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 The structurally complex DNA polymerase III holoenzyme is
responsible for replication of the majority of the chromosome in Escherichia coli. The polymerase of the enzyme and 3' Both the Interactions between the The Clearly, Strains--
E. coli DH5 Chemicals and Reagents--
All proteases were purchased from
Roche Molecular Biochemicals or Sigma. d-Biotin was
purchased from Sigma. SDS-polyacrylamide gel electrophoresis protein
standards were obtained from Amersham Pharmacia Biotech, and prestained
molecular mass markers were from Bio-Rad or Life Technologies, Inc.
Ni2+-NTA1 resin,
QIAquick Gel extraction kits, QIAquick PCR purification kits, and
plasmid preparation kits were purchased from Qiagen (Valencia, CA). The
Coomassie Plus Protein Assay Reagent and ImmunoPure Streptavidin are
vended by Pierce. CM5 sensor chips (research grade), P-20 surfactant,
NHS, EDC, and ethanolamine hydrochloride were obtained from BIAcore,
Inc. (Piscataway, NJ).
Construction of the Fusion Plasmids--
The N- and C-terminal
fusion vectors pPA1-N0 and pPA1-C0 were
constructed as previously described (23). The fusion peptides contained
a short 13-amino acid biotinylation sequence, a hexahistidine sequence,
and a thrombin cleavage site. The induced fusion proteins are under the
control of either the T7 promoter of pET-11C vector (Novagen, Madison,
WI) or the PA1/04/03 promoter/operator (referred to as
PA1). PA1 is a semi-synthetic E. coli RNA polymerase-dependent promoter containing two
lac operators (23, 24). The dnaX gene was derived
from the pRT610A plasmid in which the dnaX gene was modified
at the frameshifting site. This modification results in the specific
expression of the
PA1-N-
Plasmid PA1-C(0)
PCR was used to generate plasmid PA1-N-
Plasmid pET11-N-
Plasmid PA1-C- Cell Growth and Induction--
For protein expression
applications, E. coli strain BL21 was transformed with
plasmids PA1-C(0) Ni2+-NTA Chromatography--
BL21 cells containing
expression plasmids PA1-C(0)
The purification procedures for C(0) SDS-Polyacrylamide Gel Electrophoresis--
Proteins were
separated by overnight electrophoresis at 65 V on a 10-17.5%
SDS-polyacrylamide gradient gel (0.75 × 18 × 16 cm). Gels
were stained with a 0.1% solution of Coomassie Brilliant Blue R-250 in
20% methanol and 10% acetic acid and then destained in a solution of
10% methanol and 10% acetic acid.
Biotin Blots--
After separation by SDS-polyacrylamide gel
electrophoresis, proteins were transferred onto polyvinylidene
difluoride membranes at 500 mA for 3 h in 25 mM
Tris-HCl, 192 mM glycine, pH 8.3, 20% methanol, and 0.01%
SDS. Membranes were dipped in methanol and then air-dried for 20 min.
Membranes were incubated with alkaline phosphatase-conjugated
streptavidin (2 µg/ml) in TBS + 0.05% Tween 20 plus 0.5% nonfat
milk for 1 h at room temperature and then washed three times in
TBS +0.05% Tween20. Blots were developed in a substrate solution
containing nitroblue tetrazolium chloride (0.33 mg/ml) and
5-bromo-4-chloro-3'-indolylphosphate p-toluidine salt (0.165 mg/ml) in 0.1 M Tris-HCl (pH 9.5), 0.1 M NaCl,
and 50 mM MgCl2. Reactions were stopped by
washing the membranes with distilled water.
Limited Proteolysis--
C(0) Protein Sequencing--
After digestion, the selected
biotinylated fragments were purified from others by using
Ni2+-NTA chromatography with the same buffers used for
N- Protein Determinations--
Concentrations of all purified
proteins were determined by UV spectroscopy using their extinction
coefficients. Concentrations of DNA Polymerization Assays--
Activities of Surface Plasmon Resonance--
A BIAcoreTM
instrument was used for protein binding analyses. CM5 research grade
sensor chips were used for all experiments. The carboxymethyl dextran
matrix of the sensor chip was activated by the NHS/EDC coupling
reaction as previously described (27). The matrix was activated using a
220-µl injection of a mixture of 0.2 M EDC and 0.05 M NHS in water to maximize the conversion of the carboxyl
groups of the sensor chip matrix to NHS esters. Streptavidin and bovine
IgG were sequentially captured onto the matrix by injecting over the
chip in 10 mM sodium acetate (pH 4.5) buffer at 0.2 and 0.1 mg/ml, respectively. IgG was used to partially block the negatively
charged carboxyl groups on the sensor chip surface. Unreacted NHS ester
groups were inactivated using 1 M ethanolamine-HCl (pH
8.5). Typically, 2000 response units (RU) of streptavidin were
immobilized. The biotinylated Expression and Purification of the C(0)
C(0) Limited Proteolysis of C(0)
Similar experiments were performed for six proteases, and optimal time
points were selected for each of them. After separation on
SDS-polyacrylamide gels, the digested products were transferred to
membranes. Biotin blot analyses were used to identify terminal fragments (Fig. 3A). Several cleavage products resulted from
each protease digestion. Several bands with similar mobilities were generated via digestion with different proteases, suggesting that certain regions of C(0)
To facilitate mapping of the cleavage sites closer to the N terminus of
Seven biotinylated fragments were selected for N-terminal amino acid
sequencing analysis for the identification of their cleavage sites. The
22- and 30-kDa chymotryptic fragments were chosen for analysis, as was
the 38-kDa fragment resulting from digestion with papain (Fig.
3A). Several products obtained from some of the more
specific proteases were also subjected to sequence analyses. These
included the 27- and 45-kDa products generated via endoproteinase Asp-N
treatment, as well as an 8-kDa product from the SV8 digestion (Fig.
3B). Further, a 20-kDa cleavage product generated by
chymotrypsin digestion after prolonged incubation time (data not shown)
was also evaluated. Eight corresponding cleavage sites were identified (Fig. 4). Cleavage sites N-terminal to
amino acid residues 106 (Asp) and 109 (Asp) corresponded to
endoproteinase Asp-N digestion products that migrated at about 45 kDa;
residue 222 (Asp) was the proteolytic site resulting in the 27-kDa
fragment (Fig. 3B). Papain cleaved between residues 382 (Ala) and 383 (Val) to generate the 38-kDa fragment (Fig.
3A). The 8-kDa fragment generated by SV8 (Fig.
3B) was due to cleavage between residues 407 (Glu) and 408 (Thr). The 30- and 22-kDa chymotryptic fragments (Fig. 3A) resulted from cleavage C-terminal to residues 413 (Leu) and 478 (Trp),
respectively. The 20-kDa chymotryptic fragment was due to cleavage
after residue 496 (Ala). These cleavage sites are consistent with the
substrate specificities of the respective proteases used.
The probable cleavage site resulting in the 26-kDa endoproteinase Asp-N
restriction product was deduced based on that the substrate specificity
of that protease and the distribution of aspartate residues in the
sequence of
The N-terminal half of Expression and Purification of Biotin and Hexahistidine-tagged
DnaX--
C-
The expression levels of C- The C-terminal Domain of
The interaction between
The interaction between
The calculated stoichiometry of N- We report here an effective approach to mapping heterologous
protein-protein interacting domains. We improved upon our previously reported mapping method (23) by combining it with limited proteolysis to more precisely identify probable interdomain linkers. This modification improves the probability that the expressed proteins will
contain intact domains. Using C-terminal biotin-tagged proteins in our
limited proteolysis approach allowed us to distinguish terminal
fragments from internal fragments by using streptavidin-conjugated detection reagents. The biotin tag also enabled rapid purification of
the terminal fragment for sequence analysis and precise localization of
the cleavage sites.
We chose eight proteases with different substrate specificities to
identify potential interdomain hinges. As exposed sequences, interdomain hinges are generally susceptible to cleavage by more than
one protease. Digestion was monitored as a function of time to help
distinguish relatively stable products. Cleavage products common to
more than one protease were selected for identification of the precise
sites of cleavage. Eleven cleavage sites were obtained following
N-terminal amino acid sequencing of the selected proteolysis products.
The cleavage sites were clustered within four regions: 106-109,
222-230, 383-413, and 478-496.
The Two cleavage sites (Glu407-Thr408 and
Leu413-Ala414) occur C-terminal to
Ala382-Val383. These cleavage sites were
identified via sequence analyses of different proteolytic fragments
(Fig. 3A). 30 residues lie between the scissile bonds at
Val383 and Leu413. The presence of at least
three cleavage sites within this 30-amino acid stretch suggests that it
is highly exposed to proteases and may exist as a nonstructured region
of the protein. Therefore, no domain was assigned to the stretch
flanked by residues 383 and 413. This analysis does not preclude the
formation of structures resulting from association with other molecules
not present in our proteolysis experiments domains IV and V were
assigned to the remaining sequence at the C terminus of Based on our proteolysis data and sequence alignment with the
homologous protein subunit dimerizes Escherichia
coli DNA polymerase III core through interactions with the
subunit. In addition to playing critical roles in the structural
organization of the holoenzyme,
mediates intersubunit
communications required for efficient replication fork function. We
identified potential structural domains of this multifunctional subunit
by limited proteolysis of C-terminal biotin-tagged
proteins. The
cleavage sites of each of eight different proteases were found to be
clustered within four regions of the
subunit. The second
susceptible region corresponds to the hinge between domain II and III
of the highly homologous
' subunit, and the third region is near the
C-terminal end of the
-
' alignment (Guenther, B., Onrust, R.,
Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). We propose a five-domain structure for the
protein.
Domains I and II are based on the crystallographic structure of
' by
Guenther and colleagues. Domains III-V are based on our protease
cleavage results. Using this information, we expressed biotin-tagged
proteins lacking specific protease-resistant domains and analyzed
their binding to the
subunit by surface plasmon resonance. Results
from these studies indicated that the
binding site of
lies
within its C-terminal 147 residues (domain V).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5'
exonuclease proofreading activities are contained within the
heterotrimeric DNA polymerase III core (
) subassembly. The
holoenzyme contains seven different auxiliary subunits (
,
,
,
',
,
, and
) that confer a number of special properties
requisite for replicative polymerase function (1-4). These properties
include a rapid elongation rate, high processivity, and the ability to
communicate with primosomal proteins at the replication fork (5-7).
The auxiliary subunits are divided between two functional assemblies: a
2 sliding clamp processivity factor, and the DnaX
complex, a multiprotein ATPase that assembles the
2
processivity factor onto the primer-template.
and
subunits of the holoenzyme are expressed from
dnaX. Translation of dnaX gene yields the
full-length
subunit (71 kDa) as well as the
subunit (47 kDa),
which corresponds to the N-terminal two-thirds of the
sequence
(8-11). The
subunit results from
1 translational frameshifting
to a frame with an early stop codon. The
subunit plays central
roles in the structure and function of the holoenzyme. Interactions
between the
and
subunits result in the formation of a dimeric
DNA polymerase III' (
)2
2
(12, 13). This dimeric polymerase effectively couples synthesis of the
leading and lagging strands (12, 14). The
subunit binds tightly to
, but the shorter dnaX translation product (
) does
not. This observation suggested that the C-terminal portion unique to
is critical for its interactions with the
subunit. Indeed, the
subunit and C-
, an OmpT proteolytic fragment corresponding to
the 215 C-terminal residues of
, bind with a 1:1 stoichiometry
(15).
subunit and DnaB helicase (DnaB) are
critical for rapid movement at the replication fork (16, 17). In
systems using the reconstituted DNA polymerase III holoenzyme,
subunit
DnaB interactions stimulate the rate of helicase unwinding more than 10-fold to levels approaching the rate of fork progression in
vivo. The C-terminal region found in
but lacking in
has been implicated in replication fork function. The C-
fragment was shown to interact with DnaB and to effectively couple the leading
strand polymerase with DnaB helicase at the replication fork (15).
subunits bind
'
to form the DnaX complex,
2
1(
'
) (18, 19). The
subunit also serves as a bridge between
and a
-SSB interaction,
strengthening the holoenzyme interactions with the single-stranded
DNA-binding protein-coated lagging strand at the replication fork (20,
21). As part of the elongation complex,
protects
2
from removal by exogenous
complex, increasing the processivity of
the replicase to the megabase range (22).
mediates its functions through interactions with other
subunits. To identify distinct structural domains that might mediate
these multiple interactions, we performed limited proteolytic digestion
of recombinant, biotin-tagged
. Based on these findings, we
constructed plasmids encoding truncated
fusion proteins lacking one
or more putative structural domains. The relative binding of each
resultant purified fusion protein to the
subunit was determined by
surface plasmon resonance. These studies enabled the identification of
domain V (147 C-terminal amino acid residues) as the
subunit-binding domain of
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and HB101 were used for
initial molecular cloning procedures and plasmid propagation. E. coli BL21 and BL21(
DE3) were employed for protein expression.
subunit; the alternative expression product,
,
is not encoded by this construct (25).
1
plasmid (see Fig. 1A) encodes the
protein lacking only the first amino acid (methionine) with an
N-terminal fusion peptide placed in frame. PA1-N-
1
was generated by replacing the
-encoding gene dnaE from
the vector used as the starting material (plasmid PA1-N0)
with dnaX (see Fig. 1A). Oligonucleotides 2782 and 2783 (Table I), which correspond to
the codons for
amino acids 2-10, were annealed and inserted into
pBluescript (KS
) to generate pDG10. The remainder of the
dnaX gene sequence was from pDG50, which was derived from
pRT610A via elimination of a PstI restriction site. The
2187-base pair NarI/HindIII restriction fragment
from pDG50 was ligated to a similarly digested pDG10 vector to generate
pDG100. The 2209-base pair PstI/SphI fragment from pDG100 replaced the corresponding fragment in the N-terminal fusion peptide-containing vector pPA1-N0 to yield
pPA1-N-
1
.
Oligonucleotides used for construction of deletion fusion proteins
encodes the intact
protein tagged
with a C-terminal fusion peptide (see Fig. 1B). The
NheI/PstI fragment at the C-terminal end of the
dnaX sequence within pRT610A was replaced with a
PCR-generated fragment to produce pRT610AM, in which the stop codon was
replaced with a SpeI cloning site. The 1963-base pair
XbaI-SpeI fragment from this plasmid was used to replace the corresponding fragment in the C-terminal fusion
peptide-expressing vector pPA1-C0 to produce
pPA1-C(0)
.
413
, which
lacks the sequences encoding the N-terminal 413 amino acids of
.
Oligonucleotides N-
413P1 and N-
413P2 (Table I) were used with
template pPA1-N-
1
to PCR amplify a truncated
dnaX fragment. N-
413P1 contained a PstI site
in the noncomplementary 5' region followed by a complementary region
extending from codons 414-419. N-
413P2 annealed to a region of
dnaX located 100 bases downstream of NheI site.
To generate pPA1-N-
413
, the amplified dnaX
fragment was ligated into pPA1-N-
1
following
digestion of the plasmid with PstI and NheI.
496
lacks the sequences encoding the N-terminal
496 amino acids of
. Primers N-
496P1 and N-
496P2 were used
with template pPA1-N-
1
to generate a partial
dnaX fragment, which contained a PstI site in the
noncomplementary 5' region and KpnI restriction site more
proximal to the 3' end. The KpnI restriction site was
located downstream of the dnaX natural stop codon (see Fig.
1A). This PCR fragment replaced the dnaE gene in
PstI/KpnI-digested pET11-N0 (23) to generate
pET11-N-
496
.
147
lacks the sequences encoding the
C-terminal 147 amino acids of
. PCR primer C-
147P1 annealed to a
dnaX sequence located 146 bases upstream of the
RsrII cloning site. The other PCR primer C-
147P2 was
complementary to the dnaX from codon 494-496, followed by a
noncomplementary SpeI cloning site. The two primers were
used with template pRT610A to generate a partial dnaX
sequence. Following RsrII and SpeI digestion,
this fragment replaced the corresponding fragment in
pPA1-C(0)
(see Fig. 1B) to generate
pPA1-C-
147
.
or PA1-N-
1
. E. coli strain BL21(
DE3) was used for each of the other
expression plasmids. E. coli bearing plasmid
PA1-N-
1
was grown at 37 °C to an optical density
of 0.8 in 6 liters of F medium (26) containing 100 µg/ml ampicillin.
Bacteria transformed with each of the other plasmids were grown to the
same density under the same conditions, except that the volume of F
medium was 2 liters. The induction process was started by the addition
of isopropyl-
-D-thio-galactoside (final concentration, 1 mM). Additional ampicillin (100 µg/ml) and
d-biotin (10 µM) were added to the media at
the same time. After 2 h of induction, cells were harvested by
centrifugation at 5860 × g for 10 min at 4 °C and
resuspended in 1 ml of Tris-sucrose buffer (50 mM Tris-HCl,
pH 7.5, and 10% sucrose)/g of cells. Cells were quickly frozen in
liquid N2 and stored at
80 °C.
or
PA1-N-
1
were lysed in the presence of lysozyme (2 mg/g of cells), 2 mM EDTA, 5 mM benzamidine,
and 1 mM PMSF for 1 h on ice followed by a 4-min
incubation at 37 °C (26). For BL21(
DE3) cells containing expression plasmids PA1-C-
147
,
PA1-N-
413
, or pET11-N-
496
, the lysis procedure
was modified by increasing the concentrations of lysozyme (2.5 mg/g of
cells) and EDTA (5 mM) and by extending the heat treatment
step to 6 min at 37 °C. Lysates were centrifuged at 23,300 × g at 4 °C for 1 h to remove debris. For purification of N-
1
, 0.226 g of ammonium sulfate was added to each milliliter of the resulting supernatant and precipitant was collected by centrifugation at 23,300 × g at 4 °C for 1 h.
Protein pellets were resuspended to ~30 mg protein/ml in Buffer L (50 mM sodium phosphate, pH 7.6, 500 mM NaCl, 10%
glycerol, 0.5 mM PMSF, 0.5 mM benzamidine, and
1 mM imidazole). Ni2+-NTA resin, previously
equilibrated with Buffer L, was added to the suspensions for binding.
Binding was conducted at 4 °C for 2 h with gentle shaking.
Slurries of Ni2+-NTA resin/
fusion protein complexes
were then packed into columns. Columns were washed with 10 column
volumes of Buffer L and then with roughly 30 column volumes of Buffer W
(50 mM sodium phosphate, pH 7.6, 500 mM NaCl,
20% glycerol, 0.5 mM PMSF, and 0.5 mM
benzamidine) plus 23 mM imidazole. Bound N-
1
protein
was eluted with 10 column volumes of a 23-150 mM imidazole
gradient in Buffer W. The peak fraction eluted at about 60 mM imidazole. Wash and elution steps were performed at
4 °C.
, C-
147
, N-
413
, and
N-
496
were the same as above, except for modifications of the precipitation, binding, and washing steps for N-
413
and
N-
496
and a simplified elution step for each of these four
proteins. For N-
413
and N-
496
, supernatant proteins were
precipitated by adding 0.36 g of ammonium sulfate to each
milliliter of cell lysates. For these two fusion proteins, the
imidazole concentrations in the binding and washing steps were 2 and 15 mM, respectively. The N-
413
, N-
496
, C(0)
,
and C-
147
proteins were each eluted in single steps with 150 mM imidazole in Buffer W.
digestions were carried out in
50 mM HEPES-KOH (pH 7.4), 150 mM NaCl,
10% glycerol, and 0.1 mM EDTA. Proteolytic digestions of
C-
213
were in 50 mM Tris-HCl (pH 7.6), 100 mM NaCl, 5% glycerol, and 10 mM
Mg(CH3CO2)2. At different time
points, 15-µl aliquots from reaction mixtures were removed, mixed
with 8 µl of stop buffer (0.18 M Tris, pH 6.8, 30%
sucrose, 6% SDS, 180 mM dithiothreitol), and then
immediately boiled for 2 min. Each aliquot contained 3 µg of protein.
Digestion products were separated by SDS-polyacrylamide gel
electrophoresis and then stained with Coomassie Brilliant Blue or
transferred onto a polyvinylidene difluoride membrane for biotin blots.
1
except that urea was added to the binding and washing buffer
at 8 M final concentration. These purified biotinylated
fragments were resolved by SDS-polyacrylamide gel electrophoresis and
transferred onto polyvinylidene difluoride membranes in 10 mM CAPS (pH 11.0) and 10% methanol at constant current
(0.4 A) for 3 h. The membranes were washed with 20% aqueous
methanol and then subjected to N-terminal sequence analysis using
standard Edman chemistry (James McManaman, University of Colorado
Cancer Center Protein Core Laboratory).
fusion proteins were determined by
the Pierce Coomassie Plus Protein Assay Reagent according to the
manufacturer's instructions. Bovine serum albumin (fat-free; Sigma)
was used as a standard.
fusion proteins
were measured by their requirement for reconstitution of holoenzyme
activity and by measuring DNA synthesis from a primed M13Gori template
(26). Assay mixtures (25 µl) contained 500 pmol of M13Gori (as
nucleotide), 165 units (40 ng) of DnaG primase, 1.6 µg of E. coli single-stranded DNA binding protein, 250 fmol each of DNA
polymerase III core (
),
,
',
, and the test
fusion protein (20-50 fmol). Reactions were performed in a buffer
containing 50 mM HEPES-KOH (pH 7.5), 10% (v/v) glycerol,
100 mM potassium glutamate, 10 mM
dithiothreitol, 10 mM
Mg(CH3CO2)2, 200 µg/ml bovine
serum albumin, 0.02% (v/v) Tween 20, 48 µM dATP, 48 µM dCTP, 48 µM dGTP, 18 µM
[3H]TTP (specific activity, 520 cpm/pmol TTP), and
200 µM rNTP. Assay mixtures were incubated at 30 °C
for 5 min, quenched by trichloroacetic acid precipitation, and then
filtered through GF/C filters (26). One unit is defined as the amount
of enzyme catalyzing the incorporation of 1 pmol of dNTPs/min at
30 °C.
proteins were then injected over the
immobilized streptavidin in HBS buffer (10 mM HEPES, pH
7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% P-20
surfactant). For kinetic analyses, less than 200 RU of
fusion
protein were immobilized. Binding studies of
to the
subunit
(12.5-50 nM) were performed at 20 °C in HKGM buffer (50 mM HEPES, pH 7.4, 100 mM potassium glutamate,
10 mM Mg(CH3CO2)2, and
0.005% P-20 surfactant). A flow rate of 25 µl/min was used for
kinetic analyses. Kinetic parameters were determined using the
BIAevaluation 2.1 software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and N-
1
Proteins--
We constructed plasmids PA1-C(0)
(Fig.
1A) and
PA1-N-
1
(Fig. 1B), which encode C(0)
and N-
1
, respectively. Our nomenclature system for truncated
fusion proteins indicates the number of terminal residues deleted
following the
; the preceding N or C indicates the terminus from
which the amino acids were deleted. The fusion peptide is located at
the truncated terminus. N-
1
, for example, indicates that one
amino acid was deleted from the N terminus of the
sequence and that
the fusion peptide was added to the new N terminus. The expressed C(0)
and N-
1
represented ~5% of the total cell protein, as
determined by densitometric scans of Coomassie-stained gels.
View larger version (12K):
[in a new window]
Fig. 1.
Construction of plasmids that express
with N- or C-terminal tags. The
oligonucleotides and PCR primers used are listed in Table I.
PA1 and Ptac are the
PA1/04/03 and tac promoter, respectively. All of
the plasmids shown contain the
-lactamase gene and
lacIq. The fusion peptide region contains a
short biotinylation sequence, a hexahistidine sequence, and a thrombin
cleavage site (23). A, construction of N-terminal
fusion
expression plasmid with PA1 promoter. B,
construction of C-terminal
fusion expression plasmid with
PA1 promoter.
and N-
1
were purified via Ni2+-NTA metal
chelating chromatography (Table II). The
hexahistidine sequence within the fusion peptide specifically interacts
with Ni2+ cheated to the column resin. Lysates (FrI) were
prepared from 21 g of C(0)
or 50 g of N-
1
expression
cells. Both C(0)
and N-
1
were recovered at >85% purity after
Ni2+-NTA chromatography. The activity peaks of the eluted
fractions of both C(0)
and N-
1
corresponded to each of their
protein peaks (data not shown). Both C(0)
and N-
1
were fully
active compared with wild-type
protein in DNA polymerization
assays. C(0)
and N-
1
were the only biotinylated proteins in
the corresponding eluted fractions examined by the biotin blot analysis
(data not shown).
Purification of C(0) and N-
1
from overproducing cells
--
Limited proteolyses were
performed to identify protease-sensitive interdomain hinges of the
protein. Eight different proteases that encompass a broad spectrum of
substrate specificities were tested: chymotrypsin, endoprotease Glu-C
(SV8), papain, subtilisin, trypsin, thermolysin, endoprotease Asp-N,
and endoprotease Lys-C. We first investigated the effects of varying
the protease: C(0)
ratios and incubation times on the observed
proteolytic products. Varying incubation times distinguished the
initial cleavage products and also established the differences between
stable and unstable fragments. Results from a typical experiment
employing chymotrypsin proteolysis are shown in Fig.
2. At short incubation times, 56-, 52-, 48-, and 24-kDa products were observed along with full-length
(Fig.
3, lanes 1-3 and
8-10). At longer incubation times, the 56- and 52-kDa
products were diminished, whereas the 48- and 24-kDa products and
several small bands (<20 kDa) became more intense (Fig. 2, lanes
4-7 and 11-14).
View larger version (75K):
[in a new window]
Fig. 2.
Proteolysis of C(0)
with chymotrypsin. C(0)
was subjected to limited
proteolysis by two concentrations of chymotrypsin for varying times.
Each lane contains 3 µg of C(0)
protein. After digestion, products
were separated by 10-17.5% SDS-polyacrylamide gel and stained with
Coomassie Blue. Arrows on the left indicate two
examples of cleavage products (48 and 24 kDa) which become more intense
with increased digestion time (arrows indicate position in
lane1; the same products migrate faster in following lanes
because of electrophoresis irregularity). Lane 15,
undigested C(0)
.
View larger version (46K):
[in a new window]
Fig. 3.
Biotin blots of C(0)
and C-
213
proteolysis products. After digestion, samples were boiled
immediately with the addition of SDS sample buffer, resolved on
10-17.5% SDS-polyacrylamide gel, and subjected to biotin blots as
described under "Experimental Procedures." A, C(0)
was subjected to limited proteolysis with six proteases. Abbreviations
used for proteases and their dilution (w/w) and digestion temperature
are: C, chymotrypsin, 1:100 at 20 °C; Th,
thermolysin, 1:2000 at 37 °C; S, subtilisin, 1:1500 at
20 °C; T, trypsin, 1:2000 at 37 °C; P,
papain, 1:500 at 20 °C; SV, endoprotease Glu-C, 1:2000 at
37 °C. Lane 13, C(0)
, no protease. Arrows
on the left indicate the bands (38 kDa, lane 9;
30 kDa, lane 1; 22 kDa, lane1) selected for
sequencing. B, C-
213
was subjected to limited
proteolysis with five proteases for several different times.
Abbreviations used for proteases and their dilution (w/w) and digestion
temperature are: Asp-N, endoprotease Asp-N, 1:500 at
37 °C; Lys-C, endoprotease Lys-C, 1:50 at 37 °C;
Glu-C, endoprotease Glu-C, 1:50 at 37 °C;
papain, 1:500 at 20 °C; chymtry, chymotrypsin,
1:300 at 20 °C. Arrows on the left indicate
the bands (45 kDa, lane 3; 27 kDa, lane 5; 8 kDa,
lane 12) selected for sequencing.
were subject to cleavage by multiple proteases. For example, bands of roughly 38 kDa were obtained by
digestion with thermolysin, papain, or subtilisin. Bands migrating at
~30 kDa were obtained with either chymotrypsin or subtilisin, and
products of about 22 kDa were obtained after digestion with chymotrypsin, SV8, or papain. These observations suggested that C(0)
contains several protease-sensitive regions.
, C-
213
, which is equivalent to the
protein plus the
fusion peptide at its C terminus, was subjected to limited proteolysis.
The conditions for each protease were optimized as described above.
Products with apparent molecular masses of 45 kDa were obtained after
digestion with endoproteinase Asp-N, endoproteinase Lys-C, or
chymotrypsin (Fig. 3B). These and other cleavage products vanished after longer endoproteinase Lys-C digestion because of excessive proteolysis. Intensely staining cleavage products of both 27 and 26 kDa were obtained by digestions with either endoproteinase Asp-N
or papain. Products of about 8 kDa were obtained after digestion with
either SV8 or chymotrypsin (Fig.
3B).
View larger version (13K):
[in a new window]
Fig. 4.
Protease susceptible sites of the
protein. Walker A (GXXXXGKT), zinc
module (CX8CXXCXXC),
Walker B (DEXX), and SRC motifs are described (30). A total
of 11 cleavage sites were obtained by N-terminal amino acid sequencing
of the selected biotinylated proteolytic products. The lower
panel shows potential structural domains of the
protein.
Domain I, 1-179 amino acids; domain II, 180-221 amino acids; domain
III, 229-382 amino acids; domain IV, 413-496 amino acids; and domain
V, 497-643 amino acids.
View larger version (30K):
[in a new window]
Fig. 5.
Purified truncated fusion proteins.
The upper panel shows the truncated fusion proteins of
used in BIAcore analysis. Both C-
147
and N-
496
are
constructed based on
proteolytic site at amino acid 496 and contain
domains I-IV and domain V, respectively. N-
413
is based on
proteolytic site at amino acid 413 and contains domain IV+V. The
rectangular box represents the fusion peptide. The
lower panel is the Coomassie Blue-stained 12.5%
SDS-polyacrylamide gel of the above proteins after Ni2+-NTA
chromatography with each lane containing approx. 1.5 µg of protein.
Lane 1, C(0)
. Lane 2, C-
147
. Lane
3, N-
413
(upper band). Lane 4,
N-
496
.
. Taking an experimental error of ±10% for molecular
mass determination via SDS-polyacrylamide gel electrophoresis into
account, the cleavage site resulting in the 27-kDa fragment would be
predicted to be roughly 9 residues C-terminal to that of the 26-kDa
fragment (Asp222). There are two aspartates C-terminal to
Asp222: those at positions 229 and 245. Thus, the 26-kDa
endoproteinase Asp-N fragment is likely due to cleavage N-terminal to
Asp229. Similarly, in consideration of the substrate
preferences of papain in light of the known cleavage site of the 27-kDa
endoproteinase Asp-N product, it seems likely that the 26-kDa papain
digestion product is due to cleavage of bond(s) involving
Gly228 and/or Gly230. These probable cleavage
sites (228, 229, and 230) together with the eight cleavage sites
determined by sequence analysis all cluster at four regions of the
subunit: amino acid residues 106-109, 222- 230, 383-413, and
478-496.
shares high sequence similarity with
'
(28-30), another component of the DnaX complex. The crystal structure
of
' contains three domains, and sequence alignment predicts a
similar three-domain structure for the N-terminal half of
(30). The
majority of the cleavage sites we observed agree and extend this
prediction to a five domain model for
(see "Discussion" for
detail): amino acid residues 1-179 as domain I, 180-221 as domain II,
230-382 as domain III, 413-496 as domain IV, and 497-643 as domain V
(Fig. 4).
, the unique C-terminal portion of
, is required for
binding to
(15). C-
contains the majority of predicted domain IV
and the entire domain V. The fusion protein N-
413
(domains IV and
V) was expressed and purified so that it could be used as a tool for
mapping the
binding domain of the
subunit. We also expressed
fusion proteins corresponding to domain V by itself (N-
496
), and
domains I-IV (C-
147
).
147
, N-
413
, and N-
496
were
~3, 1, and 5% of total cell protein, respectively. All
three of these fusion proteins were soluble. After purification by
Ni2+-NTA chromatography, C-
147
and N-
496
were
obtained at greater than 80% purity, and N-
413
was obtained at
over 65% purity as determined by scanning densitometry (Fig. 5). 11.5 mg of C-
147
, 8 mg of N-
496
, and 5.3 mg of N-
413
were
purified from 1250, 330, and 1200 mg of total protein from cell
lysates, respectively. C-
147
, N-
413
, and N-
496
were
the only biotinylated proteins in the corresponding eluted fractions
examined by the biotin blot analysis. Thus, the biotinylated
fusion
proteins were presumed to be the only proteins captured onto the
BIAcore sensor chip during the immobilization
step.2 C-
147
is as
active as C(0)
in DNA polymerization assays.
Binds to
--
The interaction
between
and C(0)
was first assessed using BIAcore methodology.
Streptavidin was chemically coupled to the CM5 sensor chip, and C(0)
was immobilized via biotin-streptavidin interaction. Dilutions of
were injected over and bound the immobilized C(0)
(Fig.
6A). The off rate
(koff) was determined after saturating
with
to eliminate artifacts arising from re-association. The calculated
Kd was ~4 nM (Table
III).
View larger version (28K):
[in a new window]
Fig. 6.
BIAcore analysis of the interaction of
with immobilized C(0)
,
N-
413
,
N-
496
,
C-
147
, and
N-
1
. Five
biotinylated
-fusion proteins were captured onto the streptavidin
chips as described under "Experimental Procedures." A streptavidin
derivatized flow cell lacking bound
protein provided the blank
control, which was subtracted from the data shown. The analysis of the
interaction with
was performed in HKGM buffer. A-C,
sensorgrams of overlays of the indicated concentrations of
injected
over the immobilized C(0)
(150 RU), N-
413
(710 RU), and
N-
496
(34 RU) flow cells for 3 min at 25 µl/min, respectively.
D, 720 RU of C(0)
and 750 RU of C-
147
were captured
onto streptavidin flow cells, respectively. 50 nM of
was injected over each of these two and the control flow cells in HKGM
buffer at 2 µl/min for 15 min. E, 150 RU of N-
1
were
captured onto streptavidin flow cell. 20 nM of
was
injected over the flow cell in HKGM buffer at 25 µl/min for 4 min.
BIAcore analyses of the binding of to
derivatives
and N-
413
was characterized by an
association rate similar to that observed for C(0)
-
binding; however, its dissociation rate was at least 1000-fold slower (Fig. 6B). This indicated that residues sufficient for specific
binding to the
subunit lie within the C-terminal 230 amino acid
residues of
. C-
147
(domains I-IV) and N-
496
(domain V)
were then immobilized on the streptavidin chips separately to further
limit the
-binding region. Binding to the
subunit was observed
with N-
496
but not C-
147
(Fig. 6, C and
D), strongly suggesting that domain V functions as the
-binding component of the
subunit. The off rates
(koff ) of both
-N-
496
and
-N-
413
interactions were extremely slow (Fig. 6, B
and C). Within the 30-min dissociation period, the response
unit changes were within the machine noise level (10 RU), precluding
the calculation of a dissociation rate constant, but permitting limits
to be placed on the off rates and the III-V domains (Table III).
and N-
1
was examined to investigate
whether the weaker interaction between C(0)
-
might be due to
interference by the proximity of the peptide tag component of C(0)
to the
-binding domain. The N-
1
-
interaction was characterized by a dissociation rate similar to that observed for the
N-
496
-
interaction but much slower than that detected for the
C(0)
-
interaction (Fig. 6E). This suggests that the C-terminal tag interferes with the
-
interaction. We also
examined the C(0)
-
interaction in the presence of the auxiliary
subunit
,
', and
and ATP. The presence of
,
', and
and ATP did not make obvious changes in the rate of C(0)
-
interaction, suggesting that the
'
-
interaction and
-
interaction are independent events.
496
:
(1:0.65) was similar to
that obtained for the full-length
standards (1:0.75 and 1:0.65 for
N-
1
and C(0)
, respectively). This indicates that domain V,
expressed alone, is properly folded and functional.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
' subunit shares a 34% sequence similarity with the N-terminal
region of both
and
and aligns to the N-terminal 370-amino acid
sequence of DnaX proteins (28, 30), which corresponds to over half of
the
sequence. There are several known motifs conserved between
these two proteins, including the Walker A
(GXXXXGKT),3
Walker B (DEXX), zinc module
(CX8CXXCXXC), and SRC
sequences (Fig. 4) (30). The
' crystal structure showed three
domains in a C-shaped configuration, suggesting that the N-terminal
half of
also contains three domains, based on their sequence
homology (30). Their alignment suggested that
amino acid residues
1-179 were domain I and that residues180-221 were domain II. Less
certain was the assignment of domain III to the residues following 226. Our results support the prediction that the N-terminal end of
and
' have similar structures. One of the
protein cleavage sites
(Asp222-Gln223) is located within the stretch
predicted to be a hinge between domains II and III. The following
cleavage site (Ala382-Val383) occurs just 13 residues C-terminal to the end of the
-
' alignment and may define
the C-terminal boundary of the third domain of
. We observed no
cleavage between domains I and II, suggesting that this hinge was not
accessible for proteolysis under the conditions employed in these
studies. Based on the most N-terminal cleavage site identified
(Arg105-Asp106), we constructed a plasmid
encoding
amino acid residues 1-105. The resultant protein was not
stable in expression cells (data not shown). Both the residue
Asp106 of
and the corresponding residue
Glu95 of
' are predicted to be in a similar position of
a helical region revealed by the PHD program (data not shown). The
residue Glu95 is on the surface of domain I of
' (30).
Above results suggest that
residues 1-105 do not form an intact
structural domain by themselves.
. Domain IV
is composed of 17 amino acid residues in common with the
translation product and 66 residues unique to the C-terminal sequence
of the
. Domain V corresponds to 147 amino acid residues of the
C-terminal end of
.
interacts with the C-terminal region unique
to
containing domains IV and V. To further limit the
binding
domain of
, BIAcore analysis was performed. Domain V alone bound
with domains III-V in the pM range. The interaction
between
and N-
1
was of a similar magnitude, indicating that
domain V contains all of the binding energy for
-
interaction.
This also suggests that our domain assignments for the C-terminal
sequence of
accurately reflect both the structural and functional
integrity of domain V. C-terminal tagged
bound
with 1000-fold
lower affinity, whereas tagging at the N terminus did not reduce
binding activity in the BIAcore assay. This observation is consistent
with localization of the
binding sites for
at or near the C
terminus of domain V.
', considered in light of the
' crystal structure, we assigned five structural domains to the
subunit. Final proof of the domain assignments awaits more complete
determination of the structure of the
-subunit.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. John Kuriyan for sharing the '
structure and coordinates with us in advance of publication and Dr.
Deborah Wilkinson-Fitzgerald for writing and editorial assistance on
this and the following two papers.
![]() |
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.M009828200
2
A portion of the same preparation of N-413
used in BIAcore experiments was chromatographed over a soft link, soft
release monomeric avidin column (Promega) equilibrated in Buffer
containing 50 mM HEPES (pH 7.4), 100 mM sodium
glutamate, 5% (v/v) glycerol, and 0.5 mM PMSF. The column
was washed with five column volumes of the equilibration buffer. The
bound protein was eluted from the column with 5 mM biotin
in equilibration buffer. The N-
413
band was the only band
detected in a Coomassie Blue-stained SDS-polyacrylamide gel
electrophoresis of the eluted fractions.
3
' sequence has a clearly derivative but
mutated Walker A motif so it cannot bind ATP.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NTA, nitrilotriacetic acid; CAPS, 3-cyclohexylamino-1-propanesulfonic acid; PCR, polymerase chain reaction; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide; PMSF, phenylmethylsulfonyl fluoride; RU, response units.
![]() |
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. |
Kornberg, A.
(1988)
J. Biol. Chem.
263,
1-4 |
4. | Kelman, Z., and O'Donnell, M. (1995) Annu. Rev. Biochem. 64, 171-200[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Fay, P. J.,
Johanson, K. O.,
McHenry, C. S.,
and Bambara, R. A.
(1981)
J. Biol. Chem.
256,
976-983 |
6. |
Wu, C. A.,
Zechner, E. L.,
Reems, J. A.,
McHenry, C. S.,
and Marians, K. J.
(1992)
J. Biol. Chem.
267,
4074-4083 |
7. |
Wu, C. A.,
Zechner, E. L.,
and Marians, K. J.
(1992)
J. Biol. Chem.
267,
4030-4044 |
8. | McHenry, C. S., Griep, M. A., Tomasiewicz, H., and Bradley, M. (1989) Molecular Mechanism in DNA Replication and Recombination , pp. 115-126, Alan R. Liss, Inc., New York |
9. | Tsuchihashi, Z., and Kornberg, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2516-2520[Abstract] |
10. | Blinkowa, A. L., and Walker, J. R. (1990) Nucleic Acids Res. 18, 1725-1729[Abstract] |
11. | Flower, A. M., and McHenry, C. S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3713-3717[Abstract] |
12. |
McHenry, C. S.
(1982)
J. Biol. Chem.
257,
2657-2663 |
13. |
Studwell-Vaughan, P. S.,
and O'Donnell, M.
(1993)
J. Biol. Chem.
268,
11785-11791 |
14. |
Kim, S.,
Dallmann, H. G.,
McHenry, C. S.,
and Marians, K. J.
(1996)
J. Biol. Chem.
271,
21406-21412 |
15. |
Dallmann, H. G.,
Kim, S.,
Marians, K. J.,
and McHenry, C. S.
(2000)
J. Biol. Chem.
275,
15512-15519 |
16. | Kim, S., Dallmann, H. G., McHenry, C. S., and Marians, K. J. (1996) Cell 84, 643-650[Medline] [Order article via Infotrieve] |
17. | Yuzhakov, A., Turner, J., and O'Donnell, M. (1996) Cell 86, 877-886[Medline] [Order article via Infotrieve] |
18. |
Glover, B. P.,
and McHenry, C. S.
(2000)
J. Biol. Chem.
275,
3017-3020 |
19. |
Pritchard, A. E.,
Dallmann, H. G.,
Glover, B. P.,
and McHenry, C. S.
(2000)
EMBO J.
19,
6536-6545 |
20. |
Kelman, Z.,
Yuzhakov, A.,
Andjelkovic, J.,
and O'Donnell, M.
(1998)
EMBO J.
17,
2436-2449 |
21. |
Glover, B. P.,
and McHenry, C. S.
(1998)
J. Biol. Chem.
273,
23476-23484 |
22. |
Kim, S.,
Dallmann, H. G.,
McHenry, C. S.,
and Marians, K. J.
(1996)
J. Biol. Chem.
271,
4315-4318 |
23. |
Kim, D. R.,
and McHenry, C. S.
(1996)
J. Biol. Chem.
271,
20690-20698 |
24. | Lanzer, M., and Bujard, H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8973-8977[Abstract] |
25. |
Dallmann, H. G.,
Thimmig, R. L.,
and McHenry, C. S.
(1995)
J. Biol. Chem.
270,
29555-29562 |
26. | Cull, M. G., and McHenry, C. S. (1995) Methods Enzymol. 262, 22-35[Medline] [Order article via Infotrieve] |
27. |
Olson, M. W.,
Dallmann, H. G.,
and McHenry, C. S.
(1995)
J. Biol. Chem.
270,
29570-29577 |
28. | Carter, J. R., Franden, M. A., Aebersold, R., and McHenry, C. S. (1993) J. Bacteriol. 175, 3812-3822[Abstract] |
29. | O'Donnell, M., Onrust, R., Dean, F. B., Chen, M., and Hurwitz, J. (1993) Nucleic Acids Res. 21, 1-3[Medline] [Order article via Infotrieve] |
30. | Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345[Medline] [Order article via Infotrieve] |