Direct Measurement of the Substrate Preference of Uracil-DNA
Glycosylase*
George
Panayotou
,
Tom
Brown§,
Tom
Barlow§,
Laurence H.
Pearl¶
, and
Renos
Savva¶**
From the
Ludwig Institute for Cancer Research,
University College London, 91 Riding House Street, London, W1P 8BT, the
§ Department of Chemistry, University of Southampton,
Highfield, Southampton, SO17 1BJ, and the ¶ Department of
Biochemistry and Molecular Biology, University College London, Gower
Street, London WC1E 6BT, United Kingdom
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ABSTRACT |
Site-directed mutants of the herpes simplex virus
type 1 uracil-DNA glycosylase lacking catalytic activity have been used to probe the substrate recognition of this highly conserved and ubiquitous class of DNA-repair enzyme utilizing surface plasmon resonance. The residues aspartic acid-88 and histidine-210, implicated in the catalytic mechanism of the enzyme (Savva, R., McAuley-Hecht, K.,
Brown, T., and Pearl, L. (1995) Nature 373, 487-493;
Slupphaug, G., Mol, C. D., Kavli, B., Arvai, A. S., Krokan,
H. E. and Tainer, J. A. (1996) Nature 384, 87-92) were separately mutated to asparagine to allow investigations
of substrate recognition in the absence of catalysis. The mutants were
shown to be correctly folded and to lack catalytic activity. Binding to
single- and double-stranded oligonucleotides, with or without uracil,
was monitored by real-time biomolecular interaction analysis using
surface plasmon resonance. Both mutants exhibited comparable rates of
binding and dissociation on the same uracil-containing substrates.
Interaction with single-stranded uracil-DNA was found to be stronger
than with double-stranded uracil-DNA, and the binding to Gua:Ura
mismatches was significantly stronger than that to Ade:Ura base pairs
suggesting that the stability of the base pair determines the
efficiency of interaction. Also, there was negligible interaction
between the mutants and single- or double-stranded DNA lacking uracil,
or with DNA containing abasic sites. These results suggest that it is
uracil in the DNA, rather than DNA itself, that is recognized by the
uracil-DNA glycosylases.
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INTRODUCTION |
Uracil-DNA glycosylases
(UDG)1 are a highly conserved
and ubiquitous class of DNA-repair enzymes that catalyze the excision of uracil bases from DNA (1). Uracil is an RNA base and does not
normally occur in DNA, though it forms a good Watson-Crick base pair
with adenine. Uracil and thymine differ in that thymine is methylated
at the 5-position of the pyrimidine ring, and this is not involved in
forming the base pair. Uracil can occur in DNA either by
misincorporation of deoxyuridine triphosphate during replication by DNA
polymerases, which apparently do not discriminate between this
nucleotide and thymidine triphosphate, or as the product of spontaneous
hydrolytic deamination of cytosine residues in DNA (1, 2). The Gua:Ura
mismatches that result from cytosine deamination are promutagenic,
leading to Gua:Cyt
Ade:Thy transition mutations unless the uracil
is repaired to the original cytosine (1, 2). As thymine methyls in
Ade:Thy base pairs are essential in sequence-specific recognition by
many regulatory DNA-binding proteins (3), Ade:Ura base pairs, although
not mutagenic, are potentially disruptive.
Recently, the structural basis for the exquisite recognition of uracil
by UDGs has been elucidated (4-6). The structures show that these
enzymes are able to accomodate sequence nonspecific DNA along a channel
and that this channel contains an active site pocket that is perfectly
tailored to admit only uracil bases. The evidence for a DNA-binding
channel is further supported by the structures of a specific peptide
inhibitor of the UDGs in complex with the enzymes. This inhibitor
prevents binding of UDG to DNA, and is seen to mimic a polynucleotide
in its interaction with the enzyme (7, 8).
The structures of DNA modification and repair enzymes in complex with
polynucleotides either show directly, or indicate the likelihood of,
the extrusion of the target base from the DNA duplex (6, 9-11). In the
case of the UDGs, there is no apparent facility for a gross
conformational change in the enzyme, and the only way that uracil can
enter into the specific pocket is for the base to become extrahelical.
This has recently been shown structurally for UDG (6).
The phenomenon of "base-flipping" has yet to be experimentally
deconvoluted from the reaction as a whole, and it is still open to
debate as to whether the extrusion of DNA bases from a duplex is
spontaneous or is actively promoted by the DNA modification or repair
enzymes (12). Structural analysis alone is unable to distinguish the
two possible mechanisms. Mutation of active site residues to yield
catalytically inactive mutant proteins coupled with careful binding
studies may well yield a satisfactory answer to this question; it is
with this question in mind that these studies were undertaken. Both of
the residues implicated in catalysis by structural studies, aspartic
acid-88 and histidine-210, were mutated to asparagine and used in
binding studies to a range of oligonucleotides using surface plasmon
resonance. This system is a powerful means of attempting to deconvolute
the mode of substrate recognition and binding by UDG in the absence of
catalysis.
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EXPERIMENTAL PROCEDURES |
Construction of Recombinant Mutants of HSV1
UDG--
Aspartic acid-88 and histidine-210 of the recombinant
HSV1 UDG were both mutated to asparagine (D88N and H210N, respectively) to abolish catalytic activity. Oligonucleotide 18-mers were designed to
change the codons in the open reading frame from aspartic acid and
histidine, respectively, to asparagine with the substitution of just
one nucleotide in each case. The oligonucleotides were designed such
that the mismatch was located in the middle of the sequence. The method
used for mutagenesis was the Altered Sites II system (Promega) and was
followed as per protocol. The mutagenesis was effected by subcloning
the HSV1 UDG open reading frame from the original expression vector
pTS106· (13) using NcoI and filling the overhang with
Klenow purification of DNA by Geneclean (Anachem) and final excision of
the open reading frame using HindIII, and inserting it into
the mutagenesis vector supplied with the kit (pALTER1) into
SmaI and HindIII, thus recreating the
NcoI site for subsequent recloning into pTrc99A following
mutagenesis. Unique restriction sites were introduced by mutagenesis
(EcoO109I for D88N, and BstBI for H210N), and
these were used to screen for positive mutant DNAs. Positively
identified mutants were cloned as NcoI to HindIII
fragments in pTrc99A (Pharmacia Biotech Inc.) cut with the same
enzymes.
Expression and Purification of Mutant Proteins--
Expression
was performed in the Escherichia coli strain BL21(DE3), and
standard assays were carried out for UDG activity (14). Inactive mutant
clones were stored as 50% glycerol stocks at
80 °C. Expression of
D88N mutant resulted in low level expression of insoluble protein under
the standard expression conditions (13), and optimum expression was
obtained by growing the bacteria to A600 nm = 0.8-1.0 at 28 °C, before inducing with 0.5 mM isopropyl-
-D thiogalactopyranoside for 36 h. The H210N mutant was expressed under the standard conditions (13). Purification was by
the following method. Following lysis of cells and partition of soluble
and insoluble fractions, (13), the clarified supernatant was applied to
a two-column system (60 ml Q-Sepharose outlet into the inlet of 60 ml
SP-Sepharose) equilibrated with 20 mM Tris-HCl, 10 mM EDTA, pH 8.3, 0.1 mM phenylmethanesulfonyl
fluoride, 1 mM dithiothreitol chilled in an ice-bath. A
wash of 800 ml of the equilibration buffer was performed subsequent to
loading, and a further 400 ml wash using the same buffer was then
performed on the SP-Sepharose column alone. A 0 to 1.6 M
sodium chloride gradient was run through the SP-sepaharose column over
7 column volumes. Fractions containing the bulk of mutant UDG, as
judged by SDS-polyacrylamide gel electrophoresis analysis, were pooled and diluted such that the final sodium chloride concentration was less
than 50 mM, using buffer B (20 mM Tris-HCl/10
mM EDTA pH 8.3, 10% glycerol, 0.1 mM
phenylmethanesulfonyl fluoride, 1 mM dithiothreitol). The
diluted protein was loaded onto a 25-ml poly-U-Sepharose column,
pre-equilibrated in buffer B. A wash of 5-column volumes of buffer B
with 100 mM sodium chloride was performed after loading,
and then a gradient of 100-250 mM sodium chloride over
5-column volumes was performed. UDG mutant protein was eluted in a 2 M sodium chloride step. Dialysis was performed twice
against 100 volumes of 10 mM Tris-HCl, pH 8.3, 0.1 mM phenylmethanesulfonyl fluoride. The proteins were shown
to be greater than 95% pure after this procedure, with D88N having a
final yield of 2 mg/liter bacterial culture and with H210N having a
final yield of 15 mg/liter bacterial culture.
Crystallization of Mutant UDG Proteins--
Proteins were
concentrated following purification in Amicon-stirred ultrafiltration
cells using PM10 membranes (Amicon Corp.). The D88N mutant was far less
soluble than H210N, and was used for crystallization trials at 15 mg/ml. The H210N mutant was used at 35 mg/ml. Purified concentrated
proteins were 0.22-mm filtered in Ultrafree MC microcentrifuge cups
(Millipore), and phenylmethanesulfonyl fluoride and sodium azide were
added to final concentrations of 0.1 mM and 0.02% (w/v),
respectively. The protein stocks were then stored at 4 °C where they
were stable for several months. Both mutants were put through
crystallization trials using the Hampton research crystal screen,
implemented as 1:1 microbatch mixtures under paraffin oil in Terazaki
plates (15). Plates were stored at 16 °C in the dark. Similar
screens were also performed for proteins mixed at ratios of 1:2
(protein:ligand) with nucleotides and oligonucleotides.
Preparation of Immobilized Ugi Protein--
The Ugi protein was
prepared as described previously (16), and immobilized on Affi-Gel 15 cationic beads (Bio-Rad) using the following protocol. Beads, stored as
supplied at
20 °C, were allowed to reach room temperature over 30 min with occasional inversion of the bottle. After thoroughly
resuspending the beads by inversion and agitation, a volume of
approximately 120 µl was withdrawn with a 1-ml micropipettor using a
wide bore tip (made by sawing off one-third of the tapered end) and
transferred to a 1.5 ml microcentrifuge tube. The tube was pulsed for
30 s at 16,000 × g to pellet the beads, and the
bulk of the isopropanol-rich medium was discarded. A volume of 500 µl
of ice-cold deionized water was added to the beads, and the tube was
vortexed for 5 s. The tube was pulsed as described, and the water
was discarded. This was repeated twice more, and then finally with 50 mM HEPES-NaOH, pH 7.0. Upon discarding the HEPES-NaOH, 400 µl of HEPES-NaOH, pH 7.0, containing 3 mg of purified Ugi protein was
added to the beads. The tube was agitated gently to suspend the beads
in the protein solution, and the agitation was continued on a shaker for 90 min at room temperature. The protein binds to the beads through
lysine
-amino groups during this step. The tube was pulsed as
described previously, and the supernatant was removed and stored. The
beads were washed three times with 500 µl of 50 mM
HEPES-NaOH, pH 7.0, as described earlier, with the supernatant being
stored after each spin. After removing the supernatant following the final spin, 10 µl of freshly prepared ethanolamine, pH 8.0, were added and mixed in by tapping the tube gently; the tube was allowed to
stand for an hour at room temperature. This step blocks any beads that
have not bound protein. The tube was pulsed as described previously,
discarding the supernatant, and was washed twice with 400 µl of 50 mM HEPES-NaOH, pH 7.0, discarding the supernatant each
time. The beads were then ready to use.
Synthesis and Purification of Oligonucleotides Used in These
Binding Studies--
The oligonucleotide sequence used in these
experiments was 5
-biotin-CCGAATCAGTTCACTTCNAGCCGAGGTATTTAGCC, for
oligonucleotides ssC and ssU, where N is C in ssC and N is U in ssU.
The nonbiotinylated oligonucleotides were
5
-GGCTAAATACCTCGGCTNGAAGTGAACTGATTCGG where N is either A or G to form
the duplexes dsU/A and dsU/G, respectively, with the biotinylated
strand ssU, and the duplex dsC/G with ssC. Oligonucleotide synthesis
was performed on an Applied Biosystems 394 DNA synthesizer on the
0.2-µm scale using cyanoethyl phosphoramidite chemistry. Standard DNA
synthesis reagents and cyanoethyl phosphoramidite monomers were
obtained from Applied Biosystems, Ltd. The biotin phosphoramidite was
obtained from Cruachem Ltd. and used as a 0.15 M solution
in anhydrous acetonitrile; the coupling time was extended to 3 min.
Stepwise coupling efficiencies were measured automatically on the
synthesizer by trityl analysis, and all monomers were coupled at
greater than 98%. Oligonucleotides were deprotected in concentrated
aqueous ammonia for 8 h at 55 °C. High performance liquid
chromatography purification was carried out on a Gilson model 306 high
performance liquid chromatography system using a Brownlee Aquapore
octyl reverse phase column (10 × 250 mm) with a flow rate of 3 ml/min and a gradient of 0-75% buffer B over a period of 30 min.
(Buffer A, 0.1 M triethylammonium acetate; buffer B, 0.1 M triethylammonium acetate with 25% acetonitrile). In the
case of oligonucleotides labeled with biotin, the correct product was
the major peak (final peak to elute from the column). After high
pressure liquid chromatography purification, the major product was
evaporated to dryness and desalted using a Pharmacia NAP 10 column
(Sephadex G25) according to the manufacturer's instructions.
Experimental Assay Procedure Using Surface Plasmon
Resonance--
The principle of operation of the BIAcore biosensor and
its use in analyzing protein-DNA interactions have been described before (17, 21). All interactions were analyzed in binding buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20) at a constant flow rate of 5 µl/min and at a constant temperature of 25 °C. Biotinylated
oligonucleotides were injected over a streptavidin-coated sensor chip
(SA5, Pharmacia Biosensor) until a suitable level was achieved (see
"Results"). For the formation of double-stranded oligonucleotides,
nonbiotinylated DNA was injected until no more increase in binding to
the immobilized single-stranded DNA could be observed. Bound protein
was eluted from the DNA by a short pulse (5 µl) of 0.05% SDS. This
regeneration procedure did not alter to any measurable extent the
ability of the immobilized DNA to bind protein in subsequent
cycles.
Analysis of the data was performed using the evaluation software
supplied with the instrument. To eliminate small "bulk" refractive change differences at the beginning and end of each injection (due to
small differences in buffer composition of the stock protein solutions), a control sensorgram obtained over a nonbinding surface was
subtracted for each protein injection. For obtaining the association and dissociation rate constants the following equations were used, respectively.
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(Eq. 1)
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and
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(Eq. 2)
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R, R0, and
Req are the response at time t,
t0, and at equilibrium, respectively;
C, the concentration of protein; ka, the
association rate constant; kd, the dissociation rate
constant; and t0, the start time of the
dissociation or association. For the determination of the equilibrium
dissociation constant (KD) from binding
experiments
|
(Eq. 3)
|
was used (where Rmax is the maximum
response level).
 |
RESULTS |
Confirmation of Tertiary Structure of Mutant Proteins--
The
integrity of the tertiary structures of the mutant proteins was
verified in the absence of catalytic activity against a standard
substrate routinely used to assay UDGs (14) by the following methods.
First, Bacillus subtilis bacteriophage PBS1 Ugi protein (16)
immobilized on Affi-Gel 15 beads, was shown by SDS-polyacrylamide gel
electrophoresis to sequester and by standard assay (14) to inactivate
wild-type HSV1 UDG. Similarly immobilized Ugi protein was shown by
SDS-polyacrylamide gel electrophoresis to sequester both mutants with
similar affinity to its capture of wild-type enzyme. To remove the UDG
or mutants from the Ugi protein, it was necessary to heat the beads in
the presence of SDS. As a control, whole protein from an E. coli whole cell lysate was shown by SDS-polyacrylamide gel
electrophoresis to bind very weakly apart from E. coli UDG,
which was retained strongly. This strongly suggests that the
DNA-binding channel and overall fold of the mutant proteins do not
significantly differ from that of the wild-type enzyme.
The second method used to indicate correct folding was using
crystallization and x-ray diffraction. Both D88N and H210N mutants were
crystallized using the Hampton research crystal screen following purification, and the latter also formed rectangular rod-like crystals
spontaneously in situ after 10 days at 4 °C, average size
80 mm thick by 200 mm long, which diffracted to 2.5 Å on a laboratory
source. The spacegroup was found to be P21 with unit cell
a = 42.9 Å, b = 61.6 Å,
c = 43.9 Å, and
= 93.05, with a solvent content of
58% and 1 molecule in the asymmetric unit. Due to the fact that this
is a monoclinic crystal form, a complete dataset has not yet been
collected, and a structure is not yet available. The needle-like
crystals of the D88N and H210N mutants obtained in crystal screens from
polyethylene glycol 4000/buffer mixtures were too small for x-ray
analysis, but co-crystals of the D88N mutant with the single-stranded
trideoxynucleotide pdTpdTpdU, resulted in six-sided plates with average
dimensions of 100-mm across by 20-50-mm thick. These crystals are seen
to diffract beyond 2.6 Å at a synchrotron radiation source. Further
characterization is in progress. The D88N mutant also yields rod-like
crystals with the nucleotides 3
-dUMP and 5
-dUMP, and both mutants
yield large orthorhombic rods with the trideoxynucleotide pdTpdTpdT identical to those obtained with the wild-type enzyme. Crystals of
macromolecules, which diffract to medium and high resolution, are a
good indication that there is inherent order and homogeneity in the
components of the lattice. Morphological changes of protein crystals in
the presence of potential ligands is a good indication that productive
binding has taken place, demonstrating that the protein is correctly
folded.
Binding of Proteins to Immobilized Oligonucleotides--
The
BIAcore biosensor was used to measure the specificity and affinity of
the interactions between mutant or wild-type enzyme and single- or
double-stranded oligonucleotides. In this system, the biotinylated
oligonucleotide is immobilized on a streptavidin-coated dextran layer
attached to a sensorchip, and the protein is injected at a constant
flow rate. The biosensor measures refractive index changes close to the
dextran layer, which in turn correspond to changes in the amount of
protein bound to the surface. The plot of bound protein (measured in
arbitrary resonance units (RU)) versus time is called a
sensorgram and can be used to derive kinetic or equilibrium constants.
This method has been used before to study other protein-DNA
interactions (17-19). To immobilize double-stranded oligonucleotides,
one of the strands was biotinylated and attached to the surface first
and then the other strand was injected at saturating amounts until no
further binding was observed (see "Experimental Procedures").
Complete formation of double-stranded DNA was verified by observing
approximate doubling of the static resonance signal after the annealing
of the second strand (Fig. 2). There was no measurable dissociation of
DNA from the surface even after many cycles of binding and
regeneration. Measurements of protein-DNA interactions were obtained by
passing protein solutions over the immobilized oligonucleotides on the
surface of the chip and analyzing the resulting sensorgrams.
Specificity of Binding--
Initial experiments were designed to
check the specificity of binding to uracil-containing DNA and therefore
the maximum possible amounts of oligonucleotides ssC and ssU (see
"Experimental Procedures") were immobilized on the
streptavidin-coated chip (approximately 1100 RU). The mutant D88N and
H210N proteins were then passed over both surfaces. As can be seen in
Fig. 1 even with these high amounts of
immobilized DNA, there was no significant binding to oligonucleotide
ssC, demonstrating the absolute requirement for uracil in specific
recognition. When the wild-type enzyme was tested, no binding was
observed to oligonucleotides ssC or ssU. The most obvious
interpretation for this observation would be the rapid removal of
uracil by the active enzyme making it impossible to detect a stable
enzyme·DNA complex. This was confirmed by testing the binding of
mutant enzyme before and after injection of the wild-type enzyme. Fig.
3 shows that following application of the active enzyme, the mutant D88N was no longer capable of interacting with the surface, indicating that almost all of the available uracil had been removed. The same result was obtained with mutant H210N
(not shown). The much lower binding displayed after removal of
the uracil by the wild-type enzyme indicates that neither the wild-type
nor the mutants have significant affinity for the abasic sites
generated in the single-stranded DNA by the uracil-excision reaction.

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Fig. 1.
Specificity of binding. Interaction of
the D88N mutant with a streptavidin-coated sensorchip saturated with
biotinylated ssC and ssU oligonucleotides. The arrows point
to the start and end of the injection.
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Fig. 2.
Generation of duplex DNA in situ.
The RU values represent the magnitude of the response compared
with the baseline before the first injection. An exact doubling of the
response was observed after injection of the second oligonucleotide.
Additional injections did not increase the response any further.
Marked points indicate the following: 1)
beginning of injection of biotinylated single-strand oligonucleotides;
2) end of injection; 3) beginning of injection of
second single-strand oligonucleotides; and 4) end of
injection (a high concentration of oligonucleotides was used, and
because the stock was dissolved in water, dilution of the buffer
occurred resulting in the sudden "jump" of the response at
point 4. This is not so obvious at point 3 because of the fast binding of the oligonucleotides).
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Fig. 3.
Uracil excision by the wild-type enzyme
abolishes UDG binding to ssDNA. Immobilized ssU oligonucleotide
was allowed to interact with D88N before and after injection of an
equal concentration of wild-type enzyme. The arrows point to
the start and end of each injection. The sharp decreases in the signal
after the interaction are due to the regeneration step to remove bound
protein (see "Experimental Procedures").
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Comparison between Single- and Double-stranded
Oligonucleotides--
Two different nonbiotinylated oligonucleotides
(ssG and ssA, see "Experimental Procedures") were used to create
double-stranded DNA (dsU/G and dsU/A) using immobilized, biotinylated
oligonucleotides ssU, and dsC/G using immobilized, biotinylated ssC. As
with ssC, dsC/G showed no indications of binding with the wild-type
enzyme nor either of the mutants (results not shown). Fig.
4 shows the relative binding of a single concentration of mutant H210N to four
surfaces coated with the same amount (approximately 200 RU of
single-strand oligonucleotides) of ssC, ssU, dsU/G and dsU/A (similar
results were obtained with both mutants, data not shown). Both
double-strand oligonucleotides bound protein with reduced affinity
compared with ssU, and binding to dsU/A was considerably reduced
compared with dsU/A. As with the single-stranded uracil-containing oligonucleotides, treatment with wild-type UDG effectively abolished subsequent binding by wild-type or mutant enzymes, indicating that
neither the wild-type nor mutant enzymes display significant affinity
for the abasic sites in double-stranded DNA produced by the
uracil-excision reaction (Fig. 5).

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Fig. 4.
Comparison of binding to ssDNA and
dsDNA. Binding of H210N to four surfaces coated with the same
quantity of the indicated oligonucleotides. Similar results were
obtained for the D88N mutant.
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Fig. 5.
Uracil excision abolishes UDG binding to
dsDNA. Binding of D88N UDG to immobilized dsU/G oligonucleotide
before and after uracil excision by wild-type UDG. In contrast with the
high affinity for the uracil-containing duplex, neither wild-type nor mutant UDGs are retained on the product oligonucleotide containing an
abasic site.
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To obtain a quantitative comparison, we took advantage of the fact that
equilibrium was reached for all these interactions, and therefore the
equilibrium dissociation constant could be determined by injecting a
range of concentrations and plotting the response at equilibrium
versus the concentration of injected protein. For these
assays as well as for kinetic analysis (see below), the level of
immobilized oligonucleotides was further reduced to approximately 40 RU
to avoid possible artifacts due to mass transport limitations. Furthermore, it has been suggested (20) that the measurement of
KD values by equilibrium binding assays is affected by the amount of "receptor" used, and considerable deviations can
be observed from the true values if the levels are too high. Whereas
the overall response was small, the data obtained in this way fitted
very well to an equation describing a simple bi-molecular interaction
obeying the law of mass action (see "Experimental Procedures"). The
results of these experiments for the mutant H210N are summarized in
Table I. An example of the fits obtained using Equation 3 is shown in Fig. 6 for
binding to the ssU oligonucleotides. This figure also demonstrates that
there is no substantial difference between the two mutant enzymes, D88N
and H210N, and similar good fits were obtained for the other
oligonucleotides (Table I).
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Table I
Kinetic and equilibrium dissociation constants of the interaction
between the H210N mutant and oligonucleotides
The estimates of the kinetic constants are shown as average ± standard deviation (S.D.) of n independent determinations
done at different concentrations of H210N protein. The calculated
equilibrium dissociation constant, KD, was obtained
from the equation KD = kd/ka for each experiment and then averaged. The experimental determination of KD
was obtained using Equation 3, and the standard error (S.E.) for the fit is indicated.
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Fig. 6.
Comparison of the binding of the two mutant
enzymes to ssU. A range of protein concentrations was injected,
and the response at equilibrium was plotted versus the
concentration. The data were analyzed using Equation 3 (see
"Experimental Procedures") to obtain estimates of the equilibrium
dissociation constant, KD.
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Estimation of Kinetic Constants--
To analyze the interaction
with the uracil-containing oligonucleotides further, the kinetic
constants were determined. An example is shown in Fig.
7 for the binding of the H210N mutant to
the dsU/G oligonucleotides. Fig. 7 shows the fit of the association rate (A) and that of the dissociation rate (B)
obtained using Equations 1 and 2 (see "Experimental Procedures"),
which describe a homogeneous single-site interaction. Data were
obtained with several different concentrations of protein and for the
three different oligonucleotides and are summarized in Table I. In all
cases the fits were statistically highly significant, and there was no
evidence of deviations due to mass transport effects, although these
were observed when higher amounts of oligonucleotides were immobilized
(not shown). The interaction with the ssU oligonucleotides is
characterized by the fastest association rate and the slowest dissociation rate. The difference in affinity between the two double-strand oligonucleotides is mainly due to an over 10-fold faster
association rate for dsU/G compared with dsU/A. From the kinetic
constants, the equilibrium dissociation constant could also be
independently estimated from the equation KD = kd/ka. As can be seen in Table I
there is very good agreement between the KD values
calculated in this way and those determined directly by the equilibrium
binding experiments described above, confirming the validity of the
fits.

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Fig. 7.
Determination of rate constants. The
"association" (A) and "dissociation" (B)
phases of a sensorgram obtained by interaction of 250 nM
H210N with immobilized dsU/G were analyzed using Equations 1 and 2,
respectively (see "Experimental Procedures"). The fits are shown
superimposed on the raw data.
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 |
DISCUSSION |
Structural studies show unambiguously that recognition of uracil
by UDGs requires insertion of the uracil base into a pocket in the
enzyme, which for dsDNA can only be achieved by "flipping" the
deoxyuridine nucleotide into an extrahelical conformation (4-6).
However, these structural results cannot explain the mechanism of this
recognition process. Two questions in particular need to be answered.
First, does the enzyme locate uracils by scanning the DNA in a
"one-dimensional" diffusion process (22) or by simple bimolecular
collision; and second, does the enzyme actively promote the flipping of
the uracil from a stacked conformation, or does it recognize
spontaneously flipped bases?
The affinity of native and mutant UDG, for single-stranded or
double-stranded uracil-free DNA, is too low to be measured using the
BIAcore system. This indicates that the residence time of the enzyme on
ordinary DNA is very short and makes it unlikely that the enzyme is
able to scan along the DNA for any appreciable length. That the rates
of association and dissociation from uracil-DNA are very well modeled
by single processes (see Equations 1 and 2 and Fig. 7) rather than by
the biphasic model required if scanning were in operation
(i.e. E + DNA(U)
E:DNA(U)
E:U(DNA)) further suggests
that direct binding of uracil in the DNA is the only process taking
place.
Whereas our observations appear to rule out facilitated diffusion as an
option for locating uracil in DNA, substrate location might be made
more efficient by electrostatic steering of the positively charged
DNA-binding face of UDG (4, 6) toward the negatively charged DNA
molecule during bimolecular collision events. Such an oriented
"hopping" process (22, 23) would certainly serve to reduce the
effective dimensionality of the search in a similar, albeit more crude,
manner than that achieved by one-dimensional diffusion. This would also
make sense of data suggesting that UDG interacts with DNA in a
"distributive" manner (24), but shows effects that would tally with
a scan length of 1.5-2 kilobases prior to dissociation from DNA (25).
A hopping mechanism is also supported by the structural data (6), which suggests that UDG would have to dissociate from DNA after a
uracil-excision event to allow the free uracil product to be released,
a requirement that would be difficult to reconcile with a sliding
mechanism.
Introduction of a single uracil base into the single- and
double-stranded oligonucleotides produced a dramatic increase in the
affinity of the catalytically inactive mutants for the single- and
double-stranded DNA over that observed for uracil-free DNA. The
relative affinity for uracil-DNA was strongly dependent on the
structural context of the uracil, in the order ssU-DNA > dsU/G-DNA
dsU/A-DNA, for both mutants. As neither the native nor
the mutant enzymes showed any significant affinity for the abasic site
that results from the uracil-excision reaction (Figs. 2 and 5), this increased affinity can be attributed to the interactions between the
enzyme and the destacked uracil base, which are essentially identical
in all three systems. This order of relative affinity can be understood
in terms of the work required to destack the uracil and make it
available to the binding pocket; being least in ssDNA where no base
pair is disrupted, more for the "wobble" G:U base pair, and most
for the fully stacked Watson-Crick A:U base pair. Affinity for uracil
in DNA rather than for any features of DNA per se is
consistent with the ability of UDG to act on ssDNA as well as dsDNA,
more or less irrespective of sequence context. The differences in
affinity for the DNA molecules between the two mutants are very small,
confirming the suggestion that both of these residues have purely
catalytic roles in the enzyme mechanism (4, 6).
Neither the native nor the mutant enzymes showed any apparent affinity
for abasic sites, and binding of the active wild-type enzyme to
uracil-DNA ligands was not detectable. Association with uracil-DNA and
its subsequent hydrolysis by the wild-type enzyme, though undetected,
must have taken place as demonstrated by the abolition of subsequent
binding of the catalytically inactive mutants. The lack of a detectable
enzyme·DNA complex during catalysis is consistent with the observed
fast on-rate and low affinity for the abasic reaction product and
suggests that the actual rate of bond hydrolysis is not limiting.
Two broad classes of base-flipping enzyme can be defined; sequence
specific DNA-modifying enzymes, and sequence-independent DNA-modifying
enzymes. A sequence-specific enzyme, such as a restriction methyltransferase, interacts with an extrahelical base within a defined
sequence context that can be recognized in a fully stacked conformation. Once bound to its cognate DNA sequence, a
sequence-specific enzyme can simply wait for the thermal breathing
motion of the DNA to flip its target base spontaneously (9, 12). In
contrast, a sequence-independent enzyme, such as uracil-DNA
glycosylase, must recognize the presence of its target base directly.
Unlike some lesions recognized by DNA repair enzymes, which cause
significant distortion in the structure of the double helix, uracil is
a potentially difficult target to detect. In a G:U mismatch, the wobble
of the uracil into the major groove might enable recognition in
situ of the presence of uracil, however, a U:A base pair, which is also efficiently repaired, is a fully stacked Watson-Crick base pair
with no protrusions. The pattern of functional groups presented by an
A:U base pair in the major groove is different from the patterns
presented by either of the normal G:C or A:T base pairs and might
therefore provide a means for in situ recognition. However, the major groove pattern for A:U is also very different from that presented by G:U, and it is difficult to imagine how an enzyme would
achieve simultaneous specific major (or minor) groove recognition for
these very different base pairs in dsDNA while also recognizing unpaired uracil in ssDNA. A much simpler model, consistent with the
relative strengths of binding we observe for uracil in these different
contexts, would suggest that UDG is recognizing "flipped out"
uracil. These findings are supported by the rates for association and
dissociation of either mutant from the different oligonucleotides. The
association rates are markedly different in the order ssU-DNA > dsU/G-DNA
dsU/A-DNA suggesting that the enzyme·U-DNA complex formation is dependent on the relative strength of any base pairing. The dissociation rates are relatively similar and suggest that once the
complex is formed, the enzyme dissociation is from an essentially
identical complex in all cases.
The limited interaction between UDG and DNA-containing bases other than
uracil is a function of the exquisitely selective specificity pocket of
the enzyme, which will only allow uracil and some close analogues to
enter and bind (4-6). The displacement of several bound water
molecules from the pocket upon binding of uracil is entropically
favorable, and the formation of three hydrogen bond contacts to uracil
in the active site (4, 6), as well as the insertion of a hydrophobic
side-chain into the space left in the duplex by the flipped nucleotide
(6), is a stable arrangement. Further stabilization will be provided by the DNA-binding channel, which makes favorable contacts with the distorted backbone geometry generated by a flipped nucleotide (6). This
suggests that molecular impact of the enzyme onto the DNA and thermal
motion of DNA, together with the stabilization of the helix distortion
provided by the DNA-binding channel of the enzyme, would be sufficient
to drive the complete process of recognition, binding, and catalysis
without penalty.
The question of whether UDGs are indeed facilitating inherent base
flipping in the duplex, or just binding to opportunistically flipped-out bases cannot be demonstrated solely from our results and
the available structural information. However, this could be addressed
by measuring energy changes on interaction using a technique such as
microcalorimetry. This approach is currently being investigated for the
mutant proteins used in this study.
 |
FOOTNOTES |
*
This work was supported by the Cancer Research Campaign.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.:
44-171-380-7372; Fax: 44-171-380-7193; E-mail:
pearl{at}bsm.biochemistry.ucl.ac.uk.
**
Present address: Dept. of Crystallography, Birkbeck College,
Malet St., London, WC1E 7HX UK.
1
The abbreviations used are: UDG, uracil-DNA
glycosylase; HSV1, herpes simplex virus type 1; ss, single-stranded;
ds, double-stranded; Ugi, uracil-DNA glycosylase inhibitor; RU,
resonance units; E+DNA(U), enzyme in the presence of uracil-DNA;
E:DNA(U), enzyme in complex with uracil-DNA; E:U(DNA), enzyme in
complex with a DNA-uracil.
 |
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