 |
INTRODUCTION |
Chromosomal replicases are highly processive machines owing to a
sliding clamp subunit that encircles and slides on DNA, acting as a
mobile tether for the replicase during synthesis (1-4). These circular
clamps require a multimeric clamp loader assembly for their opening and
closure around DNA in a process that consumes ATP. In Escherichia
coli the clamp is the 
dimer, formed from two crescent-shaped
protomers (5), and the ring is opened and closed by the 
complex
clamp loader (
'
). Once on DNA, acts as a mobile
tether for the replicase, DNA polymerase III holoenzyme, holding it to
DNA for highly processive synthesis (1). In fact, the subunit can
also couple with all the other E. coli DNA polymerases (DNA
polymerases I, II, IV, and V) (6-9) and with DNA ligase and MutS (10).
The eukaryotic system is similar (11). Here, the
RFC1 clamp loader assembles
the ring-shaped PCNA clamp onto DNA for processive DNA polymerase
action (12, 13). PCNA is also known to interact with several other
proteins indicating that, like , these clamps serve multiple roles
in cellular DNA metabolism (14).
This report is part of a continuing study on the mechanism of the
E. coli complex clamp loader. The complex consists
of five different subunits (') (15), three of which
(') are essential to clamp loading action (16). One copy each
of the and subunits bind the ' core but are not
essential to clamp loading activity (17). The crystal structure of the ' complex has recently been solved, and it shows that there are three subunits and one each of and ' in a circular
pentameric arrangement (18). A protein in the holoenzyme known as 
is encoded by the same gene as (dnaX) and therefore is
essentially identical to except for an extra C-terminal section in
(19, 20). Fully active clamp loading complexes can be reconstituted and are composed of one each of , ', and either 3
or 3, or mixtures of and (i.e.
12 and 21)
(17, 21). The unique C terminus of , lacking in , binds to the
DNA polymerase III core and DnaB, thereby acting to organize the
replisome machinery (22, 23).
The () subunit of the clamp loader is the only one that binds
and hydrolyzes ATP, and thus is the motor of the clamp loader (19). The
subunit of complex forms a strong attachment to and, in
fact, opens or destabilizes one of the dimer interfaces (25-27).
No ATP is required for this ( does not bind ATP); therefore, the
energy for ring opening is derived from protein-protein interaction between and . The recent crystal structure analysis of in complex with a monomer of provides detailed insight into action (28). The way in which the subunit binds leads to disruption of
one of the dimer interfaces, preventing the ring from closing. Further,
monomeric forms a shallower crescent than each protomer in the
dimer, and thus the subunit structure would appear strained to bend
into a half-circle shape upon partnering with another protomer to
form a closed ring. Hence, upon cracking one dimer interface, the
strain is released in the two halves, allowing them to adopt
shallower crescent shapes and resulting in significant widening of the
gap at the open interface.
The energy for clamp opening is supplied by the protein-protein
interaction between and . However, ATP is required by complex. What is the role of ATP if it is not required for clamp opening? Our studies on this subject reveal that the subunit is
buried within complex such that its interaction with is weak
compared with the ·B complex (26). Upon ATP binding to the subunits, however, the complex undergoes a conformational change
that exposes for interaction with (26, 29). Only in the
presence of ATP and does the ATP· complex· composite show
appreciable affinity for ssDNA (i.e. a site for DNA binding becomes exposed) (29, 30). Upon binding to DNA, especially a primed
site, ATP is hydrolyzed and the connection between and complex
is severed (25). At a primed site, this process results in a closed ring encircling DNA.
Docking of onto the subunit of the crystal structure of
' (by replacing in the ' structure with the
- structure) suggests that may also bind to (18). Our
previous studies utilized gel filtration to detect the relatively
strong interaction of subunit with , but failed to detect an
interaction of with or any other subunit of complex (26).
Hence, we reexamined complex subunits for interaction with in
such a manner that we could detect even very weak interactions.
This report reveals that, in addition to , and also interact
with , and possibly ' as well. Further, like , the subunit
can open , as inferred from its ability to increase its rate of
dissociation from circular DNA. However, binds weaker than and is ~20-fold less efficient in unloading from DNA compared
with (kunloading = 0.42 min
1; kunloading = 0.016 min1). The ' subunit does not appear to unload the ring from DNA, but it binds and prevents from unloading .
' also inhibits the unloading activity. Interestingly, the
'-mediated inhibition of , and of , is relieved upon adding
ATP. Hence, ' is essential for coupling ATP to the action of and
with , even though ATP binds and not '. These
interactions between complex subunits among themselves and , and
their regulation by ' and ATP, are discussed in terms of a molecular
model of complex mechanism.
| |
EXPERIMENTAL PROCEDURES |
Proteins and Other Reagents
Radioactive nucleotides were purchased from PerkinElmer Life
Sciences. Unlabeled nucleotides were purchased from Amersham Pharmacia
Biotech. Bio-Gel A-15m gel was purchased from Bio-Rad. Oligodeoxyribonucleotides were synthesized by Life Technologies, Inc.
Singly nicked plasmid DNA was prepared as described (21) using M13 gpII
endonuclease and pBluscript plasmid DNA, which is nicked once at the
M13 origin by gpII. Proteins were purified as described: 
, 
, and
(31); and ' (32); and (33); 
(34); and SSB (31).
The complex was reconstituted from pure subunits and purified from
unassociated proteins as described in our earlier study (17). The
monomer (I272A, L273A) was purified as described
(35). 32P-, a derivative of containing six
C-terminal amino acid residues (NH2-RRASVP-COOH) that serve
as an efficient substrate for cAMP-dependent protein
kinase, was labeled using [-32P]ATP as described (36,
37). 32P- used in this study had a specific activity of
150 dpm/fmol. The catalytic subunit of cAMP-dependent
protein kinase produced in E. coli was a gift from Dr. Susan
Taylor (University of California, San Diego, CA).
Buffers
Buffer A is 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 2 mM DTT, 4% (v/v) glycerol, 1 mM ATP, and 8 mM MgCl2. Buffer B is
20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 100 µg/ml bovine serum albumin (Sigma), 2 mM DTT, 4% (v/v)
glycerol, 8 mM MgCl2. Buffer C is Buffer B, but
contains 2 mM MgCl2 and lacks ATP. 6× sample
loading dye contains 0.25% bromophenol blue, 15% Ficoll, and 0.25%
xylene cyanol. Buffer D contains 8.9 mM Tris, 8.9 mM sodium borate, and 0.2 mM EDTA. Surface
plasmon resonance (SPR) buffer contains 10 mM Hepes-NaOH
(pH 7.4), 150 mM NaCl, 3.4 mM EDTA, and 0.005%
Tween 20. Replication Buffer contained 20 mM Tris-HCl (pH
7.5), 4% glycerol, 0.1 mM EDTA, 40 g/ml bovine serum
albumin, 5 mM DTT, 1 mM ATP, and 8 mM MgCl2.
Surface Plasmon Resonance
The monomer was immobilized on a
carboxymethyldextran matrix-coated sensor chip CM5 using carbodiimide
covalent linkage in 10 mM sodium acetate (pH 5.5) to yield
a final value of ~7000 response units (RU) of immobilized
monomer. The mobile phase (SPR buffer) contained 1 µM , 1 µM , 1 µM ,
or 1 µM ' or 1 µM . SPR buffer
containing protein was passed over the immobilized at a flow rate
of 6 µl/min for 5 min, after which SPR buffer lacking protein was
injected over the chip.
Preparation of ·DNA Complex
The ·DNA complex was prepared as substrate for clamp
loading assays as follows. 32P- (1.5 pmol) was incubated
with 1 pmol of complex and 1.25 pmol of gpII nicked pBluescript
plasmid DNA at 37 °C for 10 min in 50 µl of Buffer A. The reaction
was applied to a 5-ml Bio-Gel A-15m gel filtration column (Bio-Rad)
equilibrated in Buffer B at room temperature, and fractions of 180 µl
were collected. Because of the large size of the DNA, the
32P-·DNA complex elutes early (usually fractions
11-14) and separates from free 32P-, complex, and
ATP (in fractions 21-31). Three peak fractions (usually 11-13)
containing 32P-·DNA (determined by scintillation
counting) were pooled for use as substrate in the unloading reactions.
Clamp Unloading Assays
Proteins were analyzed for ability to unload clamps by mixing
0.4 nM 32P-·DNA substrate in 25 µl of
Buffer B on ice with 25 µl of Buffer C containing , ', or
3 (0.5-3.0 µM, as indicated in the plots or figure legend), and then the reaction was shifted to 37 °C for
incubation. Reactions were quenched at various times (5-180 min, as
indicated in the plots or figure legend) upon addition of 5 µM monomer (3 µl of 82 µM
monomer), and then the quenched reaction was immediately
placed on ice. We have shown previously that monomer
effectively quenches -mediated unloading (35) and we find that
it also quenches -mediated unloading of (as discussed under
"Results" and shown in Fig. 3). Next, 8 µl of 6× sample loading
dye was added to the quenched reactions, followed by analysis in a
1.5% neutral agarose gel, which separates free 32P-
from 32P-·DNA complex. Electrophoresis was for 1 h (100 V) at room temperature in Buffer D. Gels were then removed,
fixed with 20% acetic acid for 10 min, and overlaid with one layer
each of DE-81 paper, nitrocellulose membrane, Whatman 3M paper, and
several paper towels, and then flattened under a lead brick until ~3
mm thick. The flattened gel was then wrapped in plastic wrap and
exposed to a phosphor screen (Amersham Pharmacia Biotech) for ~12 h.
Bands corresponding to 32P- on and off DNA were
visualized using a PhosphorImager (Amersham Pharmacia Biotech), and the
amount of 32P- in each band was quantitated using
ImageQuant (Amersham Pharmacia Biotech). The fraction of on DNA at
each time point was calculated as the ratio of
32P-·DNA complex to total (summation of free
32P- and 32P-·DNA). This value was then
normalized to 1.0 by dividing by the fraction of on DNA at time 0 ([ on DNA]t/[ on DNA]t=0). Typically the percentage of total on DNA at time 0 was 70-95%; the variability is likely the result of some spontaneous loss of from DNA between the initial isolation of
the substrate 32P-·DNA, and the unloading experiment.
Overall, clamp unloading reactions are second order, but because the
concentration of catalyst in the reaction (e.g. or )
is much higher than the substrate (32P-·DNA), the
reaction becomes pseudo first-order and the unloading rate
(kunloading) can be obtained at any particular
or subunit concentration using the first-order equation:
([·DNA]t/[ on
DNA]t=0) = e(kunloading)(t),
where t = time. Data points from unloading time courses
were fit to this equation to obtain the observed
kunloading value at a given concentration of
protein subunit (i.e. 3, , ', or
combination thereof). The kunloading values were
then plotted versus the concentration of the protein
subunit used as the catalyst for unloading and then the best
fit to the hyperbolic equation: kunloading=
(kunloading(max))([3])/([3] + Kd) was determined to obtain the apparent maximal
unloading rate (kunloading(max)) and the
apparent Kd value for interaction of the catalyst
with the ·DNA complex.
Assays that examine subunit mixtures for 32P-
unloading activity were performed upon mixing 0.4 nM
32P-·DNA in 25 µl of Buffer B with 25 µl of Buffer
C containing some combination of (0.2-1.0 µM),
3 (0.5-3 µM), and ' (0.5-5 µM). Subunit mixtures were preincubated for 10 min on ice
before addition to the assay. Specifics are as follows. Assays that
examined the effect of and/or ' on the ability of to unload
at a fixed time point (3 min) all contained 0.4 nM
32P-·DNA in Buffer B (25 µl) to which was added 25 µl of a mixture containing 1 µM and either (0.5, 1, 2, or 3 µM) or ' (0.5, 1, 2, 4, or 5 µM). Time courses of unloading assays (0, 5, 15, and
30 min) performed using mixtures of either or ' contained 0.2 µM that was preincubated (25 µl) with either 1 µM 3 or 1 µM ' before
addition to the 32P-·DNA substrate. Reactions
containing three subunits were performed by preincubating 0.2 µM with either 2 µM each
3 and ' or 3 µM each 3
and ', in the presence or absence of 1 mM ATP in 25 µl
of Buffer B. Reactions were incubated at 37 °C for the indicated times or as described in the legend, and then quenched using
monomer and analyzed on an agarose gel as described
above. In reactions containing ATP, 1 mM final
concentration of ATP was included in the protein pre-incubation reaction.
monomer Inhibition Reactions
DNA Synthesis--
In assays examining monomer
inhibition of DNA synthesis, reactions contained 420 nM SSB
(as tetramer), 1.4 nM M13mp18 ssDNA primed with a DNA
30-mer, and 0.3 µM complex in 25 µl of Replication Buffer. Following this, monomer was added to reactions
on ice at 0, 0.2, 0.4, 0.8, 1.2, 1.6, or 2.0 µM
concentration. Next, a mixture was added yielding final concentrations
of 60 µM each of dATP, dGTP, and dCTP; 20 µM [-32P]dTTP; 4.8 nM core
(); and 8 nM . The mixture was shifted to
37 °C for 3 min, and then quenched upon addition of 25 µl of 1%
SDS, 40 mM EDTA. Reactions were spotted onto DE81 filters, washed as described (38), and analyzed by liquid scintillation counting.
Clamp Loading--
The effect of monomer on clamp loading by the complex was performed in 100 µl of Buffer A
containing 3 µM SSB (as tetramer), 10 nM
M13mp18 ssDNA primed with a DNA 30-mer, 8 nM complex, and 5 µM monomer (when present). This
reaction was brought to room temperature for 5 min, 32P-
was added to a final concentration of 10 nM, and the
reaction was shifted to 37 °C for 5 min. Reactions were analyzed by
gel filtration on a 5-ml Bio-Gel A15m column equilibrated in Buffer A
as described above for preparation of the 32P- clamp
unloading substrate.
Clamp Unloading--
In assays that examine the effect of
monomer on clamp unloading by and , the
50-µl reactions contained 0.2 nM
32P-·DNA, and reactions were initiated by the addition
of either 0.5 µM or a mixture of 0.5 µM
and 2 µM monomer. Reactions were
incubated at 37 °C for 3 min and then were analyzed in a native
agarose gel as described above for the clamp unloading assay. In assays
that examine the effect of monomer on clamp unloading by , procedures were as described above except that 1 µM , or a mixture of 1 µM and 5 µM monomer, was added and reactions were
incubated at 37 °C for 80 min before analysis in a native agarose gel.
| |
RESULTS |
Interaction of Complex Subunits with --
To
increase the possibility of detecting weak complex subunit
interactions with , we employed the SPR technique. In the SPR
experiment of Fig. 1, we immobilized on the chip surface. For this study we utilized a mutant that
contains two amino acid replacements, which disrupt the dimer interface
resulting in a monomer (27). Use of monomer provides
a stabile base line in SPR, compared with immobilized dimer, which
drifts down over time, probably because of slow dissociation of the
protomer that is not directly cross-linked to the chip. In Fig.
1A, was passed over the immobilized and the
formation of a - complex was indicated by the resulting increase
in mass (recorded as RU). Following the protein injection, buffer
lacking was passed over the chip, resulting in dissociation of from as indicated by loss of the signal. The time courses of mass
accumulation, and mass loss provide information from which the rates of
association and dissociation of - complex can be calculated. The
equilibrium constant calculated from these rates is ~0.03
µM. In Fig. 1B, passage of over also
demonstrated an interaction between them with an approximate
Kd of 0.9 µM (assuming as a
trimer). appears to be a tetramer in solution (39), but it is
trimeric when in association with and ' (40). Therefore, for
ease in comparing kinetic constants obtained using alone, and with and ', we have calculated the concentration of as a trimer for consistency throughout this report. If is considered a
tetramer, the calculated equilibrium constant for the experiment in
Fig. 1B is ~25% lower.
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Fig. 1.
Interaction of complex subunits with the clamp.
Interaction between complex subunits and immobilized were
examined using SPR. The open arrow in each
sensorgram indicates the start of an injection of the indicated subunit
over the immobilized . The solid arrow
indicates the end of the protein injection and the start of an
injection of buffer. The following subunits were injected over
immobilized : panel A, ; panel B, ;
panel C, '; panel D, ; panel E,
. Complex formation with immobilized is indicated by an
increase in mass, registered as RU. Each panel shows a pair
of sensorgrams. In each panel, the lower sensorgram shows
the result of injecting the indicated complex subunit over a sensor
chip that lacked immobilized .
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Fig. 1C examines ' for interaction with ; however,
only very slight, or no, interaction was detected. Fig. 1D
demonstrates that interacts with (~Kd = 1.1 µM). This -to- interaction is not explored
further in this report because neither the nor subunits are
required for clamp loading (41-43). Further, we did not detect a
significant effect of in the experiments of this report. The subunit is not soluble, which prevented us from analyzing for a
-to- interaction. However, the subunit is soluble as a
complex (34, 44). Fig. 1E indicates that complex
binding to gives a similar signal as alone
(~Kd = 1.0 µM), suggesting that forms the major contact to and that may not make a significant
contribution to the interaction between and . However, we
cannot rigorously exclude the possibility of a - interaction from
this data.
The SPR analysis in Fig. 1 was performed using immobilized
monomer because use of dimer in SPR analysis is
limited by dissociation of 2 during the experiment.
However, it is important to keep in mind that interacts with
monomer much tighter than dimer. We do not know at
present whether other complex subunits bind the
monomer and dimer with different affinities.
We have reexamined ability to detect these complexes by gel filtration
analysis using a Superose 12 column. Although we routinely detect
- complex by this method (26, 27), we have not been successful in
detecting an interaction between and either , ', , ,
, or ' by gel
filtration.2 As gel
filtration is not an equilibrium technique, we presume that these
complexes are too weak, and dissociate too fast, to be detected by this
method. Inability to detect -, -, and - complex by
gel filtration is consistent with the high Kd values
for these complexes determined from the SPR experiments of Fig.
1.
Catalyzes Unloading--
Our previous studies showed that
can open the ring (25, 27, 35). Ring opening by was deduced
from experiments in which was first assembled onto circular DNA,
then adding subunit. Ability of to open is detected by
removal of the ring from the circular DNA. To follow dissociation from DNA, this assay utilizes a kinase-tagged version of
, which can be radiolabeled using [-32P]ATP and
protein kinase. The 32P- is then placed onto circular
DNA using complex and ATP, followed by gel filtration to isolate
the 32P-·DNA complex from free and complex/ATP. The 32P-·DNA complex is then used as a
substrate to examine complex and for unloading activity. If
32P- is unloaded from DNA, the amount of
32P- unloaded from DNA can be determined by analysis of
the reaction in a native agarose gel (or by a second gel filtration
column analysis). The 32P- on DNA comigrates with the
DNA substrate in the agarose gel and resolves from the free
32P-, which migrates faster through the gel. Using this
procedure, we showed earlier that 32P- has a half-life
for spontaneous dissociation from DNA of ~120 min at 37 °C, but if
the ring is opened by or complex, the 32P- is
unloaded from DNA much quicker (19). In Fig.
2, this assay was used to examine for
ability to open .
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Fig. 2.
The subunit has
weak unloading activity. The indicated
amounts of (A), (B), or + 1 mM ATP (C) were incubated with
32P-·DNA for 5, 15, or 30 min before being stopped and
analyzed by native agarose gel electrophoresis. The autoradiograms of
the gels are shown to the left in each panel. The
top band is 32P- on DNA; the
bottom band is free 32P-. Observed
off rates of 32P- dissociation from DNA were obtained by
fitting data to a first order decay process. These rates were as
follows. Panel A, no added, 0.005 min1;
0.2 µM , 0.11 min1; 0.3 µM
, 0.16 min1, 0.5 µM , 0.18 min1; 1 µM , 0.27 min1.
Panel B, no , 0.005 min1; 0.5 µM 3, 0.006 min1, 1.0 µM 3, 0.016 min1 2 µM 3, 0.014 min1; 3 µM 3, 0.016 min1.
Panel C, no 3 + 1 mM ATP, 0.003 min1; 0.5 µM 3 + 1 mM ATP 0.004 min1, 1.0 µM
3 + 1 mM ATP, 0.010 min1; 2 µM 3 + 1 mM ATP, 0.008 min1; 3 µM 3 + 1 mM ATP, 0.010 min1. Replots of the observed
off rates versus subunit concentration are shown
at the bottom right in each panel. The
best fit to the data yielded the following maximum unloading rate
(kunloading) and apparent Kd,
respectively: panel A for , 0.42 min1, 0.53 µM; panel B for , 0.023 min1,
1.6 µM; panel C for + ATP, 0.016 min1, 1.5 µM.
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First, in Fig. 2A, is a control reaction using the subunit. In the absence of added , spontaneously dissociates
from DNA with a half-life of ~140 min. Addition of to the assay
results in much more rapid dissociation of 32P- from the
DNA (t1/2 = 5 min or less). This experiment was
repeated using 0.2-1.0 µM , and the autoradiograms of
the agarose gels are shown to the left of Fig. 2A. From the amount of 32P- on and off the
DNA, the ratio of remaining on DNA can be obtained at each time
point and is plotted in the top right of Fig.
2A. The data points were fit to a model of this kinetic
process (see "Experimental Procedures") to obtain the observed
rates of dissociation from DNA at each concentration of . A
replot of the observed rates versus subunit
concentration (Fig. 2A, bottom right)
yields an apparent maximal kunloading value
of 0.42 min1.
Next, we examined the effect of increasing concentrations of on the
stability of 32P- on DNA. Previous studies using this
assay did not detect opening by , but only a low concentration
of was used in that study and the incubation time was limited to 10 min (25). The assay in Fig. 2B utilizes several different
concentrations of and extends the incubation time with
32P-·DNA for up to 120 min. The results show that, in
the presence of 1 µM , the dissociation time of
32P- from DNA is reduced to 75 min and is further
decreased to 40 min at the highest concentration of tested (3 µM ). A replot of these observed rates
versus subunit concentration indicate an apparent
maximal rate of unloading (kunloading) of
0.023 min1. Hence, can unload from DNA, but is
less efficient compared with .
These experiments were performed in the absence of ATP, yet is an
ATP interactive protein. Does ATP alter the results? We examined the
effect of ATP on -catalyzed unloading in Fig. 2C, but
the results were essentially the same as those observed in the absence
of ATP.
In these unloading experiments, time points are removed from a
reaction, placed on ice, and quenched from further unloading by adding
the monomer mutant, which acts as a competitor and prevents further - or -mediated unloading of
32P-2. SDS can not be used to quench the
reaction because it would simply denature , causing all the
32P-2 to be released from DNA. We have shown
previously that monomer (1) stops
-mediated clamp unloading (35), and this is demonstrated in Fig.
3A for as well. In fact,
the monomer also inhibits complex-mediated clamp
loading, as illustrated in Fig. 3B. In the Fig.
3B experiment, complex is used to assemble
32P-2 onto DNA, then the reaction is
filtered over a Bio-Gel A15m column, which resolves the large
32P-2·DNA complex (fractions 12-16) from
free 32P-2 that is unattached to DNA. The
monomer prevents complex from loading the
32P-2 onto DNA. We have also tested the
monomer for an effect on DNA synthesis using
2, complex, and core polymerase. The results, in
Fig. 3C, demonstrate that monomer inhibits
DNA synthesis. What underlies ability of monomer to
inhibit in each of these assays? We have demonstrated previously that
binds monomer at least 50-fold tighter than the
dimer (26). Hence, formation of dead end complexes between
monomer and (or or complex) likely underlies
the mechanism of inhibition in each of the assays of Fig. 3, as
diagrammed in the schemes to the left of each
panel.
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Fig. 3.
monomer inhibits
clamp unloading, clamp loading, and replication. The
schemes at the left illustrate how a dead end
complex may form between monomer and , , or
complex, which could explain the observed inhibition by
monomer of unloading, loading, and DNA
synthesis. Panel A, effect of monomer on unloading by and . Reactions contained 32P-·DNA
complex and were incubated 3 min at 37 °C with: lane 1,
no addition; lane 2, 0.5 µM ; lane
3, 0.5 µM + 2 µM
monomer; lane 4, 1 µM
3; lane 5, 1 µM
3 + 5 µM monomer. The
positions of 32P-·DNA complex (top
band) and free 32P- (bottom
band) are indicated to the right of the
autoradiogram of the agarose gel. Panel B,
monomer inhibits clamp loading by complex. Reactions
contained 32P-, complex, SSB-coated primed M13mp18
ssDNA in the presence (squares) or absence
(circles) of monomer. After incubation at
37 °C for 5 min, reactions were analyzed for 32P- on
DNA by gel filtration as described under "Experimental Procedures,"
which resolves 32P-·DNA complex (fractions 12-16)
from free 32P- in solution (fractions 20-32).
Panel C, monomer inhibits DNA synthesis. The
complex was preincubated with DNA, , and increasing
concentrations of monomer, and then core polymerase was
added to initiate primer extension around the primed M13mp18 ssDNA
template. DNA synthesis was monitored by following the incorporation of
radioactive dNTPs.
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The finding that monomer is a potent inhibitor of
replication in vitro begs the question of whether it may do
so in vivo as well. However, the Kd of
the dimer to monomer equilibrium is below 50 pM (45),
and its concentration in the cell is ~500 nM (35). Hence,
there should be very little of the monomeric species of in the
cell, especially relative to the amount of dimer.
Inhibits -Mediated Unloading--
Next, we studied the
effect of a mixture of and on the stability of
32P- on DNA. In the experiment of Fig.
4 (A and B), we
added 1 µM and various concentrations of subunit
to 32P-2·DNA complex. Reactions were then
incubated for 3 min at 37 °C before quenching with
monomer and analysis in an agarose gel. If and act independently, the expected rate of unloading using the mixture
would be approximately the same rate as using without , because
1 µM is much more efficient at unloading than
even 3 µM . However, we observed a markedly different result; the presence of with resulted in a marked decrease in
the rate at which unloaded . In Fig. 4C, the time
course of inhibition of , using at 0.2 µM and
at 3 µM, yielded a t1/2 ~ 35 min
for unloading, ~7 times slower than the rate of dissociation in the presence of by itself.
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Fig. 4.
blocks from unloading clamps from DNA.
The scheme at the top shows bound to an open
ring and suggests that interaction with may block the
- interaction, thereby largely preventing from opening and unloading it from DNA. Panel A, 1 µM was incubated with the indicated amounts of 3 and
32P-·DNA complex at 37 °C for 3 min, then quenched
with monomer and analyzed on a native agarose gel.
Panel B, the bar plot is a
quantitation of the autoradiogram shown in panel A. Panel C, the effect of ATP on ability of to inhibit in clamp unloading was examined. The control reaction contained 0.2 µM , and the best fit to the data
(diamonds) yields
kunloading = 0.135 min1. In
the presence of both 3 µM 3 and 0.2 µM , the rate was decreased to
kunloading = 0.021 min1 in the
absence of ATP (circles) and 0.022 min1 in the
presence of ATP (squares).
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This apparent dilemma, in which a mix of two unloading proteins results
in slower unloading of from DNA compared with the rate observed
using subunit separately, may be explained by at least two
different mechanisms. In one case, interaction with may be
competitive with , thereby preventing the more effective from
even binding . This seems unlikely, given the higher affinity of for compared with for . Another possibility is that, when
binds , it occludes the sites on and from interaction
with . This last possibility is illustrated in Fig. 4.
Next, we examined the / reaction for an effect of ATP. Fig.
4C shows a comparison of the / activity in
32P- unloading in the presence and absence of ATP. The
result shows that ATP has no significant effect on the reaction. We
have examined this reaction at 0.2 µM and several
different concentrations of (0.5, 1, 2, and 3 µM) but
have not detected a significant difference plus or minus ATP. This
result is quite interesting given the fact that complex is an
efficient unloader only when ATP is present (25, 35, 45). Hence, it
would appear that ' (which is not present in the reactions of Fig.
4) must be present for ATP to stimulate clamp unloading, even though
, and not ', is the ATP binding subunit.
' Blocks from Unloading --
In Fig.
5 we utilized the unloading assay to
examine ' for ability to open and unload it from DNA, and for
ability of ' to block in unloading. The experiment in Fig.
5A compares the stability of 32P- on DNA in
the presence and absence of 3 µM '. The results show
that ' exerts no apparent effect, positive or negative, on the
stability of 32P- on DNA. This result is consistent with
the very low affinity interaction, or no interaction, between ' and
in the SPR experiment of Fig. 1.
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Fig. 5.
' inhibits unloading by . The
scheme at the top indicates that binds and
opens , but ' binds to , blocking its ability to interact with
, thereby preventing from unloading from DNA. Panel
A, stability of 32P- on DNA is compared in the
presence and absence of 3 µM '. The autoradiograms of
the neutral agarose gels are shown to the left, and the data
are quantitated in the plot to the right. Curve fitting
yields kunloading values of 0.005 min1 in both the absence (circles) and
presence (squares) of '. Panel B, the presence
of ' inhibits unloading by subunit. Unloading reactions
contained 0.2 µM in the absence or presence of 3 µM '. The autoradiograms of the gels are to the
left, and the quantitation to the right yields
kunloading values of 0.133 min1
for alone (circles) and 0.009 min1 for plus ' (squares). Panel C shows a titration of
' into meditated clamp unloading reactions. Lane 1 in
the autoradiogram of the gel, to the left, is a reaction
lacking added protein that was incubated the same amount of time (3 min
at 37 °C) as the rest of the reactions. Lanes 2-7 are
reactions that contain 1 µM and the indicated amount
of '. Results are quantitated in the bar plot
to the right.
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Does the presence of ' influence the activity of in unloading
from DNA? The next experiment demonstrates that ', like
Sliding Clamp of Escherichia coli DNA Polymerase III
Holoenzyme*
and
TOP
ABSTRACT
INTRODUCTION
