From the Department of Biochemistry, School of Hygiene and Public
Health, The Johns Hopkins University, Baltimore, Maryland 21205
The DNA-dependent ATPase activity of
UvrB is required to support preincision steps in nucleotide excision
repair in Escherichia coli. This activity is, however,
cryptic. Elicited in nucleotide excision repair by association with the
UvrA protein, it may also be unmasked by a specific proteolysis
eliminating the C-terminal domain of UvrB (generating UvrB*). We
introduced fluorescent reporter groups (tryptophan replacing
Phe47 or Asn51) into the ATP binding motif of
UvrB, without significant alteration of behavior, to study both
nucleotide binding and those conformational changes expected to be
essential to function. The inserted tryptophans occupy moderately
hydrophobic, although potentially heterogeneous, environments as
evidenced by fluorescence emission and time-resolved decay
characteristics, yet are accessible to the diffusible quencher acrylamide. Activation, via specific proteolysis, is accompanied by
conformational change at the ATP binding site, with multiple changes in
emission spectra and a greater shielding of the tryptophans from
diffusible quencher. Titration of tryptophan fluorescence with ATP has
revealed that, although catalytically incompetent, UvrB can bind ATP
and bind with an affinity equal to that of the active UvrB* form
(Kd of ~1 mM). The ATP binding site of UvrB is therefore functional and accessible, suggesting that conformational change either brings amino acid residues into proper alignment for catalysis and/or enables response to effector DNA.
 |
INTRODUCTION |
Two challenges to our understanding of nucleotide excision repair
in Escherichia coli are 1) the remarkable breadth of
structural damage to DNA recognized by the Uvr repair proteins and 2)
the complexity and energetics of assembly of an incision complex at the
damaged site (reviewed in Refs. 1-3). The extent of linkage between
these may become more clear as the mechanisms of this multistep process
are revealed in greater detail.
One of the greater enigmas remaining in our attempt to understand these
mechanistic steps is that of the multiple roles of ATP binding and
hydrolysis by the UvrA and, especially, UvrB proteins. Both recognition
of damage and incision (an energetically favorable reaction) are
accomplished by other, specific endonucleases without the need for ATP
(1). UvrA is a DNA-independent ATPase, with two ATP binding sites, both
essential in nucleotide excision repair (4-6). UvrB, although it
contains one Walker type A ATP motif (7-9), is catalytically silent in
isolation. A cryptic, DNA-dependent ATPase activity is
elicited, however, by interaction with UvrA (10, 11), and this
UvrB-associated activity is required for ensuing steps in damage
location and in formation of the preincision complex.
The obligate nucleotide cofactor ATP has multiple roles in the
succession of macromolecular association/dissociation reactions that
lead to dual endonucleolytic incisions flanking a damaged site in DNA.
The binding of ATP promotes dimerization of UvrA (12, 13) and the
interaction of UvrA2 with UvrB and DNA (6, 14). It is
significant that initial formation of this nucleoprotein complex, which
is accompanied by a localized unwinding of the DNA (15), occurs at an
undamaged site (13, 16). The ability to disengage from the nonspecific
loading site, and thus to locate damage, is dependent on ATP hydrolysis
by the UvrB component of the repair complex. Mutation of a conserved
lysine in the UvrB ATP motif (K45A), which knocks out the UvrB ATPase,
results in formation of a very stable, damage-insensitive
UvrA2-UvrB(K45A)-DNA complex (16). Once released from the
loading site, ATP hydrolysis by UvrB may also be required to support
the search for damage along DNA via a tracking mechanism (1)
and/or to promote the conformational changes in DNA and/or in UvrB that
lead to formation of the stable UvrB-damaged DNA preincision complex,
from which UvrA dissociates (14, 17-22). With the addition of UvrC,
dual incision ensues, with the 3' cut apparently dependent on the
binding although not on hydrolysis of ATP by UvrB (23-25).
The mechanistic role of the UvrB ATPase may be, at least in part, to
power a UvrA2-UvrB helicase activity. UvrB itself has the
full complement of conserved helicase motifs (26, 27); yet strand
displacement activity, 5' to 3' and specific for short oligomers or
D-loops (28, 29), requires association with UvrA. The K45A mutation in
motif I (ATP binding site), cited above, eliminates the strand
displacement activity (16), as well as the ability of the
UvrA2-UvrB complex to generate supercoiling in plasmid DNA
(30), suggesting a role in complex translocation. Mutations in other
helicase motifs (V and VI) have substantiated the role of the strand
displacement and ATPase activities associated with UvrB in formation of
the preincision complex (27).
Conformational changes, to convey allosteric communications among
binding and catalytic domains and to couple the energy of nucleotide
binding or hydrolysis to physical displacements (work), are at the
heart of proposed helicase mechanisms (31). To fulfill its role in
repair, it is postulated that the UvrB protein must undergo such
multiple conformational changes: to transduce allosteric signals
associated with UvrA or UvrC binding and association with double or
single-stranded DNA (with or without damage), and to couple binding or
hydrolysis to movement. In this paper, we begin to examine the
conformational dynamics of UvrB by use of fluorescent reporter groups
that provide a signal responsive to their local environment, and that
can report on changes on the nanosecond time scale. The focus of this
study is on the initial activation of the UvrB ATPase, using as a model
the serendipitous activation by a specific proteolysis carried out by
the ompT gene product. Truncation of either 43 (8) or 66-68
amino acids (32) from the C terminus of UvrB, which deletes no
chromophores from the protein, yields an active
DNA-dependent ATPase, denoted UvrB* (10). UvrB* can
interact with UvrA and form a preincision complex, but one that is less
stable and with greatly reduced capacity for incision with added UvrC.
Tryptophan (absent in the wild-type protein) has been introduced
without significant alteration of function into the UvrB ATP binding
motif, both at a site shown earlier to be mutable without phenotypic
effect (Asn51) and at a residue nearer the essential and
conserved lysine (Lys45) of the Walker motif
(Phe47). Both "reporter mutants" reveal conformational
changes, localized to the binding site, that accompany proteolytic
activation. Furthermore, the tryptophan signal may be titrated to
characterize nucleotide binding properties of UvrB, information
previously unobtainable.
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EXPERIMENTAL PROCEDURES |
Materials--
Acrylamide (ultrapure), NATA, and ATP were
purchased from Sigma. Restriction enzymes and their buffers were
obtained from New England Biolabs. The plasmid pTZuvrB, constructed by
T. Seeley in this laboratory, was used as template for mutagenesis of
the uvrB gene and for protein expression (16). A
SacI-PstI fragment of ~4.5 kilobase pairs from
pUCuvrB (11), containing the endogenous promoters and complete coding
sequence of the wild-type uvrB gene of E. coli
(from strain AB1157), was cloned into the polylinker site of pTZ19R
(obtained from Amersham Pharmacia Biotech), generating an
ApR vector of approximately 7.4 kilobase pairs. E. coli hosts for pTZuvrB included JM109 (recA1,
endA1, gyrA96, thi
,
hsdR17, supE44, relA1, 
,
(lac-proAB), F
(traD36, proAB,
lacIq Z
M15)), for long term storage; DH5
(F
, endA1, his R17 (rk
,
mk+) sup E44, thi
,

, rec A1, gyr A96, rel A1,
(arg, F
, lac zya) U169,
80
lac Z
M15), obtained from Life Technologies, Inc. and
used for transformation with putative mutants; and the uvrB
deletion strain N364 (W3110 gal+, sup0,
F
,
(attB-bio-uvrB)), obtained from M. Gottesman (Columbia University).
Site-specific Mutagenesis--
A "cassette" mutagenesis
strategy was used to introduce tryptophan into the ATP binding motif of
UvrB, replacing 43 nucleotides excised as an
XmnI-NcoI restriction fragment (nucleotides
577-624) with a synthetic duplex (sequence and numbering from Ref. 8). The oligonucleotides (Fig. 1) bore the following codon changes to the
published wild-type sequence: for mutant F47W, TTC to TGG; N51W, AAT to
TGG. The 43-mer was annealed to its synthetic complement (following
5'-phosphorylation of both using T4 polynucleotide kinase), a 47-mer
extended by four bases at the complement's 5'-end to allow
regeneration of the NcoI restriction site. Oligonucleotides were prepared using a MilliGen/Biosearch 7500 DNA synthesizer.
Double digestion of plasmid pTZuvrB DNA with SacI and
NcoI yielded two fragments, of ~6485 and 865 nucleotides.
The shorter fragment, encompassing the 5'-end of the coding sequence,
was further digested with XmnI. The fragment of ~815
nucleotides was retained, and the wild-type equivalent of the mutagenic
synthon was discarded (fragments purified by electroelution). A
three-fragment ligation (8:2:1 molar ratio of 43-base pair mutagenic
oligonucleotide to 815-base pair SacI-XmnI to
6485-base pair SacI-NcoI, respectively) generated the mutant pTZuvrB vectors (see Fig. 1). Ligation products were transformed into DH5
for screening by dideoxy sequencing (Sequenase version 2.0, Amersham Pharmacia Biotech; primers synthesized using the MilliGen/Biosearch 7500 DNA synthesizer).
UV Resistance Screening--
Wild-type and mutant pTZuvrB
plasmids were transformed into the uvrB deletion strain
N364. Cells in early exponential growth were irradiated at varying
doses with ultraviolet light and assayed for survival by colony counts,
as described by Seeley and Grossman (11).
Proteins--
Mutant and wild-type UvrB proteins were purified
from the appropriate N364 (
uvrB)/pTZuvrB strain in late
exponential growth. Cells, resuspended in lysis buffer (100 mM Tris-Cl (pH 7.5), 20% sucrose, 5 mM EDTA, 2 mM DTT)1 were
lysed by a 1-h incubation with lysozyme (0.25 mg/ml), followed by the
addition of NaCl to 0.3 M. Purification then followed the published protocol (33) with the following modifications: fast protein
liquid chromatography (FPLC) using Butyl Sepharose 4 Fast Flow
(Amersham Pharmacia Biotech) supplanted phenyl-agarose in the
third chromatography step; Sephacryl S300 (Amersham Pharmacia Biotech) was used in place of Sephadex G-150; and FPLC using a Mono Q
column (Amersham Pharmacia Biotech) was added as a final step. The
butyl-Sepharose column was packed and equilibrated in a loading buffer
composed of 1 M ammonium sulfate, 50 mM Tris-Cl (pH 7.5), 25 mM KCl, 1 mM DTT, 5% glycerol.
Ammonium sulfate was added to the fractions pooled from the
DEAE-Sephacel column to a concentration of 1 M prior to
loading. Protein was eluted with a linear gradient from 1 to 0 M ammonium sulfate, and other buffer components were
unchanged. UvrB (or mutant)-containing fractions were concentrated by
ultrafiltration (Amicon Diaflo YM30 membrane) before loading on a 4.9 cm2 × 100-cm Sephacryl S300 sizing column, equilibrated
and eluted at 0.6-0.75 ml/min with a buffer composed of 50 mM Tris-Cl (pH 7.5), 300 mM KCl, 15% glycerol,
1 mM DTT. The Mono Q column (HR10/10) was equilibrated in a
buffer of 1 M KCl in 20 mM Tris·Cl (pH 7.5), 5% glycerol, 1 mM DTT. Protein was loaded at this KCl
concentration and eluted in a linear gradient from 1 M to
25 mM KCl. The purified UvrB proteins (KCl concentration of
the pooled Mono Q fractions was approximately 300 mM) were
concentrated by ultrafiltration, supplemented with glycerol to 30%,
and frozen in liquid nitrogen for storage at
80 °C. The
purification of UvrA and UvrC proteins has been described (33).
The proteolytically truncated proteins, UvrB* and its mutant
counterparts, were produced and purified as described (16), using the
ompT-expressing E. coli strain UT5600/pML19
(34).
The concentration of protein in stock solutions was determined
spectrophotometrically by absorbance at 280 nm in a buffer of 6 M guanidine hydrochloride, 20 mM phosphate, pH
6.5 (35). Molar extinction coefficients of 29,560 M
1 cm
1 for wild type and 35,250 M
1 cm
1 for both Trp reporter
mutants were calculated from the amino acid sequence (36). The protein
assay kit of Bio-Rad, based on the Bradford assay (37), using bovine
serum albumin as a standard, was used for comparison among diluted
samples.
Incision Assay--
The comparative ability of mutant and
wild-type UvrB proteins to complement UvrA and UvrC in the incision of
UV-irradiated DNA was assayed by conversion of supercoil to nicked
plasmid, as resolved by agarose gel electrophoresis. Reaction mixtures included 25 ng of 3H-labeled plasmid (pHE6; 3996 base pairs
at 105 cpm/µg, exposed to 750 J m
2 at 254 nm (11)) and UvrA and UvrC proteins at saturating concentrations in 40 mM K+-MOPS (pH 7.6), 85 mM KCl, 1 mM EDTA, 1 mM DTT, 15 mM
MgSO4, 2 mM ATP, and bovine serum albumin (to
50 µg/ml). UvrB was added at varying concentrations prior to a 5-min
incubation at 37 °C. Reactions were stopped by the addition of
sodium dodecyl sulfate (to 0.2%) and EDTA (to 20 mM), and
DNA species were separated by electrophoresis on a 0.8% agarose gel,
1 × TBE buffer. Ethidium bromide staining of outer lanes
containing marker DNA was used to locate substrate and product bands.
These were excised and solubilized in 21% hydrogen peroxide, 17%
perchloric acid, and activity was measured by liquid scintillation
counting (Biosafe II mixture, Research Products International). The
extent of reaction was calculated by use of the Poisson
distribution.
ATPase Assays--
Comparisons of mutant and wild-type UvrB
ATPase properties, in association with UvrA protein, or of the
proteolyzed form of UvrB in isolation were performed essentially as
described (11, 16). The reaction, using
-32P-labeled
ATP, in 50 mM K+-MOPS (pH 7.5), 100 mM KCl, 15 mM MgCl2, 1 mM DTT and bovine serum albumin (to 50 µg/ml), was
monitored by thin layer chromatography. Chromatograms were scanned by a
phosphor imager (Fujix BAS 1000), using MacBas version 2.0 for
intensity quantitation. UvrB was added to varying concentrations either
to 70 nM UvrA in assays omitting DNA or to 15 nM UvrA in the presence of UV-irradiated plasmid DNA
(pTZ18R at 30 µM nucleotide, irradiated at 254 nm to 750 J m
2). The kinetic properties of UvrB* and the
proteolyzed mutants, in the absence of UvrA, were determined using
single-stranded DNA (ssDNA) as effector (
X174 phage DNA at 200 µg/ml).
Steady State Fluorescence--
Steady state emission spectra and
intensity measurements were obtained with a Perkin-Elmer LS-50
luminescence spectrometer, with cuvette temperature controlled by a
Lauda circulating bath. All spectra and intensity measurements were
corrected for background emission and scatter and, where appropriate,
inner filter effects. Spectra are not corrected for
wavelength-dependent instrument effects (output and
photomultiplier response), but their stability was monitored during all
assays by use of a p-terphenyl standard (available from
Perkin-Elmer embedded in polymethylmethacrylate; excitation at 295 nm,
emission at 340 nm). Quantum yields were estimated by direct comparison
of emission intensities of protein samples to equimolar aqueous
solutions of tryptophan, using a value of 0.14 for the quantum yield of
tryptophan in water (38).
Quenching experiments with acrylamide were performed in stirred cells,
at 25 °C, titrating from a stock of 3 M acrylamide. UvrB
Trp reporter mutants were at 1 µM concentration in 25 mM HEPES (pH 7.5), 100 mM KCl. Tryptophan
emission, monitored at 340 nm, was selectively observed using 295-nm
excitation. Intensity data following quencher additions were averaged
over a 10-s collection and corrected for background emission (paired
control lacking protein), dilution, and inner filter effect (calculated
from acrylamide absorbance at 295 nm as described by Lakowicz (39)).
Intensities, F, at given quencher concentration, [Q], were
then analyzed using the Stern-Volmer equation,
|
(Eq. 1)
|
where F0 is the emission intensity of the
protein in the absence of quencher, and KS-V is
the Stern-Volmer constant for quenching, given by the slope when data
are plotted as F0/F versus [Q]
(40). Where there is an apparent static component to the quenching
mechanism, a modified form of the Stern-Volmer equation is used to
describe total quenching and estimate the contributions of dynamic and static interactions (41),
|
(Eq. 2)
|
where the additional term, V, is a descriptor of the
probability of an instantaneous interaction with a nearby quencher.
Fluorescence emission was similarly titrated for study of ATP binding.
Protein, at 1 µM, was assayed in 25 mM HEPES
(pH 7.5), 85 mM KCl, 15 mM MgCl2, 1 mM DTT, 10% glycerol in stirred cells at the indicated
temperature. Excitation again was at 295 nm, with emission spectra
collected in triplicate, and averaged for each addition of ATP. A
paired titration substituting N-acetyltryptophanamide (NATA)
for protein was performed to empirically correct for the significant
inner filter effects arising from ATP absorbance as well as from
dilution (at the maximum ATP concentration, 8 mM, the
measured reduction in light intensity, (I0
I)/I0, as gauged by the decrease in
NATA emission, (F0
F)/F0, was 0.35 ± 0.02).
To obtain binding data from the ATP titrations, data were fit to the
model,
|
(Eq. 3)
|
where
F is the observed change in fluorescence
intensity (observed minus initial, corrected), Kd is
the association constant, and L is the concentration of free
ligand (approximated by the total ATP concentration). This model
assumes a single binding site per protein molecule, with no interaction
among proteins, and represents a trivial derivation from a standard
binding expression (Eadie). In this model, simple yet appropriate to
the published characteristics of UvrB, fluorescence quenching from
F0 (no ligand bound) to
Fmax (100% of protein binding one equivalent
of ATP) is linearly related to occupancy of the binding site.
Fluorescence Lifetime Measurements--
Time-resolved
fluorescence measurements were performed in collaboration with Drs.
Ludwig Brand and Dmitri Toptygin (Johns Hopkins University).
Instrumentation, data collection, and analysis have been described (42,
43). Briefly, excitation was by the pulse method, using a picosecond
dye laser pumped by a mode-locked Nd:YAG laser (Spectra-Physics 3000),
with dye laser output frequency doubled to provide 295-nm pulses.
Emission data, at 340 nm, were collected in duplicate using single
photon counting detected by a Hamamatsu R1564U-06 microchannel plate
photomultiplier. Parallel collections were made with a buffer control
and a Ludox scattering solution, the latter for characterization of
lamp output. Protein concentrations and buffer, which varied, are
described in Table II and accompanying text. Data collection was at
25 °C.
 |
RESULTS |
Preparation of "Tryptophan Reporter" Mutants and
Proteins--
Residues within the ATPase motif of UvrB targeted for
replacement by the fluorescent amino acid tryptophan included
phenylalanine 47 and asparagine 51. It has been demonstrated that the
latter position could be mutated without significant phenotypic effect, either by conservative or nonconservative substitutions (N51A, N51K;
Ref. 11). A calculated risk was taken with the Phe47
position, due to the conservative nature of the substitution and
because the residue at this position within the motif is not conserved
(see Fig. 1 in Ref. 11).

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Fig. 1.
The pTZuvrB phagemid used for mutagenesis and
protein expression. The larger SacI-PstI
fragment, containing the complete uvrB gene, with endogenous
promoters, was cloned from the E. coli K-12 strain AB1157
into the vector pTZ19R, which contributes the -lactamase gene
(Ap), and the pBR322 and phage f1 origins of replication.
Shown above the phagemid map are the two synthetic
oligonucleotide duplexes used in cassette mutagenesis; one of these was
used to replace the XmnI (576) to NcoI (624)
restriction fragment that spans the ATP binding motif of
uvrB. The codons mutated are underlined.
Numbering of the restriction sites within the uvrB gene is
from the published gene sequence (see Ref. 8) rather than by location
within the plasmid.
|
|
Employing cassette mutagenesis generated the desired mutants with
75-100% efficiency. The sequences of selected mutant clones were
verified by reading at least 10 nucleotides beyond each ligation junction. We note that a commonly used oligo-directed mutagenesis approach (44) was not effective with template DNA produced from the
full-length pTZuvrB construct. We have since been able to use
oligo-directed mutagenesis following the cited protocol, by cloning
only a fragment of the uvrB gene (EcoRI to
BglII) into the pTZ19R vector. The same gene fragment,
inserted into an M13 phage vector, was used previously in this
laboratory for mutagenesis (11). We have also encountered difficulty in
sequencing the 5' terminus of the gene and regions upstream of the
promoters from the ssDNA template; these problems were obviated by
performing sequencing reactions at elevated temperatures. We speculate
that, when unpaired, DNA sequence(s) 5' to the uvrB gene may
adopt stable secondary structures that could interfere with
hybridization of the probe or provide alternative initiation sites for
DNA replication.
An additional peripheral note arises from a discrepancy noted among
published sequences of the E. coli uvrB gene. We find, for
the uvrB gene derived originally from E. coli
strain AB1157, an A rather than a G at nucleotide position 1875 (sequence published in Ref. 8). Our result is in agreement with the
sequence of Backendorf et al. (9) and with that recently
published for the E. coli genome (45). This suggests that
amino acid residue 477 is a histidine (CAC codon) rather than an
arginine (CGC). Histidine occupies this position in the UvrB sequences
of Micrococcus luteus (46), Streptococcus
pneumoniae (47), and Neisseria gonorrheae (48).
Each of the Trp reporter mutants, expressed from the pTZ19R-derived
phagemids, could fully complement deletion of the E. coli host uvrB gene (strain N364). Survival (Fig.
2) following UV irradiation did not
differ significantly from that accorded by plasmid encoding the
wild-type gene (D37 estimates, the dose at which survival is reduced to 37%, are 27 J m
2 for the wild-type
uvrB plasmid, 25.8 J m
2 for
uvrB-F47W, and 34.7 J m
2 for
uvrB-N51W). With the finding that the reporter mutants
exhibit no apparent change in phenotype, although based on expression from high copy number plasmids, we proceeded to purify the mutant and
wild-type proteins for further characterization. No difference in
binding or elution behavior was observed among the proteins in any
purification step. The final column (Mono Q) was added as a routine
precaution because it quantitatively eliminated a tryptophan-containing
contaminant that co-purified with wild-type UvrB on one occasion
(notable for a low level of UvrB expression). Purity of all
preparations exceeded 97%, as judged from densitometric scans of
Coomassie Brilliant Blue-stained SDS-polyacrylamide gel electrophoresis
gels, loaded with 5 µg of total protein.

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Fig. 2.
UV survival of plasmid-encoded Trp reporter
mutants in a uvrB E. coli host. Mean survival is
plotted, expressed as percentage of zero-exposure control, calculated
from three experiments. Cells in exponential growth were resuspended in
M9 salts prior to UV irradiation and then plated in darkness on rich
agar. , N364/pF47W; , N364/pN51W; ×, N364/pUvrB; ,
N364/pTZ18R (vector control).
|
|
Molecular Phenotype of Trp Reporter Mutant Proteins--
Assays to
characterize function of the purified reporter proteins included
incision of UV-irradiated plasmid DNA, association of UvrB with UvrA
protein as evidenced by inhibition of the DNA-independent ATPase
activity of UvrA, the DNA-dependent ATPase activity of UvrA-UvrB complex (manifestation of the cryptic UvrB ATPase), and
kinetics of the ssDNA-dependent ATPase of UvrB activated by the specific ompT proteolysis (UvrB*).
In concert with UvrA and UvrC proteins, the Trp reporter mutants
execute ATP-dependent incision of damaged DNA at levels
comparable with that with wild-type UvrB (Fig.
3a). At the greatest level of
addition of UvrB (24 ng, or 315 fmol), the UV-irradiated plasmid was
nicked to 81 ± 7% completion. This represents the mean for wild-type and mutant proteins, among which differences were not statistically significant (analysis of variance, p = 0.338). The data do suggest, however, that the F47W substitution may
have led to a slight reduction in activity. This can be seen more
clearly in a time course for incision (Fig. 3b), with
UvrB-F47W slower early on in the introduction of nicks (rate from the
1- to the 4-min time point was 47% that of the wild-type protein). In
either assay design, the N51W protein supported incision equally to
wild-type UvrB.

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Fig. 3.
In vitro incision assays. The
nicked products of incision were resolved from the substrate
supercoiled plasmid (tritium-labeled, UV-irradiated to 720 J
m 1) by agarose gel electrophoresis, followed by liquid
scintillation counting of excised bands. UvrA and UvrC proteins were in
excess, with ATP provided at 2 mM. a, titration
with wild-type and mutant UvrB proteins, incubated for 10 min at
37 °C. Means from four experiments are expressed as the total yield
of incisions (above that seen in UvrA plus UvrC-only controls),
calculated as the product of plasmid present (15 fmol) and the average
nicks per plasmid, derived from a Poisson distribution. UvrB proteins
are F47W ( ), N51W ( ), and UvrB wild-type (×). b, time
course of incision with UvrB proteins, present at 150 fmol. Assay
components minus UvrC and ATP were preincubated 5 min at 37 °C; the
addition of ATP and UvrC defined time 0. Means from three experiments
are shown, with proteins as identified in a. The initial
level of nicks in plasmid ranged from 5 to 17% (0.75-2.0-fmol nicks);
the maximum adventitious nicking seen in controls lacking only UvrB was
1.0 fmol.
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|
As described above, dual incision is the ultimate product of multiple
enzymatic activities and a succession of macromolecular interactions,
some of which are moderated by nucleotide binding. An alteration in a
specific and essential UvrB function, resulting from mutation, could
conceivably go undetected in an incision or repair assay if that
activity were not diminished to the extent that it became
rate-limiting. We therefore continued characterization of the Trp
reporter mutants by looking at earlier steps in the pathway and
specific enzymatic activities.
An early step preceding incision is the association of UvrA and UvrB
proteins. It is unclear whether this protein-protein interaction occurs
first or if UvrB is recruited to a UvrA2-DNA complex, but
it has been shown that an ATP-dependent association of UvrA
and UvrB can occur in solution (14). In such an interaction, the
DNA-independent ATPase activity of UvrA is partially inhibited, while
that of UvrB remains suppressed (10, 12, 16). In Fig. 4a, it can be seen that both
Trp reporter UvrB mutants inhibit the ATPase activity associated with
UvrA (in the absence of DNA) and to an extent that is indistinguishable
from that effected by wild-type UvrB. Fitting this data to an
exponential decay model (16), it can be estimated that 28 nM wild-type UvrB was sufficient for 50% inhibition
(I 50), with saturable inhibition to 12% of the
initial activity. The corresponding calculations for the Trp reporter
mutants are I50 = 39 and 29 nM, with
saturation at 22 and 15% initial activity for the F47W and N51W
species, respectively.

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Fig. 4.
ATPase activity of the UvrA-UvrB complex.
a, inhibition of ATPase activity in the absence of DNA. UvrB
was added at the indicated concentrations to 70 nM UvrA,
with ATP at 150 µM; ATP hydrolysis was monitored using a
thin layer chromatography assay. Data points are the averages from
duplicate experiments, representing the fraction of activity exhibited
by UvrA alone, which averaged 50 mol min 1
mol 1 UvrA. The curves are first-order decay fits to the
data. Proteins are F47W ( ), N51W ( ), and UvrB wild-type (×).
B, stimulation of ATP hydrolysis of the UvrA-UvrB complex in
the presence of UV-irradiated DNA (plasmid DNA at 30 µM
nucleotide, 720 J m 2 exposure). UvrA was present at 15 nM; UvrB was also at a fixed concentration, in excess, at
75 nM, with ATP at 150 µM. Mean initial
rates, from three experiments, are shown. , UvrA alone; , UvrA
with added N364/pF47W; , UvrA with added N364/pN51W; ×, UvrA with
added N364/pUvrB (wild type).
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With the addition of UV-irradiated DNA to UvrA and UvrB proteins in an
ATPase supporting buffer, the overall ATPase activity of the complex is
stimulated to levels exceeding that of UvrA alone. This reflects an
unmasking of the cryptic activity of UvrB (10-12). This characteristic
behavior by wild-type and Trp reporter UvrB constructs is shown in Fig.
4b. The addition of wild-type UvrB (to excess) leads to a
3.5-fold stimulation of ATP hydrolysis over that with UvrA alone. With
all UvrB species at saturating concentrations, the rate of hydrolysis
observed with the F47W mutant is close to that manifested by unaltered
UvrB, at 86%. Surprisingly, activity with UvrB-N51W surpasses that
seen with wild-type, by 2.0-fold.
Caron and Grossman (10) first reported that the cryptic
ssDNA-dependent ATPase of UvrB could be activated by a
specific proteolytic hydrolysis of the C terminus. The truncated
protein, UvrB*, may serve as a model for the active conformation of the protein and allow a more direct examination of the consequences of
tryptophan substitutions within the ATPase motif. The kinetic parameters of the UvrB* species, presented in Table
I, were obtained from assays done within
24 h of ompT-mediated proteolysis and subsequent
purification. Although UvrB is a stable protein, known to withstand
prolonged storage in the freezer (49), it has been our experience (not
yet formally addressed) that UvrB* is prone to loss in activity
(kcat decreases although Km
remains stable) with freeze-thaw cycles or extended storage on ice.
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Table I
ssDNA-dependent ATPase of ompT-truncated UvrB proteins
Initial rates of ATP hydrolysis were determined at 37 °C over the
substrate range 0.25-4 mM for proteolytically activated
(UvrB*) wild-type and Trp substitution mutants, in the presence of
single-stranded phage DNA ( X174) at 200 µg/ml. The parameters
below were obtained from Lineweaver-Burk plots.
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The two fluorescent derivatives of UvrB do show statistically
significant deviations from wild-type, and each other, in kinetic parameters of the B* fragment. The F47W protein exhibits a slight decrease in apparent affinity for substrate, with Km increasing from 0.8 mM for the wild type to 2.2 mM. The effect on function is compounded by a decrease in
kcat, to 65% that of wild-type. An increase is
also seen in the Km for UvrB-N51W, to 3.1 mM, but the effect on turnover number is nearly negated by
a 3.6-fold acceleration in the catalytic rate. We may conclude that the
tryptophan substitutions, for phenylalanine 47 and asparagine 51, do
result in measurable alterations in the catalytic properties of UvrB.
These changes, however, can be viewed as minor ones, resulting in no
substantial loss in function. Gross changes in the structure of the
active site would therefore be considered to be improbable.
Fluorescence Characterization of the Inserted Tryptophan
Environments--
Tryptophan has now been engineered into the ATPase
motif of functional UvrB proteins, and specific proteolysis of these
proteins leads to activation of an ssDNA-dependent ATP
hydrolase, as in the wild-type protein. What can we now learn from
diverse fluorescence parameters about the tryptophan environments, and
by inference, the binding/catalytic site? More specifically, what is
the structural basis for the inactivity of intact UvrB, and does
proteolytic activation lead to measurable conformational changes at the
presumed active site?
The steady state fluorescence emission spectra of the introduced
tryptophans are shown in Fig. 5.
Wild-type UvrB, as expected, exhibits tyrosine emission only (at 305 nm) with excitation at 280 nm (Fig. 5a) and is "dark"
with 295-nm excitation (Fig. 5b), a wavelength at which
there is significant absorbance by tryptophan but not tyrosine. The
spectra obtained with 280-nm excitation are shown primarily to note an
unusual property of the F47W and N51W UvrB derivatives, that being the
persistence of tyrosine emission in a Trp-containing protein.
Quantitative quenching of tyrosine emission in proteins with as few as
one Trp residue is observed with few exceptions (50). The intensity of
the tyrosine emission in the UvrB derivatives is not only relatively
strong, but it varies in relation to that of Trp between the two
reporter mutants and in both cases is abolished by proteolysis to the
B* form (no tyrosine residues are eliminated in cleavage).

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Fig. 5.
Fluorescence emission spectra of UvrB and the
Trp reporter mutant proteins. Spectra are shown for wild-type UvrB
(wt), the intact Trp reporter derivatives UvrB-F47W and
UvrB-N51W, and the ompT-proteolyzed reporter proteins (F47W*
and N51W*) in 10 mM Tris·Cl (pH 7.5), 85 mM
KCl, 1 mM DTT, 10% glycerol at 10 °C. All spectra are
corrected for scatter and background emission. a, spectra
obtained with excitation at 280 nm. Proteins were at 1 µM, with 3 nm excitation and emission slits.
b, spectra obtained with excitation at 295 nm. Proteins were
at 1.4 µM, with 5-nm excitation and emission slits.
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As noted above, it is feasible to selectively observe the tryptophan
signal by using 295-nm excitation (Fig. 5b). Emission spectra of both UvrB mutants show a blue shift in
max,
either relative to tryptophan in water (355 nm), or compared with
spectra obtained following denaturation of the proteins in a 6 M guanidine hydrochloride buffer (yielding a 356-nm maximum
for both mutants; spectra not shown). The shift, generally indicative
of a more hydrophobic environment, is greater for Trp47
(338.5 nm) than for Trp51 (345 nm). The quantum yield for
Trp47, 0.15, is slightly greater than that for tryptophan
in water, 0.14 (38), and comparable with the difference seen in
emission intensity between native and guanidine-denatured UvrB-F47W
(12% greater in native buffer). Though not as blue-shifted as the
emission from Trp47, that of Trp51 is
significantly more intense, with an estimated quantum yield of 0.24. These data suggest that, although both environments are relatively
shielded from polar solvent, Trp47 may be partially
quenched by contact with neighboring amino acid side chains. With
proteolysis, small shifts in
max of emission can be seen
in both proteins, although in opposite directions, from 338.5 to 340 nm
in UvrB-F47W and from 345 to 342 nm in UvrB-N51W. Emission intensities
also change for both proteins with proteolysis, increasing in both
cases, by 42% with proteolysis of UvrB-F47W, and by 19% for
UvrB-N51W.
We were able, in the laboratory of Dr. Ludwig Brand, and under the
direction of Dr. Dmitri Toptygin (Department of Biology and the
McCollum-Pratt Institute, The Johns Hopkins University), to compare the
fluorescence decay characteristics of the two reporter proteins. The
fluorescence decay of Trp47 is shown in Fig.
6. Four decay times were required, for
both proteins, to provide an adequate fit to the data, free of
systematic error as indicated by the residual and autocorrelation
plots. The multiple decay parameter values, presented in Table
II, are suggestive of heterogeneity in
either tryptophan environment, although the two shorter lifetimes
contribute little to emission intensity (0.4-1% for
values of
0.11-0.15 ns, and 2-8% for
= 0.7 ns). Emission intensity is
dominated in both proteins, more so in N51W, by a relatively long
lifetime, 5.8-6.4 ns, with intensity-weighted average lifetimes of 5.3 ns for Trp47 and 6.1 ns for Trp51. Tryptophan
in water, for comparison, has a lifetime of approximately 3 ns (3.0 ns
for the peptidyl-Trp analog NATA; Ref. 51). A comparison was also made
of the decay characteristics of intact and proteolyzed UvrB-F47W. To
maximize the stability of the B* form, however, glycerol was added to
the buffer (to 15%). No contaminating fluorescence signal was detected
from the glycerol (or its aromatic contaminants) in buffer controls,
and a stable signal over the data collection period indicated
negligible photobleaching at the higher viscosity. We do note that the
data analysis indicated the need for a fifth lifetime to obtain
completely random residuals, 4 ps for UvrB-F47W and 40 ps for the
proteolyzed protein. These are not reported in Table II (their omission
does not affect the average lifetime calculation) because the
contribution to intensity was minor (0.6-1.5%) and because it is
unlikely that such lifetimes, if real, could be resolved given the
timing calibrations of 13.301 ± 0.0085 ps/channel. Neither the
average lifetime, 4.8 ns for UvrB-F47W and 4.9 ns for UvrB*-F47W, nor
the distribution of lifetimes changed significantly with
proteolysis.

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Fig. 6.
Fluorescence decay of UvrB-F47W, showing fit
to four exponential terms. The fitted curve is
superimposed on data points obtained for 5 µM protein in
10 mM Tris·Cl (pH 7.5), 85 mM KCl, 1 mM DTT, at 25 °C. Excitation was at 296 nm, and emission
was at 340 nm. Residuals, and residual autocorrelation plots are shown
as testament to the lack of systematic error.
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Table II
Fluorescence decay parameters of UvrB derivatives F47W and N51W
Exponential decay parameters calculated from time-resolved data are
given for two comparisons: between UvrB-F47W and UvrB-N51W and between
UvrB-F47W and UvrB*-F47W, the latter comparison done in a glycerol and
Mg2+-supplemented buffer. Proteins were at 5 µM,
in 10 mM Tris · Cl (pH 7.5)/85 mM KCl/1
mM DTT, at 25 °C. 2, goodness of fit
parameter (reduced -square); and , lifetime (ns) and
associated fractional contribution to total emission intensity,
respectively; av, intensity-weighted average fluorescence
lifetime (52).a
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Accessibility of the Trp reporters to small, neutral molecules in
solution was directly addressed by steady state fluorescence quenching
experiments with acrylamide. Quenching occurred without shifts in the
wavelength maxima for emission (spectra not shown). Stern-Volmer plots
(Fig. 7) reveal that the tryptophans of
both derivatives of UvrB are less susceptible to quenching than the model compound NATA (in neutral aqueous solution) and that quenching is
further and significantly reduced for both proteins after proteolysis. Upward curvature in the plots for NATA and the intact proteins indicates an apparent static component to the quenching mechanism (40).
A modification of the Stern-Volmer equation (see "Experimental Procedures") was therefore used to obtain the estimates tabulated (Table III) for the dynamic
(KS-V) and static (V) quenching
constants for these species. After proteolysis, the Stern-Volmer plots
were linear, with a decrease in slope for both proteins. Plots for B*-F47W and B*-N51W were virtually superimposable, with
KS-V values of 2.96 and 2.86 M
1, respectively, as compared with 5.05 and
6.34 M
1, respectively, for the intact F47W
and N51W proteins, and 16.8 M
1 for NATA.
Using the intensity-weighted average lifetimes from Table II, the
bimolecular rate constants for quenching can be estimated
(KS-V =
× kq) for Trp in
UvrB-F47W as kq = 1.05 × 109
M
1 s
1; in UvrB-N51W, 1.04 × 109 M
1 s
1; and
for the proteolyzed F47W protein, 0.60 × 109
M
1 s
1. For comparison,
kq = 5.60 × 109
M
1 s
1 was calculated for NATA
(using the lifetime data from Ref. 51).

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Fig. 7.
Quenching by acrylamide of the steady state
fluorescence of UvrB Trp reporter proteins. Stern-Volmer plots of
tryptophan or NATA fluorescence titrated with acrylamide (0 to 400 mM) are shown. F, the emission intensity at the
given acrylamide concentration; Fo, the intensity with no
added quencher. Data points are the mean Fo/F
ratios from three trials; lines were fit either by linear
(UvrB* forms) or nonlinear regression (modified Stern-Volmer equation,
for intact proteins and NATA). Intensities at 25 °C were read at the
appropriate maxima, 340 nm for UvrB-F47W ( ) and UvrB*-F47W ( );
345 nm for UvrB-N51W ( ) and UvrB*-N51W ( ); and 355 nm for NATA
(×). Excitation was at 295 nm.
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Table III
Quenching constants for the attenuation by acrylamide of steady
state tryptophan fluorescence in UvrB derivatives
The Stern-Volmer constant (KS-V) and bimolecular
rate constant for collisional quenching (kq) are
given where the Stern-Volmer plots are linear (Fig. 7). Where there is
upward curvature, a modified Stern-Volmer equation (as described under
"Experimental Procedures") was used to estimate dynamic and
apparent static components (V) to the quenching mechanism.
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Quenching of Trp Fluorescence by ATP--
The acrylamide quenching
results suggest that amino acid residues comprising the ATPase motif of
UvrB are accessible to solute molecules and, therefore, presumably to
the substrate ATP. If binding occurs in UvrB, or following proteolysis,
quenching of the reporter Trp fluorescence may occur from its proximity
to bound ATP, and either quenching or enhancement of emission may occur
as a result of conformational change. In Fig.
8a, a quench of the steady
state fluorescence intensity of UvrB-F47W by MgATP at 37 °C is shown
(corrected for dilution, emission, and scatter by buffer and ATP and
for the inner filter effect of ATP). Quenching of Trp by ATP in either
protein, intact or proteolyzed, did not result in a measurable change
in
max of emission. Quenching also occurred, and to the
same extent, when magnesium was omitted from the buffer or with the
chelator EDTA added in excess of the magnesium concentration (data not
shown).

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Fig. 8.
ATP binding by the Trp reporter mutants of
UvrB, as shown by quenching of tryptophan emission. a, the
fluorescence emission spectra of UvrB-F47W at varying ATP
concentrations. A representative titration was performed at 37 °C,
with protein at 1 µM in 25 mM HEPES (pH 7.6),
85 mM KCl, 15 mM MgCl2, 1 mM DTT, 10% glycerol; ex = 295 nm. Spectra
are corrected for buffer scatter, dilution, and inner filter effect.
B, titration results for the intact and proteolyzed forms of
both Trp reporter mutants of UvrB. The mean quench value is plotted at
any given ATP concentration, calculated from at least three
determinations, and expressed as the change in intensity
( F) divided by the initial value (Fo, the
intensity at zero added ATP). Assay conditions were as noted in Fig.
8a. Proteins are UvrB-F47W ( ), UvrB*-F47W ( ),
UvrB-N51W ( ), and UvrB*-N51W ( ). C, data from Fig.
8b replotted according to the binding model for a monomeric
protein with one binding site: F = Fmax Kd·( F/[ATP]). Substitution of
quench ( F/Fo) for F in this
equation simply allows direct estimation of the maximum relative quench
(fractional change at saturated binding).
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It is evident from the quenching plots in Fig. 8b, that
proteolysis is not required for quenching by ATP in UvrB. Trp reporters in both the F47W and N51W proteins are quenched and to a greater degree
than the Trp in the truncated UvrB*-F47W. If it is assumed that there
is but one binding site in UvrB and that its properties could not be
altered by self-association (14), then the data can be replotted (as
described under "Experimental Procedures") to obtain estimates for
the ATP binding constants, Kd, as shown in Fig.
8c. Although the maximal extent of quenching differed,
notably between intact and proteolyzed forms
(
Fmax = 44% for UvrB-F47W and 36% for
UvrB-N51W, as compared with 17% for UvrB*-F47W and 6% for
UvrB*-N51W), the estimated dissociation constants were comparable for
all species (mean Kd = 1.0 ± 0.1 mM, range 0.86 mM for NW* to 1.14 for FW*). An
environment more sheltered from a diffusible quencher for the B*-Trp is
consistent with the acrylamide studies. More significantly, it is clear
that the ATP binding site in UvrB is functionally configured and
accessible prior to proteolytic activation of catalytic function.
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DISCUSSION |
Activation of the UvrB ATPase is expected to involve the initial
(of potentially several) conformational changes requisite for UvrB
function. We envisioned four possibilities for structural changes that
alone or in combination could unmask the cryptic UvrB ATPase: 1)
removal or displacement of a steric block to the ATP binding site
(presumably the C-terminal domain), 2) realignment to configure the ATP
binding site, 3) realignment to bring catalytic residues into position,
and/or 4) structural changes permitting recognition or response to the
cofactor ssDNA. Through the introduction of fluorescent reporter groups
into the ATPase motif of UvrB, we have obtained data in this study that
would eliminate the first two as possibilities for the structural basis
of masked activity. Tryptophan residues within the conserved motif are
accessible to a diffusible quencher of fluorescence and, moreover, are
quenched by the binding of ATP even in the catalytically silent intact protein. Characterization of the tryptophan environments indicate a
solvent exposure intermediate between that of a surface residue and one
buried within a hydrophobic core, i.e. partially shielded, as could occur within a cleft between domains of the protein. This
environment is perturbed by ompT proteolysis of UvrB,
suggesting that a conformational change does occur in proximity to the
ATPase site with activation, with one measurable consequence being the greater shielding of the Trp reporters from a diffusible quencher. Titration of the tryptophan emission with nucleotide has allowed, for
the first time, a direct demonstration of the binding of ATP by UvrB.
Estimates of the binding constants, Kd, do not differ significantly between UvrB and UvrB* forms of the protein or
between the different reporter constructs, with a mean value of
1.0 ± 0.1 mM. These values are close to the
Km values determined for ssDNA-dependent
ATPase activity of the proteolytically activated proteins (2.2-3.1
mM).
Positioning of the Fluorescent Probe and Its Phenotypic
Effects--
Scant structural information was available to guide
introduction of a tryptophan reporter to an ideal site, i.e.
one that would not disrupt structure nor function but yet would be
responsive to both conformational change at the presumed ATP binding
site and to the presence of substrate. It had been shown in this
laboratory (11, 16) that residues of a hydrophobic sheet adjoining the conserved phosphate binding loop of the Walker ATP binding motif could
be mutated without a discernible effect on the overall repair capacity
in in vivo UV challenges. The one such mutant protein selected for further study, UvrB-N51A, also performed at wild-type efficiency in in vitro incision assays, although its ATPase
activity was reduced to 65% of wild-type levels for the
DNA-dependent activity of the Uvr AB complex, and to ~6%
of the wild-type turnover number for the proteolyzed protein. In the
present study, the two Trp reporter mutants, likewise, functioned at
the wild-type level in the in vivo complementation assays.
And likewise, the ATPase activities of both Trp reporter mutants were
diminished, but to a lesser extent than seen with N51A. The F47W
substitution appears to be the more taxing of the two Trp
substitutions, with a turnover number
(kcat/Km) reduced to 24%
that of wild type for the B* assay, while that of the N51W construct
was close to wild type (94%). An unexpected observation, given the
decrease in kcat reported for N51A, is the
apparent stimulation of the catalytic rate in N51W,
counterbalanced to a degree by a roughly 3-fold increase in the
Km. Opinion in general is that helicase motif I
mutations affect substrate binding, with catalysis governed by motif II
(26, 53). Recent structure determinations for proteins with the
conserved helicase motifs described by Gorbalenya and Koonin (26),
Bacillus stearothermophilus PcrA (54) and E. coli
Rep (55), and located in UvrB by Moolenaar et al. (27) have
led to the prediction that motifs I and II would be closely apposed in
UvrB (56), together creating the binding/catalytic site. A mutation
such as N51W in motif I could conceivably alter the structure of, or
the structure supporting that of, motif II as well. Another possibility
could involve rate acceleration by facilitating product (ADP)
release.
The utility of the Trp reporter mutants may most prudently be dictated
by the level of detail asked of them. They function in repair at the
cellular level indistinguishably from wild type and at near wild-type
efficiency in in vitro incision assays. The slightly reduced
efficiency of the F47W construct in incision serves to underscore the
dependence of the repair pathway on expression of the UvrB ATP
hydrolase. These properties and others (unaltered expression and
purification behavior, interaction with UvrA) indicate that no gross
alteration of structure or function resulted from the introduction of
the tryptophans at positions 47 and 51. With these reporters proximal
to the active site, information was obtained regarding solvent
accessibility to the binding site in the unactivated protein and
regarding conformational changes at this site accompanying activation.
This information would probably be unobtainable from a distant probe
that responded to more global conformational changes. The cost for such
information is set by the alterations, up to severalfold, in apparent
kinetic constants for ATPase activity. This would place limitations on
the degree of detail to which one could examine binding, catalytic site
geometry, and perhaps substrate specificity.
Tryptophan substitutions in UvrB have a lesser impact on ATPase
activity when judged by assay of the UvrA·UvrB·UV-DNA complex rather than by the activity of the proteolyzed UvrB with ssDNA. We can
speculate that the association with UvrA could both stabilize the
active conformation of UvrB and impose greater constraints on the
structure that would not exist in the proteolytically activated protein. It is expected that use of the Trp reporter mutants of UvrB
for structural or nucleotide binding studies of the
UvrA2-UvrB complex would be limited by the fact that UvrA
contains three tryptophan residues (4). Preliminary work (not shown)
has shown that the emission of Trp47 is discernible in the
spectra of UvrA-UvrB complexes. A more promising approach, however,
could employ tryptophan analogues incorporated biosynthetically into
the UvrB protein (57).
The Tryptophan Environments and Conformational
Changes--
The steady state fluorescence data taken
together provide a description of the tryptophan reporters'
environments as being moderately hydrophobic, or partially shielded
from polar solvent. The blue shifts in emission maxima are significant
but do not approach those reported for Trp residues known to be buried
within a hydrophobic core (to ~310-320 nm; Ref. 39). The intensity of emission also increases in the native protein, slightly for Trp47 and to a greater extent in Trp51. Some
degree of Trp quenching occurs in the folded protein, by its
accessibility to polar solvent, and, more likely in the case of
Trp47, by interaction with neighboring side chains.
Accessibility to solvent was unambiguously demonstrated by the
vulnerability of both Trp reporters to quenching by acrylamide, a polar
and uncharged solute. Again, accessibility is moderate but sufficient
for there to be a static as well as dynamic component. The estimated
bimolecular quenching constants (kq) are
intermediate between values reported (41) for a deeply buried residue
(<0.05 × 10
9 M
1
s
1, in azurin) to near maximum exposure as in a randomly
coiled peptide, such as adrenocorticotropin (4.2 × 10
9 M
1 s
1), or
for the free model compound NATA (5.6 × 10
9
M
1 s
1; this study). It is
unlikely, therefore, that the inactivity of UvrB as an ATPase results
simply from positioning of the C terminus (or any other domain or loop)
as a steric block to substrate access.
Several lines of evidence indicate that conformational changes
accompany the proteolytic elimination of the C terminus and associated
activation of the UvrB DNA-dependent ATPase. Tyrosine emission, seen in both of the intact UvrB-Trp mutants when excited at
280 nm, is uncommon in Trp-containing proteins and is quenched in the
B* peptides. This provides a ready spectroscopic handle to distinguish
the forms but yields little information as to the nature of the
structural alteration. The UvrB to UvrB* transition, in that it does
lead to suppression of the tyrosine signal, may be of future interest
to spectroscopists in the continuing effort (39) to understand
intrinsic protein emission, if the structure of UvrB can be solved.
Changes in the tryptophan emission with proteolysis provide information
characterizing the conformational changes accompanying activation that
can be better localized to the active site. Shifts in emission maxima
are small but significant, and in the opposite direction for the two
reporters. These wavelength shifts are accompanied by increases in
emission intensity that may reflect a greater degree of shielding from
solvent. This was more directly shown by the decreases in
susceptibility to quenching by acrylamide, with the dynamic quenching
rate constants decreasing for both proteins to identical values and
with static components apparently eliminated. Together, these data
indicate that both residues 47 and 51 are shielded from solvent to a
greater extent in the activated conformation and that the reporters'
environments are more similar, whether coincidental or owing to greater
structural constraints on the binding site in the B* form.
Looking in greater detail at the tryptophan environments, the necessity
to invoke a multiexponential decay model for both proteins may suggest
a considerable degree of heterogeneity or flexibility in the vicinity
of the putative binding site. Both tryptophans were described best by
four lifetimes, but each was dominated by the two longer terms. Of
these, the greater term (68-84.5% of total intensity for F47W and
N51W, respectively) was roughly twice the lifetime of tryptophan in
water, with the second term (contributing 24-12.5% of total
intensity) similar or equal to the value expected in aqueous solution.
The interpretation of multiple lifetimes is still not clear (58, 59),
and one cannot, with assurance, simply associate each lifetime with a discrete conformer. However, it would be prudent to bear in mind that
the steady state results may represent the averaged behavior of
multiple conformers. And although we noted above an increase in
similarity of the tryptophan environments in the proteolyzed proteins,
if there were greater constraints on the architecture of the site, they
do not appear to impose greater homogeneity on the distribution of
lifetimes, at least for B*-F47W (lifetimes were not determined for
B*-N51W).
Binding of Nucleotide--
Quenching of tryptophan fluorescence by
acrylamide revealed that residues near the presumed binding site were
accessible to solvent. Quenching by ATP further revealed that the
binding site is not only accessible in the intact protein but also
functional. The conformational change that accompanies ompT
proteolysis reduces the extent of quenching by ATP, as it did toward
acrylamide, but the apparent affinity of binding between B and B* forms
is altered little if at all.
The mechanism of quenching of tryptophan emission by ATP is not clear.
Without a change in the wavelength of emission, there is no evidence
that binding itself induces a conformational change. The addition of
ATP in one preliminary experiment, to 1 mM, in fact did not
alter the average lifetime or the distribution of decay terms of
UvrB-F47W or of UvrB*-F47W (data not shown). This suggests that the
substrate may be close enough to the tryptophans to quench by a static
mechanism.
It remains to be seen whether ATP binding in itself alters or regulates
any relevant behavior of UvrB, as it does with UvrA (6, 13, 60), or
whether the binding site undergoes further conformational adjustments.
Studies on the UvrB-DNA preincision complex suggest that ATP may be
bound at an apparent affinity in the micromolar range (20). A
conformational change has been postulated as necessary to confer upon
UvrB the ability to stably bind DNA at a damaged site, following the
departure of UvrA from the complex (17). A concomitant refinement of
the ATP binding site would not be unreasonable.
We referred above to recent successes in structure determination for
other proteins with the helicase motif manifold found in UvrB. As seen
initially for RecA, complexed with ADP (61), the ATP binding motif is
likely to be found at the base of a deep cleft between domains, closely
linked to a juncture that could transduce conformational changes
resulting from ATP binding and/or hydrolysis to adjoining DNA binding
domains. Recently, a multidomain structure for the UvrB protein of
Thermus thermophilus was described (56). Two domains, N
(residues 2-105, containing the Walker ATP binding motif) and C1
(residues 456-590) were required for ATPase activity, with C1 also
required for DNA binding. Based on the domain structure and location of
the helicase motifs, a model structure similar to those of PcrA and Rep
was proposed. Our description of the tryptophan reporter environments
would be fully consistent with a position in such a cleft. We further predict that the conformational change noted with proteolytic activation is correlated with a rearrangement of UvrB domains to
complete the architecture of the catalytic site and/or to permit interaction with the ssDNA co-effector or transduction of this signal
to the catalytic site. Recalling an early observation (10) that the
velocity of ATP hydrolysis in UvrB* increased without saturation in
response to increasing ssDNA concentration, it would seem likely that
responsiveness to effector DNA is coupled to catalytic efficiency via
such a co-dependence on conformational change.