From the Department of Biochemistry and Molecular Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262
Received for publication, October 27, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The DnaX complex of the DNA polymerase holoenzyme
assembles the Processive and efficient replication of genomic DNA in both
prokaryotes and eukaryotes is facilitated by three conserved functional components: a DNA polymerase, a sliding clamp processivity factor and a
multiple-subunit complex that loads the processivity factor onto a
primed-template. In Escherichia coli, the DNA polymerase III
holoenzyme is responsible for duplication of the genome in a rapid and
processive manner. The holoenzyme contains ten different types of
subunits and is composed of two DNA polymerase III cores ( The dnaX gene produces two distinct proteins, Functional homomeric DnaX complexes ( Strains--
E. coli strains DH5 Materials and Buffers--
CM5 sensor chips (research grade),
P-20 surfactant, N-hydroxysuccinimide,
1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide, and ethanolamine
hydrochloride were obtained from BIAcore Inc. Ni2+-NTA1 resin,
the QIAquick Gel extraction kit, QIAquick PCR purification kit, and the
plasmid preparation kit were purchased from Qiagen. SDS-polyacrylamide gel electrophoresis protein standards were from Life Technologies, Inc. d-Biotin was obtained from
Sigma. Coomassie Plus protein assay reagent and Immunopure streptavidin were from Pierce. Buffer L, Buffer W, HBS buffer, and HKGM buffer were
prepared as previously described (19).
Construction of the Fusion Plasmids--
Plasmid
PA1-N-
Plasmid PA1-C-
Plasmid pET11-C- Cell Growth and Induction--
Plasmids
PA1-C- Protein Purification--
C(0) Surface Plasmon Resonance--
A BIAcoreTM
instrument was used for protein-protein binding analyses. CM5 research
grade sensor chips were used for all experiments. Streptavidin was
coupled to the sensor chip surface by the
N-hydroxysuccinimide/1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide coupling (19). Biotin-tagged SDS-Polyacrylamide Gel Electrophoresis--
Proteins were
separated by electrophoresis at constant current (20 mA) on 12.5%
SDS-polyacrylamide mini-gels. The gels were stained and destained as
previously described (19).
DNA Polymerization Assays--
Activities of Expression and Purification of the Truncated DnaX Fusion
Proteins--
We constructed three plasmids, each encoding specific
structural domains of DnaX Domain III Interacts with
The interactions between No Observable Binding of
Next, the truncated DnaX proteins N- Domain III of
When injected over immobilized C(0)
Samples of
10 µM of the Domain III of DnaX Is Sufficient for
Under similar experimental conditions, The DnaX complex in E. coli functions to assemble the
Among the truncated In studies directed toward mapping the The presence of Previous studies indicate that the presence of Domain III of DnaX not only contains the The DnaX complex functions to load the Results from our studies shed light on some of the functional
differences between the structurally similar subunits, 2 processivity factor onto the
primed template enabling highly processive replication. The key ATPases
within this complex are
and
, alternative frameshift products of
the dnaX gene. Of the five domains of
, I-III are
shared with
In vivo,
binds the auxiliary
subunits
' and
(Glover, B. P., and McHenry, C. S. (2000) J. Biol. Chem. 275, 3017-3020). To localize
' and
binding domains within
domains I-III, we
measured the binding of purified biotin-tagged DnaX proteins lacking
specific domains to
' and
by surface plasmon resonance.
Fusion proteins containing either DnaX domains I-III or domains III-V
bound
' and
subunits. A DnaX protein only containing
domains I and II did not bind
' or
. The binding affinity
of
for DnaX domains I-III and domains III-V was the same as
that of
for full-length
, indicating that domain III
contained all structural elements required for
binding. Domain
III of
also contained
' binding sites, although the
interaction between
' and domains III-V of
was 10-fold
weaker than the interaction between
' and full length
. The
presence of both
and
strengthened the
'-C(0)
interaction by at least 15-fold. Domain III was the only domain common
to all of
fusion proteins whose interaction with
' was enhanced in the presence of
and
. Thus, domain III of the DnaX
proteins not only contains the
' and
binding sites but
also contains the elements required for the positive cooperative
assembly of the DnaX complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
): two
2 sliding clamp processivity factors
and the clamp-loading DnaX complex. The DnaX complex contains
the DnaX proteins plus auxiliary subunits
,
',
, and
. The
distributive DNA polymerase III core becomes highly processive when the
DnaX complex assembles the
processivity factor around DNA in an
ATP-dependent process (for reviews see Refs. 1-3).
and
,
which have differential interactions with replication proteins in the cell (4, 5). Results presented in the first two reports in this series
demonstrate that it is the C-terminal portion of
, absent in the
protein, that allows the full-length DnaX gene product to interact with
both the DnaB helicase and the DNA polymerase III core. These
-mediated interactions impart rapid fork movement and coordinated
leading and lagging strand synthesis, respectively (6-10). The focus
of this investigation is the protein sequence common to both
and
.
complex,
3
1
'1
1
1,
and
complex,
3
1
'1
1
1)
can be assembled in vitro (11, 12). Thus, the N-terminal 430 residues common to both
and
have the minimal protein sequence
necessary not only to bind the auxiliary subunits
,
',
, and
but also to load the
processivity factor onto a primed template
in an ATP-dependent manner. Within the DnaX complex,
'
and
bind directly to
;
binds
', and
binds
(13,
14).
and
' form a 1:1 complex and function with DnaX to load
onto primed templates in an ATP-dependent manner (10, 15).
The
and
subunits also form a 1:1 complex and increase the
affinity of DnaX for
and
' so that a functional DnaX complex can
be assembled at physiological subunit concentrations (16). The
subunit also interacts with SSB, consistent with the finding that
-
-DnaX bridges strengthen the interactions between the holoenzyme
and the SSB-coated lagging strand at the replication fork (17, 18). In
the preceding studies, five structural domains were assigned to the
protein. The focus of this report is to determine which structural
domain(s) within the portion of DnaX common to both
and
(domains I-III) are responsible for binding the auxiliary subunits. To
this end, the relative binding of
' and
to a series of
truncated DnaX proteins lacking specific structural domains was
measured using surface plasmon resonance.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and HB101 were
used for initial molecular cloning procedures and plasmid propagation.
E. coli strain BL21(
DE3) was used for protein expression.
221
encodes the fusion protein N-
221
. The
starting material for construction of plasmid
PA1-N-
221
was plasmid PA1-N-
1
,
which encodes the
protein with the initiating methionine replaced
by an N-terminal fusion peptide. PCR primer N-221p1 contained a
PstI sequence at the noncomplementary 5'-region followed by
a complementary sequence extending from codons 222 to 228 of dnaX
(Table I). Primer N-221p2 was complimentary to a region located
102 bases downstream of the NheI site within dnaX. The resultant PCR fragment was digested with
PstI and NheI and ligated into the linearized
pPA1-N-
1
to generate plasmid PA1-N-
221
.
261
encodes the truncated fusion
protein C-
261
. To construct the plasmid
PA1-C-
261
, a partial dnaX gene sequence
encoding the C-terminal 261 amino acids of
was deleted from the
previously constructed plasmid PA1-C(0)
, which encodes the C-terminal tagged full-length
protein (19). PCR primer C-
261P1 was complementary to a region of dnaX located 430 bases upstream of the internal RsrII site. Primer C-
261P2
was complementary to the dnaX from codons 380 to 382 followed by a noncomplementary SpeI cloning site (Table I).
After digestion with RsrII and SpeI, the
resultant PCR fragment was ligated into the linearized
pPA1-C(0)
to generate pPA1-C-
261
.
422
, which lacked the sequence encoding the
C-terminal 422 amino acids of
, was constructed as follows. pET11-C(0)
was digested with AflII and SpeI to
delete a dnaX sequence encoding from residue 218 to the 3'
end (residue 643) of
. The annealed oligonucleotides C-422p1
and C-422p2 containing the sequence encoding
amino acid residues
218-221 were ligated into the linearized pET11-C(0)
vector to
generate pET11-C-
422
(Table I).
Oligonucleotides used for construction of truncated fusion proteins
261
, PA1-N-
221
, and
pET11-C-
422
were transformed into E. coli strain BL21
(
DE3) for expression of proteins C-
261
, N-
221
, and
C-
422
, respectively. The transformed BL21 (
DE3) cells were
grown at 37 °C in 2 liters of F medium (20) containing 100 µg/ml
ampicillin. Cells were induced, biotin-treated, and harvested as
previously described (19).
, N-
1
, and the other
holoenzyme subunits were expressed and purified as previously described
(19). Induced BL21 cells containing the expression plasmids introduced
in this study were lysed in the presence of lysozyme (2.5 mg/g of
cells), 5 mM EDTA, 5 mM benzamidine, and
1 mM phenylmethylsulfonyl fluoride. The expressed proteins
C-
261
, N-
221
, and C-
422
were precipitated from their
corresponding lysate supernatants by addition of 0.226, 0.258, and
0.361 g of ammonium sulfate to each milliliter of the lysates,
respectively. The fusion proteins were purified using Ni2+-NTA affinity chromatography as previously described
for PA1-N-
1
(19), except that the bound C-
261
was eluted stepwise in Buffer W containing 150 mM
imidazole. For purification of C-
422
, the imidazole concentration
was 2 mM instead of 1 mM in the binding buffer
and 15 mM instead of 23 mM in the washing
buffer; the bound proteins were eluted stepwise as for C-
261
. For
purification of N-
221
, the imidazole concentration was 15 mM in the washing buffer; the bound N-
221
was eluted
with 10 column volumes of 15-100 mM imidazole gradient in
buffer W. The imidazole concentration in the peak fraction of
N-
221
was ~55 mM.
fusion proteins (700-1600 RU) were then captured onto sensor chips via streptavidin-biotin
interaction.
-
binding studies were conducted in HKGM buffer
at a flow rate of 25 µl/min at 20 °C.
-
' and
-
'
binding studies were performed in HKGM buffer containing 2% glycerol
and 2 mM dithiothreitol at a flow rate of 10 µl/min at
20 °C. In these studies,
and
' were preincubated for 10 min
at room temperature before injecting over the
derivatized sensor
chips. Kinetic parameters were determined using the BIAevaluation 2.1 software unless indicated otherwise.
fusion proteins
were measured by their requirement for reconstitution of holoenzyme
activity as previously described (19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
under control of an inducible promoter.
Plasmid PA1-N-
221
encoded protein N-
221
(domains III-V), plasmid PA1-C-
261
encoded protein
C-
261
(domains I-III), and plasmid pET11-C-
422
encoded
protein C-
422
(domains I-II) (Fig.
1). Each of these proteins contained a
hexahistidine sequence to facilitate purification using
Ni2+-NTA metal affinity chromatography and a short
biotinylation sequence to enable immobilization on streptavidin-coated
BIAcore sensor chips. The biotin tag also enabled detection of fusion
proteins using biotin blots (21). The expression levels of C-
261
,
N-
221
, and C-
422
were ~3%, 2, and 0.5% of the total
cell protein, respectively. After one round of Ni2+-NTA
chromatography, N-
221
and C-
261
preparations at greater than 80% of purity were obtained; the purity of C-
422
was
~30%, as determined by densitometric scanning of the
SDS-polyacrylamide gels of the eluted proteins fractions (Fig. 1).
Biotin blots verified that these fusion proteins were the only
biotinylated proteins in the eluted fractions (results not shown). The
biotinylated proteins are presumably the only proteins captured onto
the streptavidin sensor chips. This assumption was verified for a
similarly purified protein (see footnote 2 in Ref. 19). The activities
of C-
261
through its purification were measured in holoenzyme
reconstitution assays. Purified C-
261
had a specific activity of
5.3×106 units/mg, which is comparable with that of C(0)
(Table II). As expected, N-
221
was
inactive in this same assay because its ATPase motif (domains I and II)
was deleted. C-
422
(domains I and II) was also inactive in this
assay, suggesting that domains I-III represent the minimum protein
required to assemble the
processivity factor onto the DNA
template.
View larger version (24K):
[in a new window]
Fig. 1.
Purified truncated
-fusion proteins. The upper panel
shows the truncated biotinylated fusion proteins of
used in BIAcore
analysis. C-
261
contains domains I-III, N-
221
contains
domains III-V, and C-
422
contains domains I and II. The
rectangular box represents the biotinylated fusion peptide.
The lower panel is the Coomassie Blue-stained 12%
SDS-polyacrylamide gel of 1.5 µg of each purified protein after
Ni2+-NTA purification. Lane 1, C(0)
;
lane 2, N-
1
; lane 3, C-
261
;
lane 4, N-
221
; lane 5, C-
422
, with
the arrow indicating the C-
422
protein (two bands are
clustered together with the lower band is the C-
422
protein which
is ~30% of the total protein of the lane; N-
1
was shown in the
first paper of this series).
Purification of deletion fusion proteins
--
The abilities of the
truncated
fusion proteins C-
261
, N-
221
, and C-
422
to interact with
were measured by using BIAcore methodology.
Because the
binding region is located within the N-terminal 430 amino acids of
, a fusion peptide tag at the remote C terminus of
would be unlikely to interfere with
binding. For this
reason, C(0)
was used as a positive control for examination of
binding to the truncated
proteins. Biotin-tagged C(0)
was
immobilized onto a streptavidin sensor chip, and various concentrations
of
(25-300 nM) were passed over immobilized C(0)
so that binding of free
to immobilized C(0)
could be measured. Representative binding curves (Fig.
2A) indicate that
bound
rapidly to C(0)
with an association rate of 2.4×105
M
1 s
1. A
dissociation rate of 2.5 × 10
3 s
1
(Table III) was obtained; this rate was
measured after saturating C(0)
with
to minimize
reassociation. The resulting
Kd2
(10 ± 1 nM) was ~5-fold higher than the
Kd reported for the native
-
interaction.
The observed difference between the Kd values could
be due to differences in experimental conditions between the two
studies. For example, the present study used biotin-streptavidin rather
than amine coupling and employed a 5-fold higher flow rate than did the
other study.
View larger version (22K):
[in a new window]
Fig. 2.
The interactions of
with immobilized
C(0)
,
N-
1
,
C-
261
,
N-
221
, and
C-
422
proteins.
Streptavidin was chemically immobilized to a CM5 sensor chip as
described under "Experimental Procedures." Biotinylated
proteins were attached to sensor chips via biotin-streptavidin
interaction. The binding analyses of
with truncated
proteins
were conducted in HKGM buffer at a flow rate of 25 µl/min.
A, Sensorgrams of C(0)
-
binding. C(0)
(1520 RU)
was attached to a sensor chip. Varying concentrations of
(50, 75, and 150 nM) were injected over the C(0)
-immobilized
sensor chip for 3 min each. B, domain III of
contains
the
binding site. The N-
1
(930 RU), C-
261
(710 RU),
N-
221
(650 RU), and C-
422
(540 RU) proteins, respectively,
were captured onto sensor chips. Solution of
(150 nM) was injected over each protein-immobilized sensor chip
for 3 min. Control injections, over streptavidin-immobilized sensor
chip, were subtracted from the curves shown.
Interactions of with truncated and full-length
fusion
proteins: kinetic and equilibrium constants
and the truncated fusion proteins
N-
1
, N-
221
, C-
422
, and C-
261
were examined
under the same experimental conditions as used for the
and
C(0)
interaction. N-
1
, N-
221
, and C-
261
bound
, but C-
422
did not (Fig. 2B), indicating that
domain III of
, the only domain shared by all of the fusion proteins
shown to bind
, is required for
binding. Similar binding
stoichiometries were obtained for the interactions of
-N-
1
and
-C-
261
compared with that of the
-C(0)
interaction (Table III). The Kd for the interaction between
-N-
221
was within 2.5-fold of that measured for the
-C(0)
interaction. These variations are likely within range of
accuracy of affinity measurements using a BIAcore and indicate that
deletion of domains I and II or deletion of domains IV and V as well as
the presence of the tag at the corresponding deletion end of the
proteins did not decrease the affinity of the
-
interaction.
Thus, domain III of
appears to be fully responsible for
binding.
' to Individual Domains of the DnaX
Protein--
The interactions between
' and the full-length
proteins with either an N- or C-terminal tag were characterized in
binding studies utilizing the BIAcore instrumentation.
' samples
were injected over the immobilized C(0)
, and the binding curves are shown in Fig. 3A. The
interaction between
' and N-
1
is characterized by weak binding
similar to that observed for the
'-C(0)
interaction (Table
IV). Although measurable, the weak
binding observed is close to the limit of BIAcore detection.
View larger version (25K):
[in a new window]
Fig. 3.
Interactions of '
with immobilized C(0)
,
N-
1
,
C-
261
,
N-
221
, and
C-
422
proteins.
Streptavidin was chemically immobilized to a CM5 sensor chip as
described under "Experimental Procedures." Biotinylated
proteins were attached to sensor chips via biotin-streptavidin
interaction. Binding analyses were conducted in HKGM buffer containing
2% glycerol and 2 mM dithiothreitol at a flow rate of 10 µl/min. A, sensorgrams of C(0)
-
' binding. C(0)
(1500 RU) was attached to a sensor chip. Varying concentrations of the
' subunit (0.5, 1, 2, and 4 µM) were injected over the
C(0)
-immobilized sensor chip for 6 min each. B,
'
binds N-
1
and C-
261
but not N-
221
or C-
422
. The
N-
1
(2300 RU), C-
261
(1600 RU), N-
221
(1470 RU), and
C-
422
(730 RU) proteins, respectively, were captured onto
individual sensor chips. For 6 min each, solutions containing 4 µM
' subunit were injected over the N-
1
and
C-
261
-immobilized sensor chips; solutions containing 5 µM
' subunit were injected over the C-
422
and
N-
221
sensor chips. Values from control injections obtained via
use of an streptavidin-immobilized sensor chip were subtracted from
each curve shown.
Interactions of ' with truncated and full-length
fusion
proteins: kinetic and equilibrium constants
221
(domains
III-V), C-
422
(domains I-II), and C-
261
(domains I-III)
were captured onto streptavidin sensor chips so that the binding of
' to these proteins could be measured. The Kd
observed for the
'subunit-C-
261
interaction was similar to
that of the C(0)
-
' interaction (Fig. 3B and Table IV).
These observations confirm that the
' binding region of
is
entirely within its N-terminal 382 amino acid residues (domains
I-III). However,
' did not bind to N-
221
or C-
422
at
concentrations of
' between 0.5-5 µM (Fig.
3B). Although we observed no interactions between
' and
either domains I and II (C-
422
) or domain III (N-
221
), a
lower limit for these Kd values could be estimated
by comparing N-
221
-
' interaction with the C(0)
-
'
interaction. When 5 µM of
' was injected over the
N-
221
-derivatized sensor chip, no binding was observed. However,
significant binding was obtained when 0.5 µM of
', a 10-fold less concentration, was injected over the C(0)
derivatized sensor chip (Fig. 3); compatible amounts of C(0)
and N-
221
were on their respective derivatized sensor chips (see legend of Fig. 3
for details). Thus, if there is an interaction between N-
221
and
', the binding affinity is at least 10-fold weaker than that of the
C(0)
-
' interaction (500 nM).
Contains the
' Binding Site and the
Sequence Required for the
Cooperativity--
The
subunit has a
positive cooperative effect on the
-
' interaction (22). Because
interactions between
' and domain III or domains I and II of DnaX
may have been too weak to be detectable by our methodology, we
re-examined binding using
' instead of
'. This enabled us to
evaluate whether the cooperative effects of
strengthen the binding
of
' to the various DnaX domain constructs to detectable levels. In
the following studies, the concentrations of
' in all of the
' samples tested were greater than the Kd of
-
' interaction,3 and
the concentrations of
were 5-10 fold higher than those of
'.
Thus, nearly all the
' in these experiments was bound to
to form
'.
(Fig.
4A),
' bound C(0)
with an association rate of 4 × 103
M
1 s
1. The dissociation of
and
' from the immobilized C(0)
was complicated because two
separate dissociation events were occurring simultaneously. One process
was the dissociation of
' from the immobilized C(0)
, and the
other was the dissociation of
from
' still bound to immobilized
C (0). Thus, the total dissociation rate measured in these studies was
actually reflective of contributions from these two different
processes. From the association and dissociation rates, the calculated
Kd was 115 nM (Table
V).
View larger version (24K):
[in a new window]
Fig. 4.
Interactions of '
with immobilized C(0)
,
N-
1
,
C-
261
,
N-
221
, and
C-
422
proteins.
Streptavidin was chemically immobilized to a CM5 sensor chip as
described under "Experimental Procedures." Binding experiments were
conducted in HKGM buffer containing 2% glycerol and 2 mM
dithiothreitol at a flow rate of 10 µl/min. A, sensorgrams
of C(0)
-
' binding. C(0)
(1550 RU) was attached to a sensor
chip.
and
' samples were mixed at three different concentrations
(0.7 µM
' + 7 µM
, 1.2 µM
' + 10 µM
, and 2 µM
' + 10 µM
), incubated for 10 min at room
temperature, and injected over a C(0)
sensor chip for 6 min.
B, domain III of
contains the
' binding site. The
N-
1
(2300 RU), C-
261
(1600 RU), N-
221
(1470 RU), and
C-
422
(720 RU) proteins, respectively, were captured onto the
streptavidin-bearing sensor chips. A mixture of 1.2 µM
' + 10 µM
was preincubated and injected over
N-
1
, N-
221
, and C-
261
-immobilized sensor chip for 6 min each. A mixture of 10 µM
' + 20 µM
was preincubated and injected over a C-
422
immobilized sensor
chip. Control values, obtained by passing injections over a sensor chip
containing immobilized streptavidin only, were subtracted from each
curve.
Interactions of ' with truncated and full-length
fusion proteins
in the presence of
subunit: kinetic and equilibrium constants
' at the same concentrations used for analysis of the
C(0)
-
' interaction were then passed over the N-
221
and
C-
422
derivatized sensor chips. No binding of C-
422
to
' was observed, but N-
221
bound
' at each
concentration of
' that was tested (Fig. 4B). These
results indicate that N-
221
contains the
' binding site.
The Kd of
'-N-
221
interaction was 1.5 µM, which is only 10-fold weaker than that of the
'-C(0)
interaction.
subunit alone was injected over the
immobilized C(0)
, C-
261
, and N-
221
. No interaction was
observed (data not shown), consistent with the report that there is no
interaction between
and
(13). In contrast, the interactions
between C(0)
-
' and C-
261
-
' could be easily detected
under the same experimental conditions. Thus, the ability to detect the
N-
221
-
' interaction resulted from the positive cooperative
effect of
on the
-
' interaction; the deletion of domains I
and II of
did not abolish this cooperativity. Thus, domain III not only binds
and
' but also contains the elements required for the positive cooperative effect of
on the
'-DnaX
interaction. This is also evidenced by similar decreases in the
Kd values of C-
261
-
' and N-
1
-
'
interactions in the presence of
(Table V). An alternative
explanation is that
can weakly interact with DnaX; even though the
interaction is too weak to be observed by itself the interaction could
lead to an increase in binding of
' to DnaX because of the
additivity of the binding energies.
to Strengthen the
'-C(0)
Interaction--
The observation that
increases
both
-
' and
-
' binding affinity indicated that the
sequence required for the positive cooperative effect of
is
localized in the
portion of DnaX (16). To identify the domain(s)
responsible for this cooperativity, C(0)
and N-
221
were
captured onto streptavidin sensor chips, and their relative affinities
for
' in the presence of
were examined. A solution of
(1 µM) was passed over the immobilized C(0)
until no further binding of
was detectable. Samples of
'
containing
were then injected over the
-saturated C(0)
, and binding was observed. Dissociation was then carried out in the
presence of the same buffer containing
. This ensured that the RU
decrease observed during the dissociation phase was only due to
dissociation of
'. The C(0)
'
complex formed faster and dissociated slower than did the C(0)
' complex (Fig.
5A). In the presence of
, the Kd was ~28 nM (Table
VI), 4-fold less than that of the the
C(0)
-
' interaction in the absence of
.
View larger version (18K):
[in a new window]
Fig. 5.
Positive cooperative assembly of the DnaX
complex on BIAcore sensor chips. C(0) and N-
221
were
immobilized to streptavidin sensor chips as described under
"Experimental Procedures." The binding study was conducted in HKGM
buffer containing 2% glycerol, 2 mM dithiothreitol, and 1 µM
at 20 °C.
' and
diluted in the above
binding buffer were preincubated for 10 min at room temperature before
injection. The injection of the
'
samples over the immobilized
C(0)
and N-
221
took 6 min at 10 µl/min. A,
sensorgram overlays of
' (1.2 µM) +
(10 µM) and
' (1.2 µM) +
(10 µM) +
(1 µM) injected over
immobilized C(0)
are shown. B, sensorgram overlays of
' (1.2 µM) +
(10 µM) and
' (1.2 µM) +
(10 µM) +
(1 µM) injected over immobilized N-
221
. The control
injections of the above samples over the streptavidin-derivatized
sensor chip were conducted and subtracted from the curves
shown.
Interactions of ' with C(0)
and N-
221
in the presence of
' +
subunits: kinetic and equilibrium constants
' samples containing
were passed over the N-
221
-derivatized sensor chip. The presence of
resulted in a 5-fold reduction in the
Kd of N-
221
-
' interaction (Fig.
5B and Table VI). This result indicates that the absence of
domains I and II did not eliminate the cooperative effect of
on
the N-
221
-
' interaction. Thus, domain III of
contains
the sequence required for
-mediated augmentation of the
DnaX-
' interaction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
processivity factor onto the primed template for processive DNA
replication. Within the complex,
' and
bind
/
with
'
bridging the
/
-
interaction and
bridging the
/
-
interaction (13). The presence of
and
strengthens the
DnaX-
' interaction (16, 23). In this study, we identified the
'
and
binding domain of the DnaX proteins by measuring the
interactions of these two subunits with truncated
proteins lacking
specific domains. Our results indicate that domain III (amino acid
residues 222-382) shared by
and
binds both
and
'.
Domain III also contains the elements required for the positive
cooperative assembly of the DnaX complex.
proteins that bound the
subunit, C(0)
(domains I-V), N-
1
(domains I-V), N-
221
(domains III-V), and C-
261
(domains I-III) showed similar affinities for
, indicating that deletion of domains I and II or domains IV and V of
did not decrease the strength of the
-
interaction. Therefore,
domain III appears to be responsible for
-
binding.
' binding domain,
' was
observed to bind C-
261
(domains I-III) and full-length
very
weakly, near the limit of detection for the BIAcore. However, binding
of
' to N-
221
(domains III-IV) or C-
422
(domains I-II)
could not be detected directly. Instead, we evaluated these interactions by measuring the binding of
' to DnaX derivatives. This exploited the increased affinity of
' for DnaX in the presence of
. In the presence of both
and
', N-
221
was observed
to bind
', but C-
422
did not, indicating that domain III of
contained the
' binding site. The Kd of
the N-
221
-
' interaction was 10-fold greater than those of
the C(0)
-
' and C-
261
-
' interactions (Table V).
N-
221
interacted less strongly with
' than did C(0)
and
C-
261
, perhaps because the N-terminal peptide tag of N-
221
slightly interfered with the binding of the fusion protein to
'.
Alternatively, deletion of domains I and II may have perturbed the
structure of domain III.
' shares a high sequence similarity to the N-terminal domains I-III
of DnaX (24, 25). Both of the DnaX proteins,
and
, are tetramers
(
4,
4) when free in solution (12, 23). The DnaX complex
(
2
1
'1
1
1
1)
contains a total of four copies of homologous subunits (
,
, and
') (12). It seems reasonable to assume that one DnaX protomer of the
tetrameric DnaX proteins is replaced by the homologous
' subunit
during the formation of the DnaX complex. It is likely that the DnaX
proteins bind each other or to the homolog
' via similar mechanisms;
the same portions of
probably mediate its binding to other
subunits and to
. We have shown that domain III of DnaX is involved
in
' binding, likely through the DnaX-
' interaction in the
presence of
. Thus, domain III is also likely involved in the
-
and
-
interactions. That is, the sequences responsible
for the tetramerization of DnaX are probably localized in domain III.
decreases the Kd of the
C(0)
-
' interaction by approximately 3-fold, as indicated by a
comparison of the Kd values of the C(0)
-
' and
C(0)
-
' interactions. The presence of
also strengthens the
C-
261
-
' interaction 3-fold. The cooperative effect of
on
the N-
221
-
' interaction could not be calculated directly
because the interaction of N-
221
, and
' in the absence of
was too weak to be detected. However, the lower boundary of the
N-
221
-
' interaction Kd was estimated to be
5 µM, based upon the comparison of the concentrations of
' and the
-derivatives used in examination of the C(0)
-
' and N-
221
-
' interactions. The effects of
on the
N-
221
-
' interaction can be estimated using the lower boundary
Kd for the N-
221
-
' interaction and the
Kd for the N-
221
-
'
interaction (Table
IV). Using these values, we calculated that
augments the
N-
221
-
' interaction ~3-fold, the same degree of enhancement
for the interactions between
' and
proteins containing domains
I-III. Therefore, domain III appears to contain all sequences required
for the full cooperative effect of
' on their interaction(s) with DnaX.
strengthens both
-
' and
-
' interactions (16). The cooperative effect of
on the DnaX-
' was examined using BIAcore technology.
The C(0)
-
' interaction is strengthened approximately 4-fold
(Table VI) in the presence of
. The absence of domains I and II
did not eliminate the cooperative effect of
on domain
III-
' binding. Rather,
decreased the Kd
of the N-
221
-
' interaction by 5-fold (Table VI). These
results indicate that domain III alone is sufficient for the positive
cooperativity of
on the
-
' interaction. The presence of
both
and
strengthens the C (0)-
interaction at least
15-fold, indicating a cooperative assembly of the DnaX complex.
' and
binding sites
but also the sequences required for cooperative assembly of the DnaX
complex. This structural arrangement suggests that the cooperativity is
a result of an allosteric effect. That is, upon interactions between
and the DnaX proteins, the DnaX proteins adopt a conformation
with higher affinity for
'; upon the interaction between
and
', the
' subunit adopts a conformation with higher affinity for
domain III of DnaX proteins. This allosteric effect is crucial for the
efficient assembly of the DnaX complex in vivo. The
interaction between
and
' is weak with a
Kd of about 100 nM, which is greater
that the 28 nM concentration of each component of the DnaX
complexes in the cell (26, 27). The affinity between
and
is
~10 nM, and the
-
subassembly can readily form
in the cell. Because of the binding of
,
adopts a higher
affinity for
' with a Kd of ~28
nM. Thus, the
-
complex efficiently recruits the
' to form
'
complex at physiological subunit concentrations.
processivity factor onto
primed template in an ATP-dependent manner. Based on their studies of the crystal structure of the highly homologous
' subunit considered in light of structural features of several ATPases, Guenther
and colleagues (25) proposed that the N-terminal three domains of
would also adapt a C-shaped conformation. They also contended that this
C-shaped region is likely to open and close in response to ATP binding
and hydrolysis by the
subunit. Experimental results also support
these hypotheses. In the absence of ATP or ATP analogs, the DnaX
complex does not bind
, presumably because the
binding partner,
, is buried in the complex. In contrast, DnaX-
interactions occur
in the presence of ATP or ATP analogs, indicating that conformational
changes occur as ATP binds to
such that
subunit becomes
exposed, enabling interaction with
(28). Our results show that the
'-binding portion of DnaX lies within
domain III and suggest
that this domain may be involved in mediating the ATP effects on the
DnaX complex and
interaction. In the absence of ATP, the C-like
arrangement of domains I-III of
is closed, and the auxiliary
subunit
, which is bridged to domain III through
', is entrapped
within the DnaX complex and not freely accessible to the processivity
factor
. In contrast, ATP binding to
at the interface of domains
I and II causes the "C" to open such that domain III of
, and
hence the bridged
' subunits are relatively exposed;
' is then
free to interact with
. Thus, domain III serves as a
"transducer" of the ATP binding signal.
and
.
is comprised of domains I-III. Domain III of
binds the auxiliary
subunit
', and
functions as the processivity factor (
2) assembly apparatus.
contains the same N-terminal
three domains, as does
plus two additional C-terminal domains
(domains IV and V). Domain V binds
(polymerase) to form the dimeric
polymerase enabling the simultaneous synthesis of the leading and
lagging strands. Domain IV interacts with the DnaB helicase to
coordinate the replicase and the primosome activities at the
replication fork. Domain III is thought to be involved in linking
to the
processivity assembly apparatus. The auxiliary subunit
binds SSB to form a tether between DnaX complex and the SSB-coated
lagging strand (18).
interacts with both the processivity assembly apparatus (via
) and the polymerase (via
). This direct
processivity assembly/polymerase link bridged by DnaX strengthens the
interactions between the holoenzyme and the SSB-coated lagging strand
at the replication fork. Interactions mediated by domains III-V of
enable this subunit to serve as a central organizer. The
subunit effectively couples the processivity assembly process, SSB binding, DnaB helicase activities, and the dimeric replicase into one
replicative complex at the replication fork.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant GM35695 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M009827200
2
Kd values were obtained by
dividing the measured dissociation rate constant by the association
rate constant of a given interaction. In most cases, the
Kd values determined in this study were not true
equilibrium Kd values but were relative values used
to compare the relative affinities of -derivatives for the same analytes.
3 M. Song, H. G. Dallmann, P. Pham, R. Schaaper, and C. S. McHenry, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NTA, nitrilotriacetic acid; SSB, single-stranded DNA-binding protein; PCR, polymerase chain reaction; RU, resonance unit.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | McHenry, C. S. (1988) Annu. Rev. Biochem. 57, 519-550[CrossRef][Medline] [Order article via Infotrieve] |
2. |
McHenry, C. S.
(1991)
J. Biol. Chem.
266,
19127-19130 |
3. | Kelman, Z., and O'Donnell, M. (1995) Annu. Rev. Biochem. 64, 171-200[CrossRef][Medline] [Order article via Infotrieve] |
4. | Kodaira, M., Biswas, S. B., and Kornberg, A. (1983) Mol. Gen. Genet. 192, 80-86[Medline] [Order article via Infotrieve] |
5. | Mullin, D. A., Woldringh, C. L., Henson, J. M., and Walker, J. R. (1983) Mol. Gen. Genet. 192, 73-79[Medline] [Order article via Infotrieve] |
6. | Kim, S., Dallmann, H. G., McHenry, C. S., and Marians, K. J. (1996) Cell 84, 643-650[Medline] [Order article via Infotrieve] |
7. |
Mok, M.,
and Marians, K. J.
(1987)
J. Biol. Chem.
262,
16644-16654 |
8. | Yuzhakov, A., Turner, J., and O'Donnell, M. (1996) Cell 86, 877-886[Medline] [Order article via Infotrieve] |
9. |
McHenry, C. S.
(1982)
J. Biol. Chem.
257,
2657-2663 |
10. |
Onrust, R.,
Finkelstein, J.,
Turner, J.,
Naktinis, V.,
and O'Donnell, M.
(1995)
J. Biol. Chem.
270,
13366-13377 |
11. |
Pritchard, A. E.,
Dallmann, H. G.,
and McHenry, C. S.
(1996)
J. Biol. Chem.
271,
10291-10298 |
12. |
Pritchard, A. E.,
Dallmann, H. G.,
Glover, B. P.,
and McHenry, C. S.
(2000)
EMBO J.
19,
6536-6545 |
13. |
Onrust, R.,
Finkelstein, J.,
Naktinis, V.,
Turner, J.,
Fang, L.,
and O'Donnell, M.
(1995)
J. Biol. Chem.
270,
13348-13357 |
14. |
Glover, B. P.,
and McHenry, C. S.
(2000)
J. Biol. Chem.
275,
3017-3020 |
15. |
Naktinis, V.,
Onrust, R.,
Fang, L.,
and O'Donnell, M.
(1995)
J. Biol. Chem.
270,
13358-13365 |
16. |
Olson, M. W.,
Dallmann, H. G.,
and McHenry, C. S.
(1995)
J. Biol. Chem.
270,
29570-29577 |
17. |
Kelman, Z.,
Yuzhakov, A.,
Andjelkovic, J.,
and O'Donnell, M.
(1998)
EMBO J.
17,
2436-2449 |
18. |
Glover, B. P.,
and McHenry, C. S.
(1998)
J. Biol. Chem.
273,
23476-23484 |
19. |
Gao, D.,
and McHenry, C. S.
(2000)
J. Biol. Chem.
276,
4433-4440 |
20. | Cull, M. G., and McHenry, C. S. (1995) Methods Enzymol. 262, 22-35[Medline] [Order article via Infotrieve] |
21. |
Kim, D. R.,
and McHenry, C. S.
(1996)
J. Biol. Chem.
271,
20690-20698 |
22. |
Dallmann, H. G.,
and McHenry, C. S.
(1995)
J. Biol. Chem.
270,
29563-29569 |
23. |
Dallmann, H. G.,
Thimmig, R. L.,
and McHenry, C. S.
(1995)
J. Biol. Chem.
270,
29555-29562 |
24. | Carter, J. R., Franden, M. A., Aebersold, R., and McHenry, C. S. (1993) J. Bacteriol. 175, 3812-3822[Abstract] |
25. | Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345[Medline] [Order article via Infotrieve] |
26. |
Wu, Y. H.,
Franden, M. A.,
Hawker, J. R. J.,
and McHenry, C. S.
(1984)
J. Biol. Chem.
259,
12117-12122 |
27. |
Hawker, J. R. J.,
and McHenry, C. S.
(1987)
J. Biol. Chem.
262,
12722-12727 |
28. |
Turner, J.,
Hingorani, M. M.,
Kelman, Z.,
and O'Donnell, M.
(1999)
EMBO J.
18,
771-783 |