From the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560 012, India
Received for publication, December 12, 2000, and in revised form, February 6, 2001
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ABSTRACT |
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Uracil DNA glycosylase (UDG), a
highly conserved DNA repair enzyme, initiates the uracil excision
repair pathway. Ugi, a bacteriophage-encoded peptide, potently inhibits
UDGs by serving as a remarkable substrate mimic. Structure
determination of UDGs has identified regions important for the
exquisite specificity in the detection and removal of uracils from DNA
and in their interaction with Ugi. In this study, we carried out
mutational analysis of the Escherichia coli UDG at
Leu191 within the
187HPSPLS192 motif (DNA intercalation loop). We
show that with the decrease in side chain length at position 191, the
stability of the UDG-Ugi complexes regresses. Further, while the L191V
and L191F mutants were as efficient as the wild type protein, the L191A
and L191G mutants retained only 10 and 1% of the enzymatic activity,
respectively. Importantly, however, substitution of Leu191
with smaller side chains had no effect on the relative efficiencies of
uracil excision from the single-stranded and a corresponding double-stranded substrate. Our results suggest that leucine within the
HPSPLS motif is crucial for the uracil excision activity of UDG, and it
contributes to the formation of a physiologically irreversible complex
with Ugi. We also envisage a role for Leu191 in stabilizing
the productive enzyme-substrate complex.
DNA in cells is unceasingly subjected to damages that occur under
normal physiological conditions and can be exacerbated by environmental
mutagens. If left unrepaired, these can be detrimental to the
maintenance of genetic integrity (1). Uracil is a natural component of
RNA but can occur in the DNA by spontaneous deamination of an
inherently unstable base, cytosine (2). Occasional incorporation of
dUMP, in place of dTMP, during DNA synthesis is yet another way by
which uracil can arise in DNA (3). If left unrepaired, in a U:G
mismatch, uracil is promutagenic and can lead to GC The crystal structures of Escherichia coli UDG
(15-17) reveal that there is a remarkable conservation of the overall
architecture and the active site geometry between the UDGs from human,
bacterial, and viral sources. However, several crucial residues
(Gln63, Asp64, and Leu191) in
E. coli UDG show subtle conformational differences when
compared with those in human UDG (15, 16). The cocrystal structures of
the human herpes simplex virus-1 and E. coli UDGs
with Ugi (15, 17-19) reveal that Ugi binds UDG at the active site face and provides one of the most outstanding examples of molecular mimicry
of the DNA substrate.
The major interactions, which render the complex between UDG and Ugi
physiologically irreversible, are defined by (i) hydrogen bonding and
packing contacts derived from the complementarity between the conserved
Leu loop (187HPSPLS192) of E. coli
UDG and eight hydrophobic residues of Ugi (Met24,
Val29, Val32, Ile33,
Val43, Met56, Leu58, and
Val71) in a cavity burrowed between the Mutational and biochemical analyses form a necessary component of
understanding the mechanism underlying the macromolecular interactions.
However, the only mutational analysis that has been carried out to
understand the mechanism of UDG-Ugi interaction has been with respect
to the seven acidic residues (Glu20, Glu27,
Glu28, Glu30, Glu31,
Asp61, and Glu78) of Ugi. With the exception of
the E20I and E20L mutants, which formed reversible complexes, the other
mutants formed irreversible complexes with E. coli UDG (20).
The mutational analysis of neither UDG nor Ugi residues that are
involved in the hydrophobic interactions has been performed.
In this study, we have mutated the Leu191 of E. coli UDG to side chains of varying lengths and investigated the
effect of these mutations on the ability of the resultant UDGs to form
complexes with Ugi under in vitro and in vivo
conditions. In addition, we utilized two of these mutants to understand
the role of Leu191 in uracil excision from the single- and
double-stranded substrates. Importantly, during these studies, we have
developed a novel urea-polyacrylamide gel system, which has allowed us
to monitor the real time dissociation of UDG-Ugi complexes.
Oligodeoxyribonucleotides--
Oligonucleotides were obtained
from Bangalore Genei or Ransom Hill Bioscience. The oligomer, SSU9,
5'-d(ctcaagtgUaggcatgcaagagct)-3', is a single-stranded 24-mer
oligonucleotide substrate containing dU at the 9th position (from the
5'-end). The substrate AU9,
5'-d(ctcaagtgUaggcatgcttttgcatgcctacacttga)-3', is a 37-mer hairpin
oligonucleotide containing dU in the stem region. In AU9, the dU is in
the same sequence context as in SSU9 except that it is located in a
double-stranded region.
32P Labeling of
Oligodeoxyribonucleotides--
Oligonucleotides (10 pmol) were
5'-32P-end labeled using 10 µCi of
[ Generation of the Leu191 E. coli UDG Mutants and
Their Overexpression Constructs--
A DNA fragment (~800 base
pairs) harboring the sequence downstream of the BamHI site
within the ung gene was excised from pTZUng4 using
BamHI (24) and subcloned into a similarly digested pTZ19R to
generate pTZUng2B'. This recombinant plasmid is similar to pTZUng2B
(24) except that it harbors the insert in the opposite orientation.
Single-stranded DNA template derived from pTZUng2B' and the mutagenic
oligomers 5'-ATG CGC CGA ACC CGG CGA CGG-3', 5'-ATG CGC CGA A (G/C) C
CGG CGA CGG-3', and 5'-ATG CGC CGA AAA CGG CGA CGG-3' were used to
obtain L191G, L191A, and L191F mutants, respectively (25). All of the
mutants were confirmed by complete nucleotide sequencing. During the
process of generating these mutations, we serendipitously obtained the
L191V mutant. To reconstitute the Leu191 mutants into the
pTrcUDG expression vector, the relevant region of the mutant pTZ19R
constructs was excised as a NruI-HindIII fragment (~500 base pairs) and subcloned into similarly digested pTrcUDG to generate pTrc99c-based expression constructs for the L191G,
L191A, L191V, and L191F mutants. To develop the T7 RNA polymerase-based
expression constructs, the ung gene sequences were amplified
by polymerase chain reaction from the pTZ19R-based recombinants using
Pfu DNA polymerase and a universal primer, 5'-GTTTTCCCAGTCACGAC-3' that anneals to the vector sequence downstream of the multiple cloning site and a gene-specific primer,
5'-CGTGAAGCTTGACGGTACGGGC-3' that anneals in the
3'-flanking region of the ung gene and harbors a
HindIII site (shown in italics). After initial heating at
95 °C for 1 min, the reactions were subjected to 24 cycles of
temperature shifts at 95 °C for 45 s, 50 °C for 30 s,
and 72 °C for 1 min. Finally, the reaction was incubated at 72 °C
for 10 min. The polymerase chain reaction products were purified and
digested with BamHI and HindIII and used to
replace a similar region of the ung gene from a pET11d
construct harboring the ung gene open reading frame by using
standard recombinant DNA methods (26). The expression constructs in
pTrc99c and pET11d thus obtained were reaffirmed by nucleotide sequencing.
Purification of Leu191 UDG Mutants--
The
pET11d-based UDG gene constructs were transformed into E. coli BL21 (DE3), and the transformants were inoculated directly into 250 ml of 2YT medium (26). Cells were harvested, and UDG was
purified by a modification of the procedure described earlier (6).
Briefly, the bacterial cell pellet was suspended in TG buffer (20 mM Tris-HCl, pH 7.4, and 10% (v/v) glycerol) and lysed by
sonication. The lysate was clarified by centrifuging at 20,000 × g for 10 min at 4 °C to obtain the S-20 supernatant.
Streptomycin sulfate was added to a final concentration of 0.9% to the
S-20 extract and subjected to centrifugation at 20,000 × g for 10 min, and the supernatant was subjected to ammonium
sulfate fractionation. The pellet from the 40-60% ammonium sulfate
saturation was dissolved in TG buffer containing 500 mM
NaCl, loaded onto a G-75 column (80 × 7.1 cm2), and
eluted with the same buffer. The fractions enriched for UDG were pooled
and concentrated by ammonium sulfate fractionation (70% saturation).
The precipitate was suspended in 5 ml of HG buffer (20 mM
Hepes, pH 7.4, and 10% (v/v) glycerol), dialyzed against the same
buffer, and loaded onto a Mono-S column (5 ml; Bio-Rad), which was
equilibrated with the HG buffer. The proteins were eluted with a linear
gradient of 0-1 M NaCl in HG buffer. The fractions
enriched for UDG were dialyzed against TG buffer, loaded onto a HiTrap
heparin-Sepharose column (FPLC; Amersham Pharmacia Biotech)
equilibrated in the same buffer, and eluted using a gradient of 0-1
M NaCl in TG buffer. The fractions containing apparently
pure UDG were pooled, dialyzed against 20 mM Tris-HCl (pH
7.5) and 50% (v/v) glycerol, quantified (27), and stored at
Time Course of Uracil Excision from SSU9--
A reaction mixture
(70 µl) was set up in UDG buffer (50 mM Tris-HCl, pH 7.4, 1 mM Na2EDTA, 1 mM dithiothreitol,
and 25 µg/ml bovine serum albumin) containing 35 pmol of SSU9. The
reaction was started by adding 5 µl of an appropriate dilution of UDG
(96.5 pg of wild type, 2250 pg of L191G, 300 pg of L191A, 72 pg of
L191V, and 49.5 pg of L191F UDGs) at 37 °C. Aliquots (10 µl) were
removed at various time points (0, 2, 4, 6, 8, and 10 min), and the
reactions were terminated by adding 5 µl of 0.2 N NaOH
and heating at 90 °C for 30 min. The reaction mixture was dried in a
SpeedVac (Savant) and taken up in 10 µl of loading dye
containing 80% formamide, 0.1% xylene cyanol FF, bromphenol blue, and
1 mM Na2EDTA, and half of the contents was
electrophoresed on 15% polyacrylamide-8 M urea gels. The
bands corresponding to substrate and product were quantitated by a
BioImage Analyser (Fuji).
Determination of Km and
Vmax--
Reactions (15 µl) containing varying amounts
of 32P-labeled substrates and appropriate concentrations of
UDG in the reaction buffer (50 mM Tris-HCl, pH 7.4, 1 mM Na2EDTA, 1 mM dithiothreitol, and 25 µg/ml bovine serum albumin) were incubated at 37 °C for 10 min and stopped by adding 5 µl of 0.2 N NaOH. Cleavage at
the abasic site was achieved by heating the contents at 90 °C for 30 min, and the reaction products were analyzed on 15% polyacrylamide-8 M urea gels. The bands corresponding to the product and the
remaining substrate were quantified by using a BioImage Analyser
(Fuji). Values of Km and Vmax
were determined from Hofstee plots (28) of at least two independent experiments.
Formation of UDG-Ugi Complexes in Vitro and Their Analysis on
Polyacrylamide Gels Containing Urea--
UDG (2.5 µg) was mixed with
Ugi (1 µg) in a 15-µl volume consisting of 20 mM
Tris-HCl, pH 7.5, and incubated for 15 min at room temperature and then
for 15 min on ice (29, 30). Subsequently, loading dye (5 µl)
consisting of 50 mM Tris-HCl, pH 6.8, and 10% (v/v)
glycerol, and 0.1% bromphenol blue was added to the reaction, and it
was subjected to electrophoresis on 10-cm-long 15% polyacrylamide (19:1 cross-linking) gels of 0.75-mm thickness without or with 2, 4, 6, or 8 M urea. The electrophoresis at ~10 V/cm was carried out using running buffer (25 mM Tris-base, 192 mM glycine, pH 8.8). The proteins were visualized by
Coomassie Blue staining of the gels.
Bicistronic Constructs for Overproduction of UDG-Ugi
Complexes--
Bicistronic constructs of various UDG mutants and Ugi
were generated in pTrc99c exactly as described (30) except that the open reading frames of the Leu191 mutants (pTrc99c
constructs) were amplified by polymerase chain reaction using
Pfu DNA polymerase and the gene-specific forward (5'-CGGAATTCCATGGCTAACGAATTAACC-3') and reverse
(5'-GGAATTCCTATTACTCACTCTCTGCC-3') primers. Template DNA (50 ng) and 20 pmol of the forward and the reverse primers were utilized in
polymerase chain reaction under the following cycling conditions. The
initial denaturation temperature was 95 °C for 1 min followed by 25 cycles of repeated denaturation at 95 °C for 45 s, annealing
for 30 s at 50 °C, and extension at 72 °C for 1 min. The
final extension was allowed at 72 °C for 10 min. To generate the
bicistronic constructs in T7 RNA polymerase-based expression vector,
NcoI-HindIII fragments harboring the complete bicistron from the pTrc99c constructs were subcloned into similarly digested pET11d (26).
Direct Analysis of the in Vivo Formed UDG-Ugi
Complexes--
Overexpression of UDG and Ugi was achieved from the
pTrc99c-based expression constructs exactly as described (30). The
cell-free extracts were analyzed on 15% polyacrylamide (19:1
cross-linking) gels with or without 6 or 8 M urea.
Purification of the UDG-Ugi Complexes--
The purification of
the complexes of Ugi with the wild type and mutant UDGs was achieved by
the protocol described earlier (30) using the pET11d-based constructs.
Due to the high level expression from the T7 RNA polymerase based
vector, chromatography on DEAE-Sephacel and Mono-Q columns was found to
be unnecessary. The purified complexes were quantified (27) and stored
in 20 mM Tris-HCl, pH 7.4, at Isothermal Denaturation of the UDG-Ugi Complexes in
Urea--
UDG, Ugi, or the in vivo formed complexes of UDG
and Ugi (1-2.5 µM) were incubated at 25 ± 2 °C
for 4 h in the absence or presence of 2, 4, 6, and 8 M
urea in 1 ml of 20 mM Tris-HCl, pH 7.4. Intrinsic
fluorescence (tryptophan) changes were measured using a Jasco FP777
spectrofluorimeter with a thermostat cell holder using a cuvette of
1-cm path length and slit widths of 5 nm for excitation and emission.
Appropriate buffer controls were also scanned and
subtracted to arrive at the fluorescence intensity values. The
excitation wavelength was 280 nm, and the corresponding emission
spectra were recorded between 300 and 400 nm.
For detailed analysis of Ugi complexes with the wild type and the L191G
UDGs, fluorescence intensities were measured at 332.5 and 326.5 nm,
respectively. These wavelengths showed maximal fluorescence difference
between the native (untreated sample) and the 8 M
urea-treated samples. The emission intensities were converted into the
relative fluorescence changes and plotted against the corresponding
urea concentration. The relative fluorescence changes were calculated as (Ff Thermal Stability--
Thermal denaturation of the various Ugi
complexes with UDGs (wild type, L191G, L191A, L191V, and L191F) was
performed in a spectrophotometer (DU600; Beckman) as described (32).
The concentration of the protein used was 1 µM and the
absorbance changes were monitored at 287 nm. The first derivative of
the thermal denaturation profile obtained using the software supplied
with the instrument was used to evaluate the apparent transition
temperatures for the proteins. The apparent denaturation temperature
(apparent Tm) is defined as the temperature at which
half of the protein is in the denatured state.
Hyperexpression and Purification of the UDG Mutants--
Our
repeated attempts to overproduce the UDG mutants at Leu191
using the pTrc99c-based expression constructs in E. coli
BW310 (ung Analysis of UDG-Ugi Complexes--
Ugi, encoded by the B. subtilis phage, PBS-1 or -2, is a proteinaceous inhibitor of UDG
and is an exceptional mimic of the DNA substrate. Co-crystals of
UDG-Ugi show that the side chain Leu191 of UDG fits into a
hydrophobic cavity of Ugi (15, 17). It was therefore of interest to us
to analyze the stability of the complexes of Ugi with the UDG mutants
with varying length of the side chains at position 191. Since the
B. subtilis UDG, which is a natural target of Ugi, contains
phenylalanine in place of the Leu191 in the E. coli UDG, we were also interested in the study of L191F for its
ability to interact with Ugi.
Fig. 2A shows electrophoretic
analysis of the in vitro formed complexes of Ugi with UDGs
on a native polyacrylamide gel. UDG (pI, 6.6; molecular mass,
25.6 kDa) migrates slowest (lane 1), and Ugi (pI,
4.2; molecular mass, 9.4 kDa) migrates fastest (lane 6). The complex of the two proteins (pI, 4.9; molecular
mass, 35 kDa) migrates with intermediate mobility (Fig. 2A,
lanes 2-6). The electrophoretic analysis of the
native gel did not reveal any differences between the complexes of Ugi
with the wild type or the mutant UDGs. The wild type UDG forms a
physiologically irreversible complex with Ugi (29). In fact, the
interaction between UDG and Ugi is so strong that, unless heated, the
complex does not dissociate even after several hours of incubation in 8 M urea. Therefore, to study the effect of mutations at
Leu191, we developed a urea-polyacrylamide gel system (Fig.
2, B-E) to discriminate the UDG-Ugi complexes based on
their relative stability. In the presence of 2 or 4 M urea,
UDG (lane 1) and Ugi (lane
7) begin to migrate as diffuse bands (Fig. 2, B
and C), suggesting urea-mediated unfolding of these
proteins. However, upon further increase in urea concentration (6 and 8 M), their mobility on the gel was relatively more compact
(Fig. 2, D and E). This is most likely, because
with the increase in urea concentration, the unfolding of the proteins
was complete. More importantly, the complex of wild type UDG with Ugi
migrated as a sharp band even on a gel containing 8 M urea
(compare lanes 2 in Fig. 2, A-E).
This observation suggests that while the UDG and Ugi are individually
susceptible to urea-mediated unfolding, the complex of the two becomes
impervious to urea under these conditions. However, under the same
conditions, the complex of Ugi with L191G UDG was more susceptible to
urea-induced perturbation, and it began to dissociate in 4 M urea gel (appearance of smear in Fig. 2C,
lane 3), and the dissociation was complete in 6 M urea gel (Fig. 2D, lane
3). Unlike the L191G mutant, the complexes of L191A and
L191V with Ugi remain intact at 4 M urea (Fig.
2C, lanes 4 and 5) but
begin to dissociate in 6 M urea (Fig. 2D,
lanes 4 and 5). On the 8 M
urea gel, while the complex of Ugi with the wild type and the L191F
UDGs showed a detectable smear, the other complexes dissociated
completely. Furthermore, in the 8 M urea gel (Fig. 2E), we observed that from among the complexes of Ugi with
the L191G, L191A, and L191V mutants, the Ugi band that resulted upon the dissociation of the respective complexes (Fig. 2E)
migrated fastest in lane 3 (L191G),
intermediately in lane 4 (L191A), and slowest in
lane 5 (L191V). On the contrary, the mobility of
the smear corresponding to UDG was maximum for the L191V, intermediate for L191A, and least for L191G. Interestingly, the Ugi band in lane 3 (L191G) migrated just slightly slower than
the Ugi control (lane 7), suggesting that the
L191G-Ugi dissociated soon after its entry into the gel. Taken
together, the use of these urea-polyacrylamide gels enabled us to
monitor the progressive real time dissociation of the UDG-Ugi
complexes, and the relative stability of the Ugi complexes with the
various UDGs could be established in the following descending order,
wild type = L191F > L191V > L191A > L191G. This order of stability of the complexes demonstrates that replacement of
Leu191 with a shorter side chain (L191A) or its abolition
(L191G) resulted in a weaker interaction with Ugi.
Analysis of the in Vivo Formed UDG-Ugi Complexes on Urea
Gels--
We described a bicistronic construct for formation of the
UDG-Ugi complexes within the cellular milieu (30). Using similar bicistronic constructs, we have analyzed the stability of the in
vivo formed complexes of Ugi with the Leu191 mutants
without subjecting them to any purification. As reported earlier (30)
and also seen from the presence of free Ugi (along with the UDG-Ugi
complexes) on the native gel (Fig. 3),
the bicistronic constructs overproduce Ugi in excess of 1:1 molar
stoichiometry with UDG. The analysis of the cell-free extracts on the
urea-polyacrylamide gels shows that, similar to the in vitro
formed complexes, the L191G complex dissociated in 6 M
urea, whereas the complexes of L191A and L191V dissociated in 8 M urea. Also, the relative mobility of the dissociated Ugi
(Fig. 3, 8 M urea) reaffirmed the relative stability of the
complexes as determined for the in vitro formed complexes
(Fig. 2). Since, in this experiment, total cell-free extracts were
used, relative mobility of different UDG bands after dissociation from
the respective complexes could not be ascertained. More importantly,
this analysis demonstrates that the urea-polyacrylamide gels afforded
direct analysis of the in vivo formed UDG-Ugi complexes from
the cell-free extracts.
Spectrofluorometric Analysis of Urea-induced Unfolding of the
UDG-Ugi Complexes--
In these experiments, we have utilized the
changes in intrinsic fluorescence (tryptophan) as a correlate to
analyze the structural changes in the proteins upon isothermal
denaturation with varying concentrations of urea. As seen in Fig.
4A, incubation of Ugi and UDG
with different concentrations of urea (2-8 M) resulted in
the successive enhancement (for Ugi) and decrease (for UDG) of the
fluorescence intensities. Concomitant with the fluorescence change,
there was a red shift in the fluorescence spectra. These changes in the
intrinsic fluorescence and the red shift of the spectra are indicative
of urea-induced unfolding of proteins. Interestingly, when the complex
of Ugi with wild type UDG was subjected to a similar analysis, no
changes in the intrinsic fluorescence were observed (Fig.
4B, panel W. T.),
suggesting that while the individual proteins were highly susceptible
to urea-mediated unfolding, the complex of the two was recalcitrant to
such a treatment. However, when the other complexes were subjected to
similar analysis, at 6 M urea, the complex with L191G
showed fluorescence quenching along with a red shift in the spectrum.
This spectrum was not affected further at 8 M urea. Similar
changes in the spectral profile for the complexes with L191A and L191V
were observed upon incubation in 8 M urea. Strikingly,
these observations are in complete accordance with the analysis of
these complexes on the urea-polyacrylamide gels (Fig. 2,
A-E) and suggest that the intrinsic fluorescence quenching
and the concomitant red shift in the spectra correlate with the
dissociation of the UDG-Ugi complex. However, the complex of Ugi with
L191F mutant showed a gradual decrease of the intrinsic fluorescence
upon incubation with urea, but this was not associated with a red shift
of the fluorescence spectra. Considering that in our
urea-polyacrylamide gel, this complex was maintained even at 8 M urea, the fluorescence differences may indicate minor
perturbations in this complex that do not result in its dissociation
(see "Discussion").
Detailed analysis of urea-induced equilibrium unfolding studies for the
Ugi complexes with wild type and the L191G UDGs was also carried out by
monitoring fluorescence changes at a single wavelength at which the
difference in the fluorescence intensity is maximum between 0 and 8 M urea concentrations. These wavelengths for the Ugi
complexes with the wild type and the L191G mutant were found to be
332.5 and 326.5 nm, respectively. As seen in Fig. 4C,
consistent with the observations made in earlier experiments, while the
wild type complex did not register a significant change and responded
as a fully folded entity at all concentrations of urea, the L191G
(UDG)-Ugi complex showed a smooth transition between the folded and the
unfolded form at around 4.5 M urea. This analysis further
supports the observations made in Figs. 2 and 4B.
Determination of Melting Temperatures of Various UDG-Ugi
Complexes--
To further analyze the effect of mutations at
Leu191, we determined the apparent Tm
values of the various UDG-Ugi complexes (Table
I). The apparent Tm
values of the Ugi complexes with the wild type and the L191F UDGs were
~80 °C. However, the apparent Tm values of the
complexes formed by the L191G, L191A, and L191V mutants of UDG were
lower (~71-74 °C). Minor differences in the Tm
values of the complexes formed by L191G, L191A, and L191V UDGs do not
allow us to discriminate among them for their relative thermal
stability. Nevertheless, the Tm analysis does
highlight the role of the two naturally occurring amino acids, leucine
(in most of the UDGs) and phenylalanine (in the UDG from B. subtilis) at position 191 (or its equivalent) in forming a highly
stable UDG-Ugi complex.
Kinetics of Uracil Excision from SSU9 and
AU9--
Three-dimensional structure determinations of the co-crystals
of UDG with Ugi suggested that Ugi is a transition state substrate mimic (17). Our analysis of the Ugi complexes with the UDG mutants such
as the L191G and L191A showed that obliteration or reduction in the
length of the Leu191 side chain resulted in a decrease in
the stability of the complex. Also, as seen in Fig. 1B,
these are also the mutants that showed compromised UDG activity.
Further, based on an earlier study which proposed the "push-pull"
mechanism for localization of the uridine into the active site pocket
of the human UDG (36), it was of interest to analyze the effect of
these two mutations at Leu191 on the kinetic parameters of
uracil excision. The two substrates harboring dU in the same sequence
context but in different structural contexts were used for this
analysis. The SSU9 substrate harbors the dU residue in the
single-stranded ("unstructured") context, and the AU9 hairpin
harbors the dU residue in the double-stranded context in the middle of
the stem region. In both substrates, dU is located at the 9th position
from the 5'-end.
As predicted from the push-pull model for target site
recognition, the kinetic parameters (Table
II) of uracil excision from SSU9 and AU9
substrates using L191G and L191A mutants showed that they were
compromised in their uracil excision activity. The ratio of relative
efficiency (Vmax/Km) by which
the uracil was excised from the single- and double-stranded DNA by the
wild type UDG was ~10.4. More importantly, the ratio of relative
efficiency of uracil excision from SSU9 and AU9 was maintained even for
the L191G and the L191A mutants (~8.7 and 9.5, respectively). Thus, while the L191G and L191A were compromised in the uracil excision, the
relative utilization of single- and double-stranded substrates by them
was similar to that of the wild type. These analyses suggest that
changes at Leu191 must be affecting a common step in the
catalysis of uracil excision from the single- and double-stranded
substrates.
The ability of uracil DNA glycosylases to use both the single- and
double-stranded DNA as substrates makes these enzymes an ideal system
to study the mechanism of damage removal from DNA. Furthermore, since
the conserved UDGs from different sources form a highly stable complex
with the B. subtilis phage (PBS-1 or -2)-encoded proteinaceous inhibitor (Ugi), these proteins have also been of interest to understand the mechanism of protein-protein interaction. Due to their small sizes, these proteins have been amenable for three-dimensional structural determinations by x-ray crystallography and NMR. Several crystal structures have shown Ugi to be a mimic of the
DNA substrate. Remarkably, the recent comparative analysis of the
UDG-Ugi structure with the UDG-DNA structure demonstrates that Ugi
mimics the transition state of the DNA bound to UDG (17). One of the
contacts that UDG establishes with Ugi is through its highly conserved
Leu191 into the hydrophobic pocket of Ugi. This pocket is
shaped by the side chains of Met24, Val29,
Val32, Ile33, Val43,
Met56, Leu58, and Val71 between the
We have investigated the effect of mutating Leu191 of
E. coli UDG on its ability to form a complex with Ugi and on
the efficiencies of uracil excision from the single- and
double-stranded DNA substrates. The mutational analysis shows that
shortening or deletion of the Leu191 side chain results in
UDGs whose complexes with Ugi are less stable than the one formed by
the wild type UDG. Both the electrophoretic analysis on urea gels
(Figs. 2 and 3) and apparent Tm determinations
(Table I) show that the L191F mutant formed a complex with Ugi, which
was as stable as that of the wild type UDG. These observations are
consistent with the prediction that bigger side chains can be
accommodated in the Ugi pocket (17). Thus, the reason(s) for slight
decrease in the intrinsic fluorescence of the L191F mutant complex with
Ugi in the presence of urea (Fig. 4B) are unclear at
present. However, it may be noted that, in E. coli UDG, the
Leu191 is differently oriented than that from human UDG,
and the context of the sequence within the DNA intercalation loop is
likely to be important for its docking into the hydrophobic cavity of
Ugi (15, 17). It would be interesting to study the urea-induced intrinsic fluorescence changes in the complex of B. subtilis
UDG, which is the biological ligate of Ugi and where
phenylalanine occurs naturally at this position. The significance of
the differently oriented HPSPLS loop is also evident from the
observation that leucine to alanine mutation in the HPSPLS motif in the
context of E. coli UDG still retained about 10% enzymatic
activity (Table II), whereas the same mutation in the context of human
UDG resulted in severe loss of the activity (1% as active as the wild
type enzyme) (37).
Cocrystal structure of an engineered human UDG (L272R/D145N)
with the in situ cleaved products led the authors to suggest the push and pull mechanism for localization of the uracil into the
active site pocket of the enzyme. It was proposed that the Leu272 side chain (L of the HPSPLS motif) inserted into the
base stack through the minor groove, causing extrahelical localization
("push") of the target uridine, which was then accommodated
("pull") into the active site pocket by establishing
uracil-specific contacts (36). However, subsequently from a high
resolution crystal structure (37), it was proposed that the uracil
localization into the active site pocket of the enzyme utilized a
concerted mechanism of "pinch, push, and pull." The serine residues
of the highly conserved HPSPLS and PPPS motifs hydrogen-bonded with the
phosphates flanking the target uridine, resulting in compression and
kinking of the DNA backbone in the vicinity of the lesion, leading to the expulsion of the target base. More interestingly, it was seen that
even in the L272A mutant containing a shorter side chain, which was
unable to reach into the base stack, the nucleotide flipping occurred,
suggesting that Leu272 was not absolutely critical for the
localization of the target uridine into the active site pocket (37).
Our results on the kinetics of uracil excision from both the single-
and double-stranded substrates by the E. coli UDG
Leu191 mutants are in complete agreement with the pinch,
push, and pull mechanism of the damage recognition. Thus, shortening of
the Leu191 side chain (L191A) or deleting it altogether
(L191G) decreased the uracil excision efficiency to ~10 and 1%,
respectively (Table II, Fig. 1B).
However, from a different perspective, the double-stranded DNA involves
a defined network of base-stacking and hydrogen-bonding interactions;
in energetic terms, the extrahelical localization of uracil from
double-stranded substrates would be more demanding compared with the
single-stranded substrates. Therefore, it may be argued that the
penalty for shortening the Leu191 side chain (L191A) or
deleting it altogether (L191G) will be higher for uracil excision from
the double-stranded substrates as compared with the single-stranded
substrate. Interestingly, we observed that although the L191A and L191G
showed decreased efficiency of uracil excision, the relative ratio of
uracil excision from the single-stranded (SSU9) and double-stranded
(AU9) substrates was unaffected (compared with wild type, the relative
ratios of uracil excision from single-stranded versus
double-stranded substrate for L191A and L191G were 0.91 and 0.84, respectively). Thus, our results point out to the role of
Leu191 at a step utilized by both the single- and
double-stranded substrates in common. These observations can be easily
rationalized if the interaction between the Ser88,
Ser189, and Ser192 and the phosphates flanking
the target uridine (37-39), which lead to the compression (the
"pinch" step) of the sugar phosphate backbone, was the first
event to take place in the substrate recognition. The kinking of
the backbone would result in destabilization of the hydrogen bonds of
the target uracil with its complement A or G. Thus, much of the
discrimination that is seen in utilization of substrates containing
uracil in different structural contexts (23, 40) should occur at the
step of interaction of UDG with the DNA backbone. And during catalysis,
if the Leu191 participated subsequent to this step, it will
not discriminate between the single- and double- stranded substrates.
Since the "push" step is not absolutely required for the
localization of the target uridine into the active site pocket of the
enzyme, based on the observation that the L191G (which lacked the side
chain) was highly compromised in its enzymatic activity, we envisage an
additional role of leucine of the HPSPLS motif. Once the uridine is
localized into the active site pocket, this leucine could be important
in the stabilization of the productive enzyme-substrate complex
by inserting its side chain into the void generated between the
flanking bases subsequent to extrahelical localization of the target
uridine. Thus, a parallel could be drawn for the role of
Leu191 in imparting stability to the UDG-Ugi complex by its
docking into the hydrophobic cavity of Ugi on one hand and
stabilization of the productive enzyme-substrate complex by
inserting its side chain between the flanking bases on the other (Fig.
5, a and b). This
stabilization would be impaired for the mutant L191A (c and d) and abolished altogether in the case of L191G
(e and f). In fact, the surface plasmon resonance
study with the wild type and the L272A of human UDG (equivalent of
L191A of E. coli UDG) did show that while the "on rates"
of enzyme binding to a nonhydrolyzable uracil (4'-S-dU) in a
double-stranded (in a U:A base pair) substrate for the wild type
and the L272A mutant were similar (ka, 0.16 versus 0.15 ×106/M/s), L272A showed
faster "off rates" as compared with the wild type UDG (0.13 versus 0.05/s) (37). The Km measurements for the L191G mutant (Table II) are also consistent with these studies.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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AT transition
mutations during the next round of replication. Further, uracil, in an
A:U pair in the DNA sequences can impend their recognition by the
cognate regulatory proteins (4). In cells, a highly efficient base
excision repair enzyme, uracil-DNA glycosylase (UDG),1 is dedicated to the
indomitable task of freeing the DNA from uracil residues (5, 6). UDGs
have been identified from a large number of prokaryotic and eukaryotic
organisms, and these enzymes show a high degree of conservation from
bacteria to viruses to humans (7, 8). UDGs also interact with a number
of proteins such as the Bacillus subtilis phage-encoded
uracil DNA glycosylase inhibitor, Ugi, and host cellular factors such
as single-stranded DNA-binding protein and proliferating cell nuclear
antigen (9-12). Thus, UDGs constitute a remarkably interesting
model system to understand the basis of catalytic prowess and
specificity associated with protein-DNA and protein-protein
interactions. The mechanism of uracil excision that has emerged from
various structural studies and mutational analyses of UDGs is that the
glycosidic bond between uracil and the sugar is cleaved by the attack
of a hydroxyl nucleophile on the deoxyribose C1' atom. This
nucleophile is generated by the activation of a water molecule by an
absolutely conserved Asp of the GQDPYH motif. Concomitant protonation
of the O2 of the uracil base by His of yet another highly conserved
motif, HPSPLS, enhances its leaving group quality (13, 14).
2
and the antiparallel
sheet of Ugi and (ii) the electrostatic
interactions between the acidic residues of the
1 edge
of Ugi with the key active site residues of E. coli UDG.
This structure also showed that the interaction between UDG and Ugi
results in the burial of about 2200 Å2 of the total
accessible surface area. The nestling of Leu191 into the
hydrophobic cavity of Ugi alone causes the exclusion of about 250 Å2 of the surface area (17).
MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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-32P]ATP (6000 Ci/mmol) and T4 polynucleotide kinase
in 10-µl reaction volumes (21) and purified by chromatography on
Sephadex G-50 minicolumns (22). This procedure routinely resulted in
labeling efficiency of ~106 cpm/pmol. For
Km and Vmax determinations,
radiolabeled oligonucleotides were mixed with cold substrates such that
the contribution from labeled substrates was much less than 1%
(23).
20 °C.
20 °C.
Fu)/Ff, where
Ff corresponds to the fluorescence intensity of the
native proteins and Fu corresponds to the
fluorescence intensity of the urea-treated proteins (31).
RESULTS
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MATERIALS AND METHODS
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DISCUSSION
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) remained unsuccessful. Therefore, we used the
T7 RNA polymerase-based expression constructs in E. coli
BL21 (DE3). The pET11d-based constructs afforded hyperexpression of UDG
even in the absence of induction. SDS-polyacrylamide gel analysis (33)
revealed that the high level expression and the use of various column
chromatography steps (see "Materials and Methods") yielded UDGs
that were purified to apparent homogeneity (Fig.
1A). Although the E. coli BL21 (DE3) is wild type for ung, it has been shown
that the UDG preparations obtained from such overexpression constructs
are essentially free from the host chromosomal background
(34).2 The T7 polymerase
based expression systems for E. coli UDG mutants have been
used in other studies as well (16, 35). Time course experiments (Fig.
1B) wherein a single-stranded DNA oligomer (SSU9) was used
as substrate showed that the wild type and the L191V and L191F mutants
were comparable in their uracil excision activity. However, compared
with the wild type UDG, the L191A and L191G mutants retained about 10 and 1% enzymatic activity, respectively.
View larger version (19K):
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Fig. 1.
A, analysis of purified UDGs (2.0 µg
each) on a 15% polyacrylamide gel containing 0.1% SDS.
Lane 1, WT; lane 2, L191G;
lane 3, L191A; lane 4,
L191V; lane 5, L191F. B, excision of
uracil from single-stranded uracil-containing oligomer (SSU9) by the
wild type and the mutant UDGs. The reactions were carried out as
described under "Materials and Methods" for the indicated times
with wild type ( ), or the mutant UDGs, L191G (
), L191A
(
), L191V (
), and L191F (
).
View larger version (73K):
[in a new window]
Fig. 2.
Analysis of the UDG-Ugi complexes on 15%
polyacrylamide gels in the absence (A) or presence of
2 M (B), 4 M
(C), 6 M (D), and 8 M (E) urea. Lane
1, E. coli UDG; lanes 2-6,
complexes of Ugi with the wild type, L191G, L191A, L191V, and L191F
UDGs, respectively; lane 7, Ugi. The various
arrows used to denote UDG, Ugi, or the complex of the two
are shown in the last panel.
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Fig. 3.
Analysis of the in vivo
formed complexes of Ugi with UDG. The cell-free extracts
from the transformants harboring the bicistronic construct for Ugi and
the various mutants of UDG were analyzed on 15% polyacrylamide gels
containing no urea or 6 or 8 M urea. Lane
1, UDG (wild type)-Ugi complex; lanes
2-5, ~10 µg of total proteins from the transformants
harboring the bicistronic construct for L191G, L191A, L191V, and L191F,
respectively; lane 6, Ugi. The various
arrows used to denote Ugi and its complex with UDG are the
same as in Fig. 2.
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Fig. 4.
A, intrinsic fluorescence (Trp) changes
in UDG, and Ugi (1 µM) in response to urea. The
excitation wavelength was 280 nm, and the emission spectra were
recorded between 300 and 400 nm. B, intrinsic fluorescence
(Trp) changes in UDG-Ugi complexes (2.5 µM) in response
to urea. Excitation wavelength was 280 nm, and the emission spectra
were recorded between 300 and 400 nm. A-E, complexes of Ugi
with WT, L191G, L191A, L191V, and L191F UDGs, respectively. The various
concentrations of urea with which they were incubated are as shown in
the lower right. C, difference
fluorescence spectra showing changes in the intrinsic fluorescence
(tryptophan) of the UDG-Ugi complexes. The complexes were either not
treated with urea or treated with the indicated concentration of urea
in 20 mM Tris-HCl (pH 7.4) at 25 ± 2 °C for 4 h before recording the changes in fluorescence emission intensities at
332.5 and 326.5 nm, respectively for complexes of Ugi with the wild
type and the L191G UDG. These wavelengths corresponded to
em at which maximum difference in fluorescence
intensities were observed between the native (0 M urea) and
treated (8 M urea) proteins. The emission intensities were
converted to relative fluorescence changes and plotted against the
corresponding denaturant concentrations. The filled and the
open circles show relative fluorescence changes
of the Ugi complexes with the wild type and the L191G UDGs,
respectively.
Apparent Tm (°C) values of wild type UDG-Ugi, L191G-Ugi,
L191A-Ugi, L191V-Ugi, and L191F-Ugi complexes
Kinetic parameters for uracil excision from SSU9 and AU9
DISCUSSION
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ABSTRACT
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DISCUSSION
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1 edge of the antiparallel
-sheet and the
2 helix. This cavity in Ugi is functionally similar to
the void generated in the DNA base stack upon extrahelical localization
of uridine.
View larger version (42K):
[in a new window]
Fig. 5.
Stereo view models highlighting the
interaction of the residue 191 (within the DNA intercalation loop) of
E. coli UDG (in red) with Ugi
(a, c, and e) and the uncleaved DNA
substrate (b, d, and f) generated by
the INSIGHTII package using the coordinates from crystal structures of
E. coli UDG-Ugi (Protein Data Bank code 1UUG; Ref. 17)
and human UDG-DNA (Protein Data Bank code 1EMH; Ref. 41). The side
chain of Leu191 is shown inserting into the hydrophobic
pocket of Ugi (a) and into the void between the neighboring
bases generated upon flipping out of the target uridine (b).
c and d, same as a and b
except that the leucine side chain has been replaced with alanine,
which results in its partial entry into the respective hydrophobic
cavities. e and f, same as a and
b except that the leucine side chain has been changed to
glycine, which results in the complete abolition of the hydrophobic
contact seen before between the Leu191 and Ugi or the
DNA. The Ugi and the target DNA strand are shown in
green. The DNA complementary to the target strand is shown
in yellow.
Finally, in this study, we have developed a polyacrylamide gel system
that has allowed us to discriminate among the UDG-Ugi complexes on the
basis of their stability in urea. The results obtained from the
urea-polyacrylamide gels were in complete agreement with those obtained
from the biophysical methods (Fig. 4). More importantly, the urea gel
analysis not only used much smaller amounts of the protein but also
permitted the analysis of the UDG-Ugi complexes directly from the
cell-free extracts. Also, the observation that the relative stability
of the complexes of Ugi with WT, L191G, L191A, L191V, and L191F UDGs
could be ascertained from a single gel (Figs. 2 and 3) highlights the
potential of this gel system in facile screening of a large number of
mutants of the two interacting proteins.
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ACKNOWLEDGEMENT |
---|
We thank K. Saikrishnan for invaluable help in generating the models shown in Fig. 5.
![]() |
FOOTNOTES |
---|
* This work was supported in part by research grants from the Council of Scientific and Industrial Research (CSIR), the Department of Biotechnology, and the Department of Science and Technology, New Delhi, India.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.
Supported by a senior research fellowship of CSIR.
§ To whom correspondence should be addressed. Tel.: 91-80-309-2686; Fax: 91-80-360-2697 or 91-80-360-0683; E-mail: varshney@ mcbl.iisc.ernet.in.
Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M011166200
2 P. Handa, S. Roy, and U. Varshney, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: UDG, uracil DNA glycosylase; WT, wild type.
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