 |
INTRODUCTION |
Mismatch repair is the primary mechanism for repair of replication
errors in Escherichia coli (1-3). The mismatch repair machinery also prevents recombination between highly divergent DNA
sequences (4, 5). Thus, an active mismatch repair system ensures the
precision of chromosomal replication and maintains genomic stability
(for reviews, see Refs. 1-3). Consistent with this idea, defects in
mismatch repair genes in human cells have been linked to genomic
instability and hereditary colon cancer, underscoring the importance of
this repair pathway (6-9).
The sequence of biochemical reactions that define the mismatch repair
pathway has been well described in E. coli, and the proteins
responsible for each step are known (for reviews, see Refs. 1-3).
Mismatch recognition is accomplished by a MutS dimer (10, 11). MutL,
also a dimer, then binds the MutS·DNA complex (12), and the DNA is
looped out in an active search for the nearest d(GATC) methylation site
either 5' or 3' of the mismatch (12, 13). Once found, the complex
stimulates MutH to generate a nick on the unmethylated (or nascent)
strand at the hemimethylated d(GATC) site (14-16). DNA helicase II and
the appropriate exonuclease then excise the error-containing DNA (17,
18), beginning at the nick and continuing past the mismatch (18). The
single-stranded DNA (ssDNA)1
gap is filled by DNA polymerase III, and DNA ligase seals the remaining
nick (19).
Importantly, the mismatch repair reaction pathway has bidirectional
capability. The nick is generated at the d(GATC) site located closest
to the mismatch and therefore could exist on either side of the
mismatch (18). However, DNA helicase II unwinds DNA with a specific 3'
to 5' polarity (20). As a result, helicase II must be loaded on the
appropriate DNA strand to ensure unwinding toward the mismatch. It is
also important to note that the d(GATC) site nearest an error may be
>1 kilobase away (19).
The precise biochemical activity associated with the MutL protein has
been a matter of debate for several years. Some groups have
demonstrated that MutL binds to both ssDNA and double-stranded DNA (21,
22), whereas others have reported that MutL does not bind DNA (23). It
is now clear that MutL catalyzes a weak ATPase reaction and that this
activity is required for mismatch repair (21, 24, 25). In addition,
MutL stimulates the biochemical activities of MutS, MutH, and helicase
II (12, 13, 23, 24, 26-29), and several lines of evidence indicate
there is a physical interaction between MutL and MutS (12, 13, 23),
MutL and MutH, and MutL and helicase II (26, 27). Thus, MutL has been suggested to function as a master coordinator or molecular matchmaker in the mismatch repair pathway (1, 29, 30).
Of particular interest is the fact that MutL specifically stimulates
the unwinding reaction catalyzed by DNA helicase II (the uvrD gene product) (27, 29). The unwinding activity of the Rep protein (40% identical to UvrD) is enhanced by MutL, but to a
significantly lower extent (29). Moreover, on a nicked circular heteroduplex DNA substrate, MutL and MutS together activate
UvrD-catalyzed unwinding, whereas there is no detectable enhancement of
unwinding by Rep helicase (29). Thus, stimulation of the UvrD-catalyzed unwinding reaction by MutL is specific and likely due to a
protein-protein interaction. This is consistent with data demonstrating
a physical interaction between these two proteins (25, 26).
Little is known about the mechanism by which the UvrD-catalyzed
unwinding reaction is enhanced by MutL. However, important details of
the reaction have been described. For example, on a nicked circular
molecule containing a mismatch, MutS, MutL, and UvrD initiate unwinding
at the nick site and begin helix opening in the direction toward the
mismatch. This reaction requires all three protein components and the
presence of a mismatch (31). In addition, although MutL dramatically
stimulates the unwinding rate by DNA helicase II, MutL does not
increase the ATP hydrolysis rate of helicase II (26).
The mechanism responsible for stimulation of UvrD-catalyzed unwinding
by MutL has been investigated. The experiments described here support a
model in which MutL loads UvrD onto the DNA substrate, increasing the
rate of initiation. In addition, MutL-directed loading of UvrD is
observed to be continuous. A model for methyl-directed mismatch repair
is presented that incorporates the observed biochemical and physical
interactions between MutL and UvrD.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Plasmids--
E. coli BL21 was
from Novagen. GE1752
mutS (28) was constructed previously
in this laboratory. Plasmids pET11d, pLysS, and pET3c were from
Novagen. Plasmids pCYB2 and pLitmus28 were from New England Biolabs
Inc. M13mp7 ssDNA was purified as described previously (32). Plasmid
pCYB2-mutL (intein fusion construct) was constructed
previously (28). The plasmid that expresses UvrD has been described
(33).
Oligonucleotides and Enzymes--
Restriction endonucleases, DNA
polymerase I (large fragment), and T4 polynucleotide kinase were from
New England Biolabs Inc. and were used as recommended by the supplier.
The 48-base oligonucleotide 5'-GGAAAAATTAGTTTCTCTTACTCTCTTTATGATATTTAAAAAAGCGGT-3' was used in
electrophoretic mobility shift assays and as a trap in some helicase assays.
Protein Purification--
UvrD was purified as described
previously (34). Purification of pCYB2-mUTL from BL21 was as
described previously (26). This protein was used for nitrocellulose
filter binding experiments.
Additional MutL was purified from a strain lacking MutS,
GE1752mutS. MutL was overexpressed prior to purification
by growing GE1752mutS containing pCYB2-mutL in
2× yeast-tryptone medium at 30 °C. Cells were grown to an
absorbance of 1.0 (600 nm). Protein expression was induced by the
addition of 0.5 mM isopropyl-
- D-thiogalactopyranoside.
For purification from GE1752mutS containing
pCYB2-mutL, 19.5 g of cells were harvested by
centrifugation. pCYB2-mutL generates the MutL protein as an
intein fusion. MutL was purified using a chitin column (Impact I
system, New England Biolabs Inc.) according to the manufacturer's
instructions. Protein was eluted with buffer containing 20 mM Tris-HCl (pH 8.0 at 25 °C), 10% (v/v) glycerol, and
0.1 mM EDTA supplemented with 0.2 M NaCl and 30 mM dithiothreitol. Pooled fractions were diluted with
buffer A (20 mM Tris-HCl (pH 7.5 at 25 °C), 10% (v/v)
glycerol, and 0.1 mM EDTA) to a conductivity equivalent to
that of buffer A + 0.1 M NaCl. The pool was loaded onto a
10-ml DEAE-Sephadex column (2.7-cm internal diameter; Sigma) that had
been equilibrated with buffer A + 0.1 M NaCl. Protein was
eluted from the column with a linear gradient of 0.1-0.6 M NaCl in buffer A. Protein eluted at a conductivity equivalent to that
of buffer A + 0.13 M NaCl. Pooled fractions (8 ml) were concentrated by solution absorption with polyethylene glycol 20,000. Fractions were placed in a dialysis bag with a molecular weight cutoff
of 3500. The dialysis bag was covered with polyethylene glycol 20,000. Then, 3 ml of concentrated protein material were extensively dialyzed
into MutL storage buffer (20 mM Tris-HCl (pH 7.5 at
25 °C), 50% (v/v) glycerol, 0.2 M NaCl, 0.1 mM EDTA, and 1 mM 2-mercaptoethanol).
The concentration of helicase II was determined using the published
extinction coefficient of 1.29 ml mg
1
cm1 (35). The concentration of MutL was
determined using the Bradford protein assay (Bio-Rad) with bovine serum
albumin as a standard.
DNA Substrates--
A 148-bp blunt duplex DNA fragment was
produced by digestion of pLitmus28 with XbaI and
PvuII and purified from an agarose gel using Geneclean (Bio
101, Inc.). The purified DNA fragment was radioactively labeled by
filling in the XbaI site with [
-32P]dCTP,
dTTP, dATP, and dGTP using DNA polymerase I (large fragment). After
phenol/chloroform extraction, the DNA fragment was separated from
unincorporated nucleotides using a Sephadex G-50 column (Sigma). The
concentration of the final product was estimated assuming an 80% yield.
The 750-bp blunt duplex DNA fragment was prepared by digestion of
pLitmus28 with DraI, followed by treatment with calf
intestinal phosphatase (Roche Molecular Biochemicals) to produce 5'-OH.
The fragment was isolated from an agarose gel using Geneclean and was
subsequently labeled with [
-32P]ATP and T4
polynucleotide kinase. The DNA fragment was purified as described above
using an A-5m column (Bio-Rad). The concentration was estimated as
indicated above.
The 20-bp partial duplex substrate used in single-turnover assays was
prepared by labeling the single-stranded 20-mer with [-32P]ATP and T4 polynucleotide kinase. The labeled
20-mer was mixed in annealing buffer (50 mM NaCl, 10 mM Tris-HCl (pH 7.5 at 22 °C), and 1 mM
MgCl2) with M13mp7 ssDNA at a 1:1 molar ratio. The annealing mixture was heated at 95 °C for 5 min, followed by
successive 20-min incubations at 65, 42, and 22 °C. The 92-bp
partial duplex used in single-turnover assays was annealed as described
above prior to labeling with [-32P]dCTP as described
previously (36). Following labeling and annealing, DNA substrates were
diluted to 100 µl in 100 mM NaCl, 10 mM
Tris-HCl (pH 7.5 at 25 °C), and 1 mM EDTA and
phenol/chloroform-extracted. Unincorporated nucleotides were removed
using a Sephadex G-50 spin column as described (37). Concentrations
were estimated assuming an 85% yield. The 851- and 92-bp partial
duplex substrates used in standard helicase and nitrocellulose filter
binding assays were prepared as described previously (36).
Nitrocellulose Filter Binding--
The binding of UvrD, MutL,
and UvrD + MutL to DNA was evaluated by measuring the retention of a
[32P]DNA ligand on nitrocellulose filters as described
previously (38, 39). Experiments were done using a
32P-labeled 90-mer (0.4 nM molecules; 36 nM nucleotide phosphate) or a 92-bp partial duplex
substrate (0.17 nM molecules; 1.2 µM nucleotide phosphate). Reaction mixtures contained 25 mM
Tris-HCl (pH 7.5 at 22 °C), 3 mM MgCl2, 20 mM NaCl, 5 mM 2-mercaptoethanol, and 50 µg/ml
bovine serum albumin (reaction buffer). These experiments contained the
indicated concentrations of UvrD and/or MutL. Proteins were diluted in
helicase II storage buffer (20 mM Tris-HCl (pH 8.3 at
22 °C), 0.2 M NaCl, 25 mM 2-mercaptoethanol,
50% (v/v) glycerol, 1 mM EDTA, and 0.5 mM EGTA).
Equilibrium binding experiments were done in 20-µl reactions. UvrD
and MutL were premixed. Reaction tubes were prewarmed for 30 s at
37 °C and initiated by the addition of premixed protein. Reactions
were incubated at 37 °C for 10 min and diluted with 1 ml of reaction
buffer without bovine serum albumin immediately before filtration. The
entire reaction was then filtered.
Reactions were filtered using the double-filter technique (40) over
nitrocellulose membranes (Millipore Corp.) and NA45-DEAE filters
(Schleicher & Schüll). Nitrocellulose filters (38) and DEAE
filters (40) were prepared as described and rinsed with 1 ml of
reaction buffer with bovine serum albumin prior to application of the
reaction mixtures. Reactions were filtered at a flow rate of ~2
ml/min. Filters were washed two times with 1 ml of reaction buffer and
dried for liquid scintillation counting. Total radioactivity was
determined for each titration or time point as the sum of radioactivity
on the nitrocellulose filter and the DEAE filter. Background values,
determined from nitrocellulose filters of no-protein controls, were
typically <1% of the total counts. Relative macroscopic
KD values were calculated from equilibrium binding
data by fitting the equation for a rectangular hyperbola to the data.
Data fitting was done using the nonlinear least-squares technique and
SigmaPlot (Jandel Scientific). Error values in binding constants were
generated by SigmaPlot.
Gel Mobility Shift Assays--
Gel shift reaction mixtures (20 µl) contained 25 mM Tris-HCl (pH 7.5 at 22 °C), 3 mM MgCl2, 20 mM NaCl, 5 mM 2-mercaptoethanol, 0.7 nM
32P-labeled 48-base oligonucleotide (32 nM
nucleotide phosphate), and 1 mM AMP-PNP. Proteins were
diluted in helicase II storage buffer, premixed, and incubated on ice
before initiation by the addition of the other reaction components. All
reactions were incubated for 20 min on ice, followed by the addition of
5 µl of 75% (v/v) glycerol to all reaction tubes and loading dyes to the control tube that contained only the oligonucleotide. Glycerol or
dyes did not alter the apparent migration of the ssDNA oligonucleotide (data not shown).
Samples were immediately loaded onto an 8% polyacrylamide gel (67:1
cross-linking ratio) containing 50 mM Tris, 50 mM borate, and 2.5 mM EDTA. Samples were
electrophoresed at constant voltage (8 V/cm) until the bromphenol blue
marker had migrated to ~1 inch from the bottom of the gel. Results
were visualized using a Storm 840 PhosphorImager (Molecular
Dynamics, Inc.).
Helicase Assays--
Standard helicase reaction mixtures (290 µl) contained 25 mM Tris-HCl (pH 7.5 at 22 °C), 3 mM MgCl2, 20 mM NaCl, 5 mM 2-mercaptoethanol, 50 µg/ml bovine serum
albumin, and the indicated [32P]DNA substrate (0.28 nM 750-bp blunt duplex, 0.4 nM 851-bp partial duplex, or 0.17 nM 92-bp partial duplex). Proteins were
diluted in helicase II storage buffer, and protein was premixed (either UvrD and storage buffer or UvrD and MutL) prior to combination with the
other reaction components. Reactions were prewarmed at 37 °C prior
to initiation with ATP (to 3 mM), and incubation was continued at 37 °C. Following initiation, 20-µl samples were
withdrawn at the indicated times and quenched by combining with 10 µl
of stop solution (37.5% glycerol, 50 mM EDTA, and 0.5%
each xylene cyanol and bromphenol blue). Reaction products were
resolved on 8% nondenaturing polyacrylamide gels. Results were
visualized and quantified using a Storm 840 PhosphorImager and
ImageQuant software (Molecular Dynamics, Inc.). Data obtained for the
initial 10 min of the reaction, avoiding points where the concentration of DNA substrate becomes limiting, were fit to Equation 1 from Amaratunga and Lohman (41),
|
(Eq. 1)
|
where A(t) is the total amplitude or
femtomoles unwound at time t, Ab is
the amplitude of the burst phase, kb is the rate constant for the burst phase, and vss is the
steady-state rate of unwinding represented by the second phase of the
curve (41). Values of kinetic constants were determined using the
nonlinear least-squares technique and SigmaPlot. Error values were
generated by SigmaPlot.
For the experiment shown in Fig. 6, the helicase reaction mixture was
assembled as indicated for standard helicase assays with the 148-bp
blunt duplex at a final concentration of 0.15 nM molecules.
Reactions were performed at 22 °C. Reactions were incubated for 1 min at 22 °C prior to initiation of the unwinding reaction with ATP
(to 3 mM) in a total reaction volume of 120 µl. After
30 s had elapsed, 1 µl of 0.5 mM 48-mer was added to the reaction mixture to trap free UvrD. Samples were withdrawn, quenched, and analyzed as indicated above.
In single-turnover helicase assays, reactions were assembled as
described above with a final DNA substrate concentration of 1 nM. Unwinding reactions were incubated at 18 °C.
Reactions were preincubated on ice for 10 min and at 18 °C for 4 min
prior to initiation of unwinding with a mixture of ATP (to 3 mM) and 48-mer (to 8 µM) for a total reaction
volume of 120 µl. Sample removal, quenching, and analysis were as
indicated above.
| |
RESULTS |
MutL has been shown to dramatically stimulate the unwinding
reaction catalyzed by UvrD as part of a reconstituted mismatch repair
reaction and in a standard helicase assay lacking the other enzymes
involved in mismatch repair (27, 29). However, the biochemical
mechanism of this stimulation is not known. To further investigate this
stimulation, MutL was purified to near homogeneity from a strain
lacking the mutS gene (see "Experimental Procedures") (data not shown). This was done to avoid any complications that might
arise from the presence of low level contamination by MutS. It should
be noted that experiments performed with MutL purified from a
uvrD strain produced results similar to those produced by
experiments performed with MutL purified from a mutS
strain (27) (data not shown).
MutL Binding to ssDNA and Partial Duplex DNA--
Previous
experiments reached contradictory conclusions regarding the ability of
MutL to bind DNA (21-23). The binding of MutL to ssDNA or to a partial
duplex DNA ligand was evaluated using a nitrocellulose filter binding
assay (Fig. 1). MutL bound to the 92-bp
partial duplex DNA with an apparent KD of 25 nM (Fig. 1A, 
), but failed to bind ssDNA
([32P]DNA 90-mer) in the absence of nucleotide at
concentrations up to 400 nM (Fig. 1B, 
). The
failure of MutL to bind to ssDNA in the absence of nucleotide was
confirmed by electrophoretic mobility shift assay (data not shown).
However, limited binding of MutL to ssDNA was observed in the presence
of ATP (data not shown). More significant binding to the ssDNA
oligonucleotide was apparent with the MutL protein in the presence of
the poorly hydrolyzed ATP analog AMP-PNP (Fig. 1B, ). The
apparent KD was 180 nM. Previous studies
have demonstrated that MutL binds AMP-PNP with a greater affinity than
ATP (24). Therefore, it is likely that the limited effect of ATP on
ssDNA binding compared with the more significant effect of AMP-PNP is
due to the greater binding affinity of MutL for AMP-PNP. It is also
apparent that a MutL·ATP complex is competent to bind ssDNA. These
experiments were repeated with other preparations of MutL, including
MutL from a uvrD strain, producing similar results (data
not shown).
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Fig. 1.
DNA-binding properties of MutL.
Nitrocellulose filter binding experiments were performed as described
under "Experimental Procedures" using the indicated concentrations
of purified protein. A, retention of a
[32P]DNA 92-bp partial duplex substrate by MutL () in
the absence of nucleotide. The equation for a rectangular hyperbola was
fit to the data (solid line). B, binding
of UvrD ( ) and MutL () to a [32P]DNA 90-mer in the
absence of nucleotide. The binding of MutL () in the presence of
AMP-PNP is also shown. The equation for a rectangular hyperbola was fit
to data from the binding of UvrD () and MutL () in the presence
of AMP-PNP (solid lines). Data points from the binding of
MutL () in the absence of nucleotide were connected (dashed
line). Data represent the averages of multiple independent
trials.
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Under the same experimental conditions, in the absence of nucleotide
cofactor, the binding of UvrD to the [32P]DNA 90-mer was
examined (Fig. 1B, ). The data for the binding of UvrD to
the ssDNA 90-mer were poorly described by the equation for a
rectangular hyperbola (unlike the binding of MutL in the presence of
nucleotide), indicating that the binding of ssDNA by UvrD in the
absence of nucleotide is more complicated than this simple saturation
model. However, using the equation for a rectangular hyperbola to
analyze the data, the apparent KD for the
interaction of UvrD with ssDNA was determined to be 200 nM.
This estimation of the KD for UvrD binding to ssDNA is likely an underestimate. Nonetheless, this value is consistent with
the values reported previously for the binding of UvrD to ssDNA in the
absence of nucleotide (38, 42).
MutL Enhances the ssDNA Binding of UvrD--
The migration of a
single-stranded [32P]DNA 48-mer on a polyacrylamide gel
was decreased by interaction with UvrD (Fig.
2, compare lanes 1 and
6). The smeared appearance of the DNA suggested that dissociation of the UvrD·48-mer complex occurred during
electrophoresis. This was evident when protein·DNA complexes were
transferred to nitrocellulose and probed using anti-UvrD antibodies.
The anti-UvrD antibodies failed to react with the smeared DNA material,
but recognized material that migrated more slowly on the gel,
consistent with dissociation of UvrD from the DNA during
electrophoresis (data not shown). The addition of 3.2 and 9.6 nM MutL also slowed the mobility of the ssDNA fragment and
formed a distinct species with retarded mobility (lanes 2 and 4). Thus, MutL was able to stably interact with the
ssDNA 48-mer in the presence of AMP-PNP, consistent with the results
obtained in the nitrocellulose filter binding assays.
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Fig. 2.
Electrophoretic mobility shift assays with
MutL, UvrD, and UvrD + MutL. Gel mobility shift assays were
performed with a 32P-labeled ssDNA 48-mer as detailed under
"Experimental Procedures." The binding of the indicated
concentrations of UvrD (lane 1), MutL (lanes 2 and 4), or UvrD + MutL (lanes 3 and 5)
to the 48-mer is shown. The 32P-labeled ssDNA 48-mer
incubated in the absence of protein is shown in lane
6.
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When 12.2 nM UvrD was incubated together with 3.2 or 9.6 nM MutL, a supershifted species was observed migrating more
slowly than the species due to either MutL alone or UvrD alone (Fig. 2,
compare lanes 2 and 4 with lanes 3 and
5). The supershifted species was observed only in the
presence of MutL + UvrD and was not apparent in the presence of either
MutL or UvrD alone even when the concentration of MutL was increased
from 3.2 to 9.6 nM (lanes 2 and 4) or
higher (data not shown) or when the concentration of UvrD was increased
(data not shown). It is likely that the supershifted species is due to
the specific interaction between MutL and UvrD, and not to UvrD and
MutL independently binding to the ssDNA for the following reasons. MutL
and UvrD physically interact both in vitro (in the absence
of DNA) and in vivo (25, 26). In addition, in the presence
of MutL + UvrD, there was a complete absence of the DNA species
associated with MutL alone (compare lanes 2 with lanes
3 and 5), indicating that MutL prefers to bind to
UvrD·DNA. Moreover, it had been observed by Western blotting that
UvrD was associated only with the supershifted species, and not with
the smeared species (lane 1) (data not shown). Thus, UvrD
alone dissociated from DNA during the course of the electrophoresis. However, after the addition of MutL, a distinct supershifted species was observed, indicating that MutL strengthens the interaction of UvrD
with DNA or that the MutL·UvrD·DNA complex has a greater affinity
for ssDNA than UvrD alone.
The data presented above suggest that MutL increases the binding of
UvrD to DNA. Both the affinity of UvrD for ssDNA and the stability of
the complex were improved in the presence of MutL. Therefore, MutL
could stimulate UvrD-catalyzed unwinding either by loading the helicase
onto the DNA substrate or by clamping the helicase onto the DNA and
acting to increase its processivity.
Single-turnover Unwinding of a 20-bp Partial Duplex--
To
address the idea that MutL enhances the UvrD-catalyzed unwinding of
duplex DNA by loading UvrD onto the DNA, we performed a series of
single-turnover experiments using a 20-bp partial duplex substrate.
Either UvrD or UvrD + MutL were preincubated with the 20-bp partial
duplex substrate to allow binding, and the reaction was initiated by
the addition of a mixture of ATP and a ssDNA 48-mer trap. The addition
of the trap eliminated further binding of UvrD to the partial duplex
substrate such that any unwinding observed was the result of UvrD bound
to the 20-bp partial duplex substrate prior to initiation of the
reaction. Given that the processivity of UvrD has been observed to be
40-50 base pairs (43), it is expected that once UvrD initiates
unwinding, it will complete the unwinding of the 20-bp region.
Stimulation of unwinding by MutL is therefore interpreted as evidence
for preloading of helicase II.
Fig. 3A shows the results of
these experiments. It should be noted that MutL alone did not catalyze
unwinding of the 20-bp partial duplex substrate (data not shown). In
the absence of MutL, UvrD (20 nM) unwound <10% of the
20-bp partial duplex substrate. This is consistent with the
KD for the binding of UvrD to a partial duplex
substrate in the absence of nucleotide (38, 44). With increasing
concentrations of MutL, the same concentration of UvrD was able to
unwind increasing amounts of the substrate (up to 85% with 160 nM MutL). This result clearly illustrates that MutL
enhances UvrD-catalyzed unwinding under single-turnover conditions,
suggesting that MutL functions to load UvrD onto the DNA prior to
initiation of unwinding.
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Fig. 3.
Single-turnover unwinding experiments with
the 20-bp partial duplex substrate. A, the unwinding
activity of 20 nM UvrD alone () or 20 nM
UvrD plus the indicated concentrations of MutL (5 nM ( ),
10 nM , 20 nM (), 40 nM
( ), 80 nM ( ), and 160 nM ( )) was
measured as described under "Experimental Procedures." The fraction
unwound was calculated for each protein concentration and time shown as
described previously (38). Data represent the average of at least three
independent experiments. Error bars are means ± S.D.
B, Equation 1 was fit to the data in A to
determine the amplitude of the burst phase (Ab)
at each MutL concentration. Amplitude is plotted against nanomolar
MutL. The equation for a rectangular hyperbola was fit to the data
(solid line).
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The amplitudes of the curves shown in Fig. 3A were
calculated using a modified form of Equation 1. Since the data from
these experiments have only an exponential phase, the constant term was
omitted from calculations. These calculated amplitudes were plotted as
a function of MutL concentration (Fig. 3B), and a saturation curve was observed. Interestingly, the MutL concentration at which the
amplitude was half its maximal value (40 ± 10 nM)
corresponded closely to the apparent KD (25 nM) for MutL binding to a partial duplex substrate (see
Fig. 1A). This finding suggests that, although MutL and UvrD
are known to physically interact (25, 26), stimulation of the
UvrD-catalyzed unwinding reaction is also related to the ability of
MutL to bind DNA. Since the fraction of the substrate unwound is
directly related to the amount of UvrD productively pre-bound to DNA,
it follows that MutL increases the affinity of UvrD for a partial
duplex substrate. This effect was not directly measurable with
nitrocellulose filter binding assays due to the ability of MutL to bind
to a partial duplex DNA ligand in the absence of ATP (see Fig.
1A).
MutL Stimulates Unwinding Catalyzed by UvrD on Long Duplex DNA
Substrates--
The results from the DNA binding data and the
single-turnover unwinding reactions suggest that MutL increases the
affinity of UvrD for ssDNA and may increase the affinity of UvrD for
single-stranded/double-stranded junctions. Therefore, MutL stimulates
UvrD-catalyzed unwinding by loading UvrD onto the DNA substrate. Since
loading is an early step in the unwinding reaction, a burst of
unwinding early in the reaction might be expected in reactions
containing both UvrD and MutL, as compared with reactions containing
UvrD alone. Therefore, the effect of MutL on the rate of unwinding by
UvrD was examined. Long DNA substrates (750-bp blunt duplex and 851-bp
partial duplex) were used in this analysis because the regions of DNA
excised in mismatch repair can be up to 1 kilobase in length (1,
19).
The extent of unwinding by UvrD was examined at multiple time points in
the presence (Fig. 4, A and
B, ) or absence () of 3.1 nM MutL using
both substrates. No unwinding was detected with MutL alone (data not
shown). Clearly, MutL stimulated the rate of unwinding by UvrD on both
the 750-bp blunt duplex (Fig. 4A) and 851-bp partial duplex
(Fig. 4B) substrates. However, the rate enhancement by MutL
was substantially greater on the 750-bp blunt duplex substrate than on
the 851-bp partial duplex substrate.
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Fig. 4.
Helicase reaction rates for UvrD and UvrD + MutL on long duplex substrates. The unwinding activity of UvrD
() or UvrD plus 3.1 nM MutL () was measured at the
time points indicated as described under "Experimental Procedures."
A, unwinding of the 750-bp blunt duplex substrate. The
concentration of UvrD was 53 nM. B, unwinding of
the 851-bp partial duplex substrate. The concentration of UvrD was 35 nM. Data represent the average of at least three
independent trials. Error bars are means ± S.D. The
fraction of unwound substrate molecules was calculated for each time
point as described (38). Solid lines are the curves
generated from the fit of equations to data as detailed under
"Results."
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The data from both the 750-bp blunt duplex and 851-bp partial duplex
substrates were analyzed using Equation 1 as described under
"Experimental Procedures." This equation describes unwinding as a
biphasic process with a burst phase (Ab(1 - e(kbt)))
and a steady-state phase (vsst). The
burst phase of unwinding was due to molecules of UvrD that were
preloaded on the DNA substrate prior to initiation of the reaction. The
steady-state phase of unwinding, or the observed second phase of the
unwinding reaction, was accomplished by those protein molecules that
were not preloaded on the DNA substrate or that had fallen off after
initiation and then rebound the substrate (41, 45).
The exponential equation did not fit the observed data for unwinding
catalyzed by UvrD alone on the 750-bp blunt duplex substrate (Fig.
4A, ). The equation that described the data well was a linear equation representing only the steady-state phase of the unwinding reaction (vsst). The rate
of UvrD-catalyzed unwinding of the 750-bp blunt duplex substrate was
determined to be 0.21 ± 0.01 fmol of substrate unwound per min
(Table I). A similar rate was determined
using another substrate preparation (data not shown). Preloading of
UvrD onto this substrate was inefficient since only the steady-state
phase of the reaction was observed in the absence of MutL. The absence
of a detectable burst phase of unwinding by UvrD alone further
indicates that even if some molecules of UvrD are preloaded on this
substrate, these molecules dissociate before completion of unwinding of
the duplex. Therefore, the only unwinding detected is due to unwinding
by protein molecules binding from solution after initiation of
unwinding, defined as the steady-state phase of the reaction (41).
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Table I
Rate constants for UvrD- and (UvrD + MutL)-catalyzed unwinding on
long DNA substrates
Rate constants were calculated from unwinding experiments shown in Fig.
4 as described under "Experimental Procedures." ND, not determined.
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On the other hand, the unwinding of the 750-bp blunt duplex by UvrD + MutL was well described by two distinct phases (Fig. 4A,
). Therefore, Equation 1 was used to analyze the data from these
experiments. Ab, kb, and
vss were calculated for UvrD-catalyzed unwinding
in the presence of MutL (Table I). The calculated values of
Ab, kb, and
vss were 3.98 ± 0.95 fmol, 0.61 ± 0.2 min1, and 0.06 ± 0.11 fmol
min1, respectively. The rate constants
calculated for a second preparation of the 750-bp substrate were nearly
identical (data not shown). Interestingly, the steady-state rate of
unwinding by UvrD in the presence of MutL (0.06 ± 0.11 fmol
min1) on this substrate was similar to, or
within error of, the steady-state rate of unwinding catalyzed by UvrD
alone (0.21 ± 0.01 fmol min1). The
slightly lower steady-state rate in the presence of MutL was likely due
to the concentration of substrate becoming limiting in the later phase
of the reaction because much of the substrate was unwound during the
burst phase of the reaction. These data support the notion that the
burst in unwinding in the presence of MutL is due to preloading of UvrD
and that the steady-state rate reflects unwinding after UvrD has
dissociated and rebound to the DNA.
Equation 1 could also be used to describe the data from rate
experiments using the 851-bp partial duplex DNA substrate (Fig. 4B). Similar results were obtained by fitting the equation
for a single exponential process to the data. However, Equation 1 described the data better since the unwinding reaction by UvrD and UvrD
in the presence of MutL had two phases. The burst amplitude (Ab) for unwinding catalyzed by UvrD alone was
2.26 ± 0.40 fmol unwound; the rate constant for the burst phase
(kb) was 0.44 ± 0.08 min1; and the steady-state rate
(vss) was 0.08 ± 0.06 fmol unwound per min
(Table I). For the unwinding reaction catalyzed by UvrD in the presence
of MutL, Ab was 3.76 ± 0.40 fmol,
kb was 0.45 ± 0.05 min1, and vss was
0.11 ± 0.06 fmol min1. Therefore, on
the 851-bp partial duplex substrate, UvrD was able to preload and
complete unwinding to produce the exponential phase of unwinding in the
absence of MutL. However, the amplitude of the burst phase was greater
in the presence of MutL. This result suggests that more molecules of
UvrD are preloaded as productive complexes in the presence of MutL.
Moreover, the rate constants for the burst phase of unwinding by UvrD
and UvrD + MutL were identical. The data analysis indicates that the
reason stimulation of unwinding by MutL is more significant on the
750-bp blunt duplex is due to the inability of UvrD, in the absence of
MutL, to efficiently load on this substrate. However, UvrD loads
efficiently onto the 851-bp base pair partial duplex substrate, and
less stimulation by MutL is observed. Nonetheless, there is stimulation
of the UvrD-catalyzed unwinding reaction that is likely due to
continued loading of UvrD by MutL (see "Discussion"). It should be
noted that efficient loading of a helicase, in the absence of any
accessory protein such as MutL, has been demonstrated previously to
directly increase the burst phase of either UvrD-catalyzed (45) or
Rep-catalyzed (41) unwinding. As the protein concentration or the
amount of preloaded protein is increased, the rate constant of the
burst phase remains constant, whereas the amplitude increases (41, 45).
Single-turnover Unwinding of a 92-bp Partial Duplex--
To
further address the possibility of continual loading of UvrD by MutL,
single-turnover experiments using a 92-bp partial duplex substrate were
performed. Since the 92-bp duplex region is beyond the reported
processivity of UvrD (40-50 bp) (45), it was expected that when the
possibility of continual loading of UvrD onto the DNA substrate was
eliminated, less unwinding would be observed than was the case using
the 20-bp partial duplex substrate (see Fig. 3). Fig.
5 shows that this is indeed the case; a
maximum of 2-3% of the substrate was unwound in the presence of 20 nM UvrD. This reflects both the KD for
binding the substrate and the low probability that a single molecule of UvrD will completely unwind a 92-bp duplex region. In the presence of
20 nM MutL, the fraction of the substrate unwound increased to ~14%, consistent with MutL acting to load more UvrD prior to initiation of unwinding. If MutL were acting as a clamp to increase the
processivity of UvrD, then similar fractions of the 20- and 92-bp
substrates should be unwound at equivalent concentrations of UvrD and
MutL. In addition, since less unwinding by the combination of UvrD and
MutL was observed on a 92-bp partial duplex substrate than on the 20-bp
partial duplex substrate, continued loading of UvrD by MutL was
required for optimal stimulation on this substrate. Moreover, the
observation of less unwinding on the 92-bp partial duplex substrate
indicates that MutL is not affecting the processivity of UvrD. The
observed 4-fold stimulation of unwinding on the 92-bp partial duplex
substrate is very similar to the 4-fold stimulation caused by the
addition of 20 nM MutL to the unwinding reaction involving
the 20-bp partial duplex substrate, indicating that the effect of
adding MutL is the same with both substrates and most likely reflects a
loading phenomenon.
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Fig. 5.
Single-turnover unwinding experiments with
the 92-bp partial duplex substrate. The unwinding activity of 20 nM UvrD () or 20 nM UvrD plus 20 nM MutL () was measured at the time points indicated as
described under "Experimental Procedures." Data represent the
average of at least three independent trials. Error bars are
means ± S.D. The fraction of unwound substrate molecules was
calculated for each time point as described (38). The equation for a
rectangular hyperbola was fit to the data (solid lines)
using SigmaPlot.
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A Test of Processivity with a 148-bp Blunt Duplex--
To more
directly address the issue of processivity, UvrD (3 nM) and
an excess of MutL (50 nM) were incubated with a 148-bp blunt duplex substrate, and unwinding was initiated with ATP. An excess
of ssDNA 48-mer trap (4 µM) was added at 30 s to
bind free UvrD and to prevent further initiation events. The results of
this experiment are shown in Fig. 6. The
addition of the trap 30 s after initiation of the unwinding
reaction has the effect of completely quenching the reaction. Titration
of the trap from 0.4 to 8 µM produced no change in the
observed effect (data not shown), indicating that the trap is not
acting to actively dissociate UvrD from the DNA. If MutL acted to
increase the processivity of UvrD, perhaps by clamping UvrD onto the
DNA, the fraction of the substrate unwound would continue to increase
after the addition of the ssDNA trap, as UvrD molecules
already in the process of unwinding continued to the end of the duplex
substrate. However, no increase was observed. In fact, the unwinding
reaction ceased immediately, suggesting that additional molecules of
UvrD must be loaded to complete unwinding of the 148-bp duplex
substrate. This is consistent with the observed low processivity of
UvrD (45) and suggests that MutL is not acting as a processivity factor.
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Fig. 6.
Processivity test of UvrD and UvrD + MutL on
the 148-bp blunt duplex substrate. Processivity test experiments
were performed as detailed under "Experimental Procedures" for 3 nM UvrD plus 50 nM MutL in the absence of added
ssDNA trap () or with the addition of the ssDNA trap after 30 s
of unwinding (). Data represent the average of at least three
independent trials. Error bars are means ± S.D. The
fraction of unwound substrate molecules was calculated for each time
point as described previously (38). The equation for a rectangular
hyperbola was fit to the data (solid lines) using
SigmaPlot.
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DISCUSSION |
Both UvrD and MutL are essential components of the E. coli mismatch repair machinery (12, 13, 17-19). A physical
interaction between these proteins has been demonstrated (25, 27), and MutL greatly stimulates the unwinding activity of UvrD (27, 29). Based
on the results presented here, we propose that MutL loads UvrD
productively onto the DNA, but does not clamp UvrD onto the DNA during
the unwinding reaction. We also suggest that loading of UvrD by MutL is
likely to be a continuous process.
The initial indication that MutL acted to load UvrD onto DNA came from
DNA binding studies showing that the addition of MutL increased the
affinity of UvrD for DNA (data not shown). This prompted an examination
of the DNA-binding properties of MutL by nitrocellulose filter binding
assays. These studies showed that MutL was able to bind a 92-bp partial
duplex DNA in the presence and absence of nucleotide. In contrast, the
binding of MutL to ssDNA required the presence of AMP-PNP. MutL binding
to ssDNA was significantly reduced when ATP was substituted for
AMP-PNP. This likely reflects the higher affinity of MutL for the ATP
analog and the fact that a MutL·ATP complex interacts with ssDNA,
whereas MutL alone does not interact with ssDNA (21, 24). The DNA binding data reported here are consistent with the most recently reported DNA-binding characteristics of MutL (21).
The binding of UvrD to both ssDNA and partial duplex DNA substrates has
been well documented, and it has been shown that the binding affinity
of UvrD for ssDNA is increased in the presence of AMP-PNP (see Figs. 1
and 2) (38, 42). The electrophoretic mobility shift assay experiments
reported here revealed that UvrD, in the presence of AMP-PNP, formed a
weak complex with ssDNA that dissociated during the course of
electrophoresis. In the presence of MutL, a supershifted
MutL·UvrD·ssDNA complex formed that was more stable than the
UvrD·ssDNA complex, suggesting that MutL + UvrD form a specific
complex that has a greater affinity for ssDNA than UvrD alone.
Since the DNA binding experiments were equilibrium experiments, it
could not be determined from these studies if the MutL-enhanced ssDNA-binding affinity of UvrD was due to an increased rate of association (on-rate) or a decreased rate of dissociation (off-rate) of
UvrD with ssDNA. If MutL decreased the UvrD dissociation
rate, one could envision MutL functioning as a clamp, keeping UvrD
tethered to ssDNA as it unwinds and effectively increasing its
processivity. Results from unwinding assays using a 148-bp blunt duplex
substrate suggested that this was not the case. In these experiments, a ssDNA trap was added after a short period of incubation to effectively prevent reloading of UvrD molecules. Unwinding ceased immediately upon
addition of the trap. If MutL were acting to increase the processivity
of UvrD, then UvrD already bound to the DNA would continue to unwind
the substrate after the addition of the trap to produce an observable
increase in unwound product formation. Single-turnover experiments with
the 92- and 20-bp partial duplex substrates also indicated that MutL
was not acting to increase the processivity of UvrD. A smaller
fraction of the 92-bp partial duplex molecules were unwound in
comparison with the 20-bp molecules. If MutL were acting to increase
the processivity of UvrD, then the same fraction of substrate would be
unwound in each case. Moreover, since the degree of stimulation was
similar on both partial duplex substrates, stimulation appeared to be
independent of substrate length. Taken together, the data described
above suggest that a clamping model is improbable.
An increased rate of association of UvrD with ssDNA would be
reflected in increased loading of UvrD onto the DNA. Preincubation of
MutL with UvrD resulted in increased product formation in
single-turnover experiments using a 20-bp partial duplex substrate.
This stimulation reflects an increase in the amount of productively
loaded UvrD. The only unwinding detected under these conditions is due
to preloaded UvrD, as shown previously (45). UvrD molecules that fail
to bind the DNA or that dissociate from the substrate are trapped by
the excess ssDNA. Importantly, as the concentration of MutL was
increased, the amplitude of the burst phase increased (see Fig.
3B). The concentration at which MutL was half-saturating (40 ± 10 nM) was similar to the KD
for the MutL-partial duplex DNA interaction (~25 nM). We
interpret this to indicate that the binding of MutL to DNA is important
for its role in stimulating the UvrD-catalyzed unwinding reaction.
Thus, two interactions involving MutL are critical for stimulation of
the UvrD-catalyzed unwinding reaction: the interaction between MutL and
DNA and that between UvrD and MutL.
Increased loading of UvrD by MutL was further investigated using long
duplex DNA substrates to model the lengths of DNA substrates likely to
be encountered in vivo. Data from helicase reactions using a
750-bp blunt duplex or 851-bp partial duplex substrate support the
notion that MutL loads UvrD onto DNA and further suggest that loading
by MutL is continuous. UvrD-catalyzed DNA unwinding has been
characterized as having multiple phases (41, 45, 46). In the data shown
here, unwinding of the 750-bp blunt duplex and 851-bp partial duplex
substrates was generally described by a burst phase followed by a
steady-state phase. The burst phase for these reactions reflects
unwinding by those UvrD molecules that were preloaded on the DNA prior
to initiation of the reaction by adding ATP, whereas the steady-state
phase reflects unwinding by those UvrD molecules (minus MutL) that
rebound to the DNA after dissociation. Comparison of the unwinding
kinetics exhibited by UvrD on the 750-bp blunt duplex DNA in the
presence or absence of MutL clearly showed there was no burst phase in
the absence of MutL. This reflects an inability of UvrD to efficiently
preload on blunt duplex substrates, as suggested previously (45). The second, or steady-state, phase of the unwinding reaction was similar in
the presence and absence of MutL, indicating that MutL does not play a
role in this stage of the unwinding reaction. UvrD-catalyzed unwinding
of the 851-bp partial duplex substrate was not as dramatically stimulated by the presence of MutL. The major difference in the kinetics of unwinding plus or minus MutL was a slight shift upward in
the amplitude during the burst phase of the reaction. This rather small
effect of MutL is indicative of the ability of UvrD, in the absence of
MutL, to efficiently preload on a substrate that has an excess of
ssDNA. Accordingly, ssDNA tails have been reported to facilitate
efficient preloading of UvrD (45). The small increase in the amplitude
of the reaction suggests that loading of UvrD in the presence of MutL
is still more productive than MutL-independent loading on this substrate.
All the data from unwinding assays suggest that more UvrD is
productively loaded on the DNA substrate in the presence of MutL. In
experiments using the 750-bp blunt duplex and 851-bp partial duplex
substrates, the increased productive loading of UvrD is likely to be
continuous over the entire course of the unwinding reaction.
Considering the reported processivity for UvrD (40-50 bp) (43),
completion of unwinding of these longer substrates and detection of the
significant burst phase in the unwinding assay with the 750-bp blunt
duplex DNA require multiple binding events by UvrD.
We propose the model shown in Fig. 7 to
explain stimulation of UvrD-catalyzed DNA unwinding by MutL. A nicked
DNA substrate is shown since this is believed to be the relevant
substrate in vivo. However, the model is the same for any
substrate with a duplex length in excess of the intrinsic
processivity of UvrD. The first step (step 1 to
step 2) is loading of UvrD onto the DNA. In the presence of
MutL (Fig. 7B), this rate is enhanced. After it is loaded
(step 2), UvrD begins to unwind the duplex (step
3). In the presence of MutL (Fig. 7B), multiple
molecules of UvrD are being loaded behind the leading molecule of UvrD. In the absence of MutL, loading of additional UvrD molecules is much
slower; and therefore, the concentration of UvrD on the DNA substrate
does not increase as rapidly (Fig. 7, A (step 3)
versus B (step 3)). The high
concentration of UvrD increases the overall rate of UvrD-catalyzed
unwinding in the presence of MutL. Eventually, the leading molecule of
UvrD will dissociate since it is known that UvrD translocates an
average of 40-50 bp before dissociating (43). In the case of UvrD
alone, the partially unwound duplex can re-anneal when the leading UvrD
molecule dissociates, and the whole process must start over (step
1). On the other hand, in the presence of MutL, multiple UvrD
molecules have been loaded, and the DNA does not re-anneal. The
additional UvrD molecules continue the unwinding reaction.
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Fig. 7.
Model for the mechanism of UvrD-catalyzed
unwinding with the addition of MutL. The model for unwinding by
UvrD (A) or UvrD + MutL (B) is shown. Details for
the model are described under "Discussion." Step
1 is preloading; step 2 is with one molecule of UvrD
loaded; step 3 is unwinding; and step 4 is after
dissociation. Gray ovals represent UvrD, and white
squares are MutL (L).
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§¶,
, and
TOP
ABSTRACT
INTRODUCTION
