the incorrect nucleotide with a lower
affinity than the correct nucleotide so that the ternary complex with
an incorrect nucleotide is not populated significantly. Although the
binding affinity for the incorrect nucleotide (dCTP) is increased about
2-fold for the L287W mutant relative to wild-type enzyme, the mutant
also binds the incorrect nucleotide with low affinity
(Kd = 360 ± 40 nM). To avoid inner
filter effects, we are unable to use higher concentrations of the
incorrect nucleotide. The dynamics of Trp-287 in binary (DNA) and
ternary (DNA, dNTP) pol
complexes are illustrated in Fig.
6.
View larger version (23K):
[in a new window]
Fig. 6.
Influence of correct dNTP binding on the
segmental motion of -helix N. In the open
pol
-DNA binary complex (left), crystallographic
structures indicate that
-helix N does not interact with the
templating strand (6, 7). The significant reduction in the amplitude of
the rapid anisotropy decay of Trp-287 upon nucleotide binding suggests
that the angular motion of
-helix N is reduced when interactions
with the nascent base pair occur (closed complex). The cone angles for
the motion of
-helix N in the open and closed states are calculated
to be 39 and 21°, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
slow here includes contributions averaged from both the lyase domain and intact enzyme. In
this view, the 8-kDa lyase domain motion in solution probably occurs on
a time scale intermediate between 3 and 8 ns.
-helix N of the N-subdomain (Trp-287). Crystallographic studies
indicate that Trp-325 is located on the surface, in a loop after
-helix O (Fig. 1B), with one side of the indole ring making van der Waals contact with His-285 and Ile-323 and the other
side exposed to solvent (solvent-accessible area = 79 Å2). This is consistent with partial shielding of this
tryptophan from acrylamide quenching. Leu-287 is situated near the
nascent base pair and is more solvent-exposed than Phe-25 or Trp-325. Modeling tryptophan residues at these positions, by selecting the best
tryptophan rotamer from a library, suggests that the solvent-accessible
surface of Trp-287 (~160 Å2) would be much greater than
Trp-25 (~42 Å2) or Trp-325. Because of the more exposed
environment of Trp-287, the indole ring is expected to experience more
rapid ring flipping motions as compared with Trp-325. In this case, the
rapid anisotropy decay of Trp-287 is far too long (0.93 ns) to
correspond to isolated indole ring flipping (<300 ns). Based on
Stern-Volmer quenching data, the binding of the correct nucleotide does
not influence the accessibility of Trp-287. If the rapid 1-ns component
simply represented fast indole ring rotation for this
tryptophan, then it would be expected that the amplitude of the short
component would remain the same as in the unliganded protein, because
the microenvironment of this tryptophan is not influenced by nucleotide binding (see Figs. 2-4 and Tables I-III). The significant reduction in the amplitude of the rapid anisotropy decay of Trp-287 upon nucleotide binding (14 to 22%) indicates an indirect restriction of
this tryptophan. An indirect restriction of Trp-287 could originate from segmental motion in
-helix N that is constrained upon
nucleotide binding. After formation of the closed conformation,
-helix N interacts with the nascent base pair, and these additional
interactions are expected to limit segmental motion in this helix.
-helix N may play important roles in DNA
synthesis. The analysis described above indicates that there is a
strong reduction of the amplitude of the
-helix N angular motion in
the closed conformation after binding a nucleotide. The cone angles for
the motion of
-helix N in the open and closed states are calculated
to be 39 and 21°, respectively (Fig. 6). The larger angular motion of
-helix N in the open state may facilitate selection of the correct
incoming nucleotide from the pool of structurally similar competing
molecules. When a correct nucleotide binds in the pol
active site
and forms Watson-Crick hydrogen bonds with the templating base,
-helix N can probe the geometry of the nascent base pair. Optimum
interactions (van der Waals and electrostatic) are expected to occur
between the correct nascent nucleotide and
-helix N thereby
restricting segmental motions of this critical helix. The closed
conformation is expected to signal correct geometry to initiate further
critical conformational changes and/or chemistry (10, 11). In contrast,
if an incorrect nucleotide binds to the pol
active site, then the
interactions with
-helix N and the nascent base pair (templating
nucleotide and incorrect incoming nucleotide) are not optimum, and the
nucleotide can diffuse rapidly out of the active site before chemistry
can occur. An illustration of these events and their postulated
relationship to polymerase "selectivity" is presented
in Fig. 7.
View larger version (15K):
[in a new window]
Fig. 7.
Relationship between the rapid angular motion
of -helix N and DNA synthesis fidelity.
The magnitude of the rapid angular motion (dashed arrows) of
-helix N in the pol
-DNA binary complex is significantly larger
than that for the ternary substrate complex with a correct incoming
nucleotide (Fig. 6). Incorrect nucleotides bind with a lower affinity
than correct nucleotides indicating that the dissociation rate constant
for the incorrect nucleotide is much more rapid than for the correct.
The failure of an incorrect nucleotide to influence the magnitude of
the angular motion of
-helix N suggests that incorrect nucleotides
can rapidly diffuse out of the polymerase active site in the open
conformation and that chemistry rarely occurs.
The restricted motion of -helix N observed upon binding a correct
nucleotide may also play a role in the motor function of the polymerase
to facilitate directional translocation. DNA polymerase translocation
(5'
3') along the growing DNA duplex during processive DNA
synthesis is kinetically assumed to be rapid; i.e.
translocation is much more rapid than nucleotide insertion.
Additionally, the importance of these motions to correct nucleotide
selection could imply that these events are coupled so that
translocation would not occur until the correct nucleotide is inserted.
The rapid segmental motions observed here are much more rapid than the
kinetic step that limits nucleotide insertion, ~3 s
1
(20 °C) (31), indicating that this segmental motion does not represent the rate-limiting step for DNA synthesis (10). Indirect experimental approaches had predicted previously (8, 10-12) that the
open to closed structural transition observed with
-helix N upon
binding a correct nucleotide was too rapid to be rate-limiting for
nucleotide insertion.
The anisotropy of Trp-25 also indicate a loss in angular extent or
"freezing" of segmental mobility in the lyase 8-kDa domain. Interestingly, unlike L287W, this loss of segmental motion occurs immediately upon DNA binding (Table III) and is not restricted further
upon nucleotide binding. Overall, the native Trp-325 is unperturbed by
substrate binding, whereas our site-selected reporters (Trp-25 and
Trp-287) yielded information about local segmental motions that
responded to catalytic cycle-dependent conformational changes, albeit in very different ways. Site-directed reporters like
tryptophan and analogs have the potential to detect flexibility changes
with functional importance in complexes.
![]() |
ACKNOWLEDGEMENT |
---|
We are grateful to Jennifer Myers for help with preparation of this manuscript.
![]() |
FOOTNOTES |
---|
* 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.
§ On sabbatical leave from the Department of Chemistry, Mokpo National University, Muan, Korea.
To whom correspondence should be addressed. Tel.:
919-541-3267; Fax: 919-541-3592; E-mail: wilson5@niehs.nih.gov.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M208472200
2
The subdomain nomenclature as originally
proposed for pol utilized the right-hand analogy (4,48); however,
it is functionally opposite to that employed for other DNA polymerases.
To eliminate possible confusion, a functionally based nomenclature is
employed as outlined in the text.
![]() |
ABBREVIATIONS |
---|
The abbreviation used is: pol, polymerase.
![]() |
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