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INTRODUCTION |
Chromosomal replicases of all cellular organisms studied thus far
are composed of three components, the DNA polymerase, a ring-shaped DNA
sliding clamp, and a clamp loader that uses ATP to assemble the sliding
clamp onto DNA (1-3). In bacteria, the sliding clamp is a homodimer
called 
(4). The ring-shaped dimer completely encircles DNA and
slides along the duplex (5). The clamp also binds the DNA
polymerase III, thereby tethering it to DNA for high processivity
(5).
This report on the Aquifex aeolicus pol
III1 replicase is part of our
continuing study of comparing and contrasting replicases from a variety
of bacteria. Most knowledge of bacterial DNA polymerase III (pol III)
structure and function has been obtained from studies of the
Escherichia coli replicase, DNA polymerase III holoenzyme (reviewed in Ref. 6). Therefore, a brief overview of its structure and
function is instructive for the comparisons to be made in this report.
In E. coli, the catalytic subunit of DNA polymerase III is
the 
subunit (129.9 kDa) encoded by dnaE; it lacks a
proofreading exonuclease (7). The proofreading 3'-5'-exonuclease
activity is contained in the 
(27.5 kDa) subunit (dnaQ)
that forms a 1:1 complex with (8, 9). The pol III 
complex
is found tightly associated to a third subunit, called 
, to form the
heterotrimeric E. coli DNA polymerase III core (10). The subunit (holE, 8.6 kDa) is not essential for growth and is
generally not conserved in bacteria (11).
The E. coli pol III subunit and pol III core subassembly
act distributively on primed ssDNA and have only low activity; they are
even further inhibited by the presence of SSB (7, 12). However,
after the clamp has been assembled onto a primed site, the
efficiency of the pol III subunit is greatly stimulated, and
· extends the primer at a rate of ~300 nucleotides/s with a
processivity of 1-3 kb (9). The pol III complex and pol III
core subassembly are even further stimulated by and extend DNA at a
rate of about 1 kb/s with a processivity that exceeds the entire 7.2-kb
M13mp18 ssDNA template (9).
The E. coli clamp loader of pol III consists of five
different subunits, 
, 
, ', 
, and 
, but only three of
them, (dnaX, 47.5 kDa), (holA, 38.7 kDa),
and '(holB, 36.9 kDa), are essential for clamp loading
activity in vitro (13). Homologues to E. coli (holC, 16.6 kDa) and (holD, 15.2 kDa)
subunits can only be identified in a few other organisms so far. The
and ' subunits are homologous to one another and are members of
the AAA+ family of proteins (14-16). The subunit shows no homology
to and ', but the ·, ' and 3'
crystal structures show that has the same three domain structure
and chain folding pattern as and ' (17-19). Crystal structure
analysis reveals that the five subunits of the
31'1 complex are arranged
as a circular pentamer (19).
Mechanistic studies have outlined the overall mechanism of the clamp
loader and are consistent with the structural analysis. The subunit
is the only subunit that interacts with ATP and therefore is the motor
of the clamp loader (20). The subunit alone can open one interface
of the dimer (21, 22). The clamp opener is sequestered within
complex via association to ' (21). ATP binding to
3 results in a conformational change, releasing the interactive site on from ' (21, 23). The ATP·3' species binds to , opens the ring, and
binds DNA (24, 25). Then hydrolysis of ATP brings back onto ',
severing connections to , allowing to close around DNA (21,
26-28).
In E. coli, the dnaX gene encoding also
encodes the 
subunit of DNA pol III holoenzyme (29-31). (71.1 kDa) is the full-length product of dnaX, whereas is
shorter (47.5 kDa), being truncated by a translational frameshift. can fully replace in the clamp loader, and the
3' complex is active in clamp loading (13). The
C-terminal sequences unique to are required for interaction with
the pol III subunit (32) and also with the replicative DnaB
helicase (33, 34). Therefore, within the holoenzyme, subunits must
replace two (or all three) of the subunits in order to connect the
two pol III core polymerases in the holoenzyme structure for
simultaneous replication of both leading and lagging strands (60).
We have undertaken the study of other bacterial replication systems in
an effort to delineate those features of prokaryotic replicases that
are general to all bacteria. Study of the Gram-negative Thermus
thermophilus dnaX gene showed that it produces both and ,
like E. coli dnaX (35, 36). However, instead of a 1 ribosomal frameshift, T. thermophilus employs a
transcriptional slippage mechanism that results in both 1 and 2
frameshifts (35, 37). We have also examined the pol III replicase of a Gram-positive organism, Streptococcus pyogenes (38). This
study showed that only one protein is produced from the S. pyogenes dnaX gene. This full-length protein (62.1 kDa) is
intermediate in length between E. coli (47.5 kDa) and
(71.1 kDa) but retains the capacity to bind the DNA polymerase,
like E. coli . However, the strength of this interaction
is weaker than in the E. coli system. Like other
Gram-positive bacteria, the S. pyogenes DNA polymerase, pol
C (~165 kDa), contains an inherent 3'-5'-exonuclease activity
instead of delegating this proofreading action to a separate subunit as observed in the E. coli system (39). As in
E. coli, the S. pyogenes, , , and '
subunits are required to load onto DNA, and the clamp endows pol C
with the same rapid speed and processivity as the entire E. coli DNA pol III holoenzyme (38). It is interesting to note that
S. pyogenes also contains a second homologue to E. coli , which we refer to as the DnaE polymerase (38). DnaE
polymerase is similar in size to E. coli (120 kDa) and
like E. coli it lacks 3'-5'-exonuclease activity (38).
The processivity of S. pyogenes DnaE polymerase is
stimulated by ' and , but its intrinsic speed (60 nucleotides/s) is unaltered (38).
This report examines the pol III replication machinery of the extreme
thermophile, A. aeolicus. By using the known genome sequence
(41), we identify A. aeolicus replicase genes and produce and isolate recombinant , , , , ', , subunits and
SSB. We then compare and contrast the function and assembly of these
replicase subunits from a thermophile to the Gram-negative E. coli replicase and to our previous studies on the Gram-positive
S. pyogenes replicase.
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EXPERIMENTAL PROCEDURES |
Materials--
Radioactive nucleotides were from PerkinElmer
Life Sciences; unlabeled nucleotides were from Amersham Biosciences and
The Upjohn Co. DNA oligonucleotides were synthesized by Invitrogen. M13mp18 ssDNA was purified from phage that was isolated by two successive bandings in cesium chloride gradients as described (42).
M13mp18 ssDNA was primed with a DNA 30-mer (map position 6817-6846) as described (9). The pET protein expression vectors and
BL21 (
DE3) protein expression strain of E. coli were
purchased from Novagen. DNA modification enzymes were from New England
Biolabs. A. aeolicus genomic DNA and A. aeolicus
cells were a gift of Dr. Robert Huber and Dr. Karl Stetter (Regensburg
University, Germany). Protein concentrations were determined using
Protein Stain (Bio-Rad) and BSA as a standard. Polyclonal antisera was
produced by rabbits injected with purified E. coli or
(5). Antibodies directed to A. aeolicus were
purified from antisera by transfer of E. coli from an
SDS gel to nitrocellulose followed by incubation with antisera and
elution of purified antibody from the nitrocellulose membrane.
Purification of A. aeolicus Encoded by dnaE--
The
A. aeolicus dnaE gene (41) was amplified from A. aeolicus genomic DNA by PCR using the following primers.
The upstream 37-mer
(5'-GTGTGTCATATGAGTAAGGATTTCGTCCACCTTCACC-3') contains an
NdeI site (underlined); the downstream 34-mer
(5'-GTGTGTGGATCCGGGGACTACTCGGAAGTAAGGG-3') contains a
BamHI site (underlined). The PCR product was digested with
NdeI and BamHI, purified, and ligated into the
pET24 NdeI and BamHI sites to produce pETAadnaE.
The pETAadnaE plasmid was transformed into the BL21 (DE3) strain of
E. coli. Cells were grown in 50 liters of LB containing 100 µg/ml kanamycin, 5 mM MgSO4 at 37 °C to
A600 = 2.0, induced with 2 mM
IPTG for 20 h at 20 °C, and then collected by centrifugation. Cells were resuspended in 400 ml of 50 mM Tris-HCl (pH
7.5), 10% sucrose, 1 M NaCl, 30 mM spermidine,
5 mM DTT, and 2 mM EDTA. The following
procedures were performed at 4 °C. Cells were lysed by passing them
twice through a French press (15,000 pounds/square inch) followed by
centrifugation at 13,000 rpm for 90 min at 4 °C. In this protein
preparation, as well as each of those that follow, the induced A. aeolicus protein was easily discernible as a large band in an
SDS-polyacrylamide gel stained with Coomassie Blue. Hence, column
fractions were assayed for the presence of the A. aeolicus
protein by SDS-PAGE analysis, which forms the basis for pooling column fractions.
The clarified cell lysate was heated to 65 °C for 30 min, and the
precipitate was removed by centrifugation at 13,000 rpm in a GSA rotor
for 1 h. The supernatant (1.4 g, 280 ml) was dialyzed against
buffer A (20 mM Tris-HCl (pH 7.5)), 10% glycerol, 0.5 mM EDTA, 5 mM DTT) overnight, and then diluted
to 320 ml with buffer A to a conductivity equal to 100 mM
NaCl. The dialysate was applied to a 150-ml fast flow Q-Sepharose
column (Amersham Biosciences) equilibrated in buffer A, and eluted with
a 1.5-liter linear gradient of 0-500 mM NaCl in buffer A. Eighty fractions were collected. Fractions 38-58 (1 g, 390 ml) were
pooled, dialyzed versus buffer A overnight, and applied to a
250-ml heparin-agarose column (Bio-Rad) equilibrated with buffer A. Protein was eluted with a 1-liter linear 0-500 mM NaCl
gradient in buffer A. One hundred fractions were collected. Fractions
69-79 (320 mg in 200 ml) were pooled and dialyzed against buffer A
containing 100 mM NaCl. The preparation was aliquoted
and stored frozen at 80 °C. The Coomassie Blue-stained
SDS-polyacrylamide gel of the final preparation is shown in
Fig. 1.
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Fig. 1.
Protein subunits of A. aeolicus
pol III holoenzyme system. A, subunit
preparations were analyzed in a 15% SDS-polyacrylamide gel stained
with Coomassie Blue. Proteins were prepared as described under
"Experimental Procedures." The positions of protein standards
analyzed in the same gel are indicated to the left. B, the
table gives the subunit gene name, mass predicted from the gene
sequence for A. aeolicus pol III subunits, and their percent
identify to corresponding subunits in the E. coli pol III
systems (aligned using ClustalX). The function of subunits, or
combinations of subunits, are given in the column at the
right.
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Purification of Encoded by dnaQ--
The A. aeolicus
dnaQ was identified in the genome sequence as a 202-residue
protein of 17,132 Da having significant homology to E. coli
dnaQ (41). A. aeolicus dnaQ was amplified by PCR using
the following oligonucleotide primers. The upstream 35-mer (5'-GTGTGTCATATGCGAGACAATCTCCTTGATGGCAG-3') contains
an NdeI site (underlined), and the downstream 44-mer
(5'-GTGTGGATCCTCAAAACTTACCCTTTTCCAGCCTTTTTAAGGAG-3') contains a BamHI site (underlined). The PCR product
was digested with NdeI and BamHI, purified, and
ligated into the pET24a vector to produce pETAadnaQ.
The pETAadnaQ plasmid was transformed into E. coli strain
BL21(DE3). A single colony was used to inoculate 12 liters of LB media supplemented with 200 µg/ml ampicillin. Cells were grown at
37 °C to A600 = 0.5 at which point 0.5 mM IPTG was added. After a 3-h induction, cells were
collected by centrifugation and resuspended in 50 mM
Tris-HCl (pH 7.5), 10% sucrose, 1 M NaCl, 30 mM spermidine, 5 mM DTT, 2 mM EDTA.
Cells were lysed by two passages through a French press (15,000 pounds/square inch) followed by centrifugation at 13,000 rpm for 30 min
at 4 °C. The resulting supernatant (1038 mg) was incubated in a
65 °C waterbath for 30 min. The solution was clarified by
centrifugation at 13,000 rpm for 30 min. The supernatant (426 mg) was
dialyzed against buffer A and loaded onto a 15-ml fast flow Q-Sepharose
column equilibrated with buffer A. The column was eluted with a 150-ml
linear gradient of 50-500 mM NaCl in buffer A; 80 fractions were collected. Peak fractions (fractions 36-44, 12.9 mg)
were pooled, dialyzed against buffer A, and then loaded onto a 10-ml
heparin-agarose column equilibrated with buffer A. The column was
eluted with a linear gradient of 0 mM to 1 M NaCl; 80 fractions were collected. Peak fractions (fractions 60-70) were pooled, aliquoted, and stored at
80 °C.
Identification of A. aeolicus holA and Purification of
--
The A. aeolicus holA gene was not identified
previously by the genome sequencing project. We identified A. aeolicus holA by searching the A. aeolicus genome with
the amino acid sequence of the E. coli subunit (encoded
by holA). Although the resulting match had too low a score
to be confident of its assignment as , the studies of this report
prove that the gene truly encodes subunit of the replicase. The
A. aeolicus holA gene was amplified by PCR using the
following primers. The upstream 36-mer
(5'-GTGTGTCATATGGAAACCACAATATTCCAGTTCCAG-3') contains an
NdeI site (underlined); the downstream 39-mer
(5'-GTGTGTGGATCCTTATCCACCATGAGAAGTATTTTTCAC-3') contains a BamHI site (underlined). The PCR product
was digested with NdeI and BamHI, purified, and
ligated into the pET24 NdeI and BamHI sites to
produce pETAaholA.
The pETAaholA plasmid was transformed into E. coli strain
BL21 (DE3). Cells were grown in 50 liters of LB media containing 100 µg/ml kanamycin. Cells were grown at 37 °C to
A600 = 2.0, then induced for 20 h upon
addition of 2 mM IPTG, and collected by centrifugation.
Cells from 25 liters of culture were lysed as described above for
purification of .
The cell lysate was heated to 65 °C for 30 min, and the precipitate
was removed by centrifugation. The supernatant (650 mg, 240 ml) was
dialyzed against buffer A, adjusted to a conductivity equal to 160 mM NaCl by addition of 40 ml of buffer A, and applied to a
220-ml heparin-agarose column equilibrated in buffer A containing 100 mM NaCl. The column was eluted with 1.0 liters linear
gradient of 150-700 mM NaCl in buffer A. One hundred and
four fractions were collected. Fractions 45-56 were pooled (250 mg,
210 ml), diluted with 230 ml buffer A to a conductivity equal to 230 mM NaCl, and then loaded onto a 100-ml fast flow
Q-Sepharose column equilibrated in buffer A containing 150 mM NaCl. The column was eluted with a 200-ml linear
gradient of 150-750 mM NaCl in buffer A; 73 fractions were
collected. Fractions 16-38 were pooled (95 mg, 40 ml), aliquoted, and
stored at 80 °C.
Identification of A. aeolicus holB and Purification of
'--
The A. aeolicus holB gene was identified by Blast
search using the E. coli ' sequence as a query. The
A. aeolicus holB gene was amplified by PCR using the
following primers. The upstream 41-mer
(5'-GTGTGTCATATGGAAAAAGTTTTTTTTGGAAAAAACTCCAG-3') contains an NdeI site (underlined); the downstream 35-mer
(5'-GTGTGTGGATCCTTAATCCGCCTGAACGGCTAACG-3') contains a
BamHI site (underlined). The PCR product was
digested with NdeI and BamHI, purified, and
ligated into the pET24 NdeI and BamHI site to
produce pETAaholB.
The pETAaholB plasmid was transformed into E. coli strain
BL21 (DE3). Cells were grown at 37 °C in 50 liters of media
containing 100 µg/ml kanamycin to A600 = 2.0 and then induced for 3 h upon addition of 2 mM IPTG.
Cells were collected by centrifugation and were lysed using lysozyme by
the heat lysis procedure. The cell lysate was heated to 65 °C for 30 min, and precipitate was removed by centrifugation. The supernatant
(2.4 g, 400 ml) was dialyzed versus buffer A and then
applied to a 220-ml fast flow Q-Sepharose column equilibrated in buffer
A. Protein was eluted with a 1-liter linear gradient of 0-500
mM NaCl in buffer A; 80 fractions were collected. Fractions
23-30 were pooled and diluted 2-fold with buffer A to a conductivity
equal to 100 mM NaCl and then loaded onto a 200-ml
heparin-agarose column equilibrated in buffer A. Protein was eluted
with a 1-liter linear gradient of 0-1.0 M NaCl in buffer
A; 84 fractions were collected. Fractions 46-66 were pooled (1.3 g,
395 ml), dialyzed versus buffer A containing 100 mM NaCl, then aliquoted, and stored frozen at
80 °C.
Purification of Encoded by dnaX--
The A. aeolicus dnaX gene was amplified by PCR from genomic DNA
using the following primers. The upstream 41-mer
(5'-GTGTGTCATATGAACTACGTTCCCTTCGCGAGAAAGTACAG-3') contains
an NdeI site (underlined); the downstream 36-mer
(5'-GTGTGTGGATCCTTAAAACAGCCTCGTCCCGCTGGA-3') contains
a BamHI site (underlined). The PCR product was digested with
NdeI and BamHI, purified, and ligated into the
pET24 NdeI and BamHI sites to produce pETAadnaX.
The pETAadnaX plasmid was transformed into E. coli strain
BL21 (DE3). Cells were grown in 50 liters of LB containing 100 µg/ml kanamycin at 37 °C to A600 = 0.6 and
then induced for 20 h at 20 °C upon addition of IPTG to 2 mM. Cells were collected by centrifugation and lysed as
described for purification of . The clarified cell lysate was heated
to 65 °C for 30 min, and the protein precipitate was removed by
centrifugation. The supernatant (1.1 g in 340 ml) was treated with
0.228 g/ml ammonium sulfate followed by centrifugation. The subunit
remained in the pellet which was dissolved in buffer B (20 mM Hepes (pH 7.5), 0.5 mM EDTA, 2 mM DTT, 10% glycerol) and dialyzed versus
buffer B to a conductivity equal to 87 mM NaCl. The
dialysate (1073 mg, 570 ml) was applied to a 200-ml fast flow
Q-Sepharose column equilibrated in buffer A. The column was eluted with
a 1.5-liter linear gradient of 0-500 mM NaCl in buffer A;
80 fractions were collected. Fractions 28-37 were pooled (289 mg, 138 ml), dialyzed against buffer A to a conductivity equal to 82 mM NaCl, and then loaded onto a 150-ml column of
heparin-agarose equilibrated in buffer A. The column was eluted with a
900-ml linear gradient of 0-500 mM NaCl in buffer A; 32 fractions were collected. Fractions 15-18 (187 mg, 110 ml) were
dialyzed versus buffer A, then aliquoted, and stored at
80 °C.
Purification of Encoded by dnaN--
The A. aeolicus
dnaN gene (41) was amplified from genomic DNA by PCR using
the following primers. The upstream 33-mer
(5'-GTGTGTCATATGCGCGTTAAGGTGGACAGGGAG-3') contains an
NdeI site (underlined); the downstream 35-mer
(5'-TGTGTCTCGAGTCATGGCTACACCCTCATCGGCAT-3') contains
an XhoI site (underlined). The PCR product was digested with
NdeI and BamHI, purified, and ligated into the
pET24 NdeI and BamHI sites to produce pETAadnaN.
The pETAadnaN plasmid was transformed into E. coli strain
BL21 (DE3). Cells were grown in 1 liter of LB containing 100 mg/ml kanamycin at 37 °C to A600 = 1.0 and then
induced for 6 h upon addition of 2 mM IPTG. Cells were
collected (7 g) and lysed as described in the purification of above. The cell lysate was heated to 65 °C for 30 min, and the
protein precipitate was removed by centrifugation. The supernatant (39 mg, 45 ml) was applied to a 10-ml DEAE-Sephacel column (Amersham
Biosciences) equilibrated in buffer A. The column was eluted with a
100-ml linear gradient of 0-500 mM NaCl in buffer A; 75 fractions were collected. Fractions 45-57 were pooled (18.7 mg),
dialyzed versus buffer A, and applied to a 30-ml
heparin-agarose column equilibrated in buffer A. The column was eluted
with a 300-ml linear gradient of 0-500 mM NaCl in buffer
A; 65 fractions were collected. Fractions 27-33 were pooled (11 mg, 28 ml) and stored at 80 °C.
Purification of SSB Encoded by ssb--
The A. aeolicus
ssb gene (41) was amplified from genomic DNA by PCR using
the following primers. The upstream 47-mer
(5'-GTGTGTCATATGCTCAATAAGGTTTTTATAATAGGAAGACTTACGGG-3') contains an NdeI site (underlined); the downstream 39-mer
(5'-GTGTGGATCCTTAAAAAGGTATTTCGTCCTCTTCATCGG-3') contains a BamHI site (underlined). The PCR product
was digested with NdeI and BamHI, purified, and
ligated into the pET16b NdeI and BamHI sites to
produce pETAassb.
The pETAassb plasmid was transformed into E. coli strain
BL21 (DE3). Cells were grown in 6 liters of LB media containing 200 µg/ml ampicillin. Cells were grown at 37 °C to
A600 = 0.6, then induced at 15 °C overnight
in the presence of 2 mM IPTG, and collected by
centrifugation. Cells were lysed as described above for purification of
except cells were resuspended in buffer C (20 mM
Tris-HCl (pH 7.9), 500 mM NaCl).
The cell lysate was heated to 65 °C for 30 min, and the resulting
precipitate was removed by centrifugation. The supernatant (1.4 g, 190 ml) was applied to a 25-ml chelating Sepharose column (Amersham
Biosciences) charged with 50 mM nickel sulfate and then equilibrated in buffer C containing 5 mM imidazole. The
column was eluted with a 300-ml linear gradient of 5-100
mM imidazole in buffer C. Fractions of 4 ml each were
collected. Fractions 81-92 were pooled (~240 mg in 48 ml) and
dialyzed overnight against 2 liters of buffer B containing 200 mM NaCl. The dialysate was diluted to a conductivity equal
to 92 mM NaCl using buffer A and then loaded onto an 8-ml
MonoQ column equilibrated in buffer A containing 100 mM
NaCl. The column was eluted with a 120-ml linear gradient of 100-500
mM imidazole in buffer A. Seventy four fractions were
collected. Fractions 57-70 were pooled (100 mg, 25 ml), aliquoted, and
stored at 80 °C.
Preparation of ' Complex--
The (30 mg) and ' (30 mg) subunits were mixed in a volume of 23 ml of buffer A (final
conductivity equivalent to 130 mM NaCl) and incubated at
24 °C for 10 min. The mixture was applied to an 8-ml MonoQ column
equilibrated in buffer A. The protein was eluted with an 80-ml gradient
of 100 mM to 500 mM NaCl; 60 fractions were
collected. The ' complex elutes later than either or '.
The ' complex was stored at 80 °C.
Constitution of ' Complex--
The reaction mixture
contained 400 µg of ' and 1.2 mg of in a volume of 200 µl
of buffer A containing 300 mM NaCl. The protein mixture was
incubated at 24 °C for 15 min and then injected onto an HR 10/30
Superose 12 column equilibrated with buffer A containing 300 mM NaCl. After the first 6.6 ml, fractions of 170 µl were
collected. Fractions were analyzed in 10% SDS-polyacrylamide gels
stained with Coomassie Blue. The controls included ' complex alone (400 µg) and alone (1.2 mg).
Quantitation of the Intracellular Concentration of A. aeolicus
--
A. aeolicus cells were a generous gift of Dr. Karl
Stetter (Regensburg University). A. aeolicus cells were
resuspended in buffer A to ~2 × 1011 cells/ml by
direct counting as follows. Serial dilutions of the cell suspension
(107, 108, 109, and
1010) were counted using a hemocytometer (Hausser
Scientific) under a high power microscope (Olympus IX70), and the
results were averaged. A 50-µl aliquot was then cracked in 100 µl
of a solution containing 0.3% SDS, 30 mM Tris base, 30 mM DTT, 0.01% bromphenol blue, and 8% glycerol. Amounts
equivalent to 2.33 × 108 and 9.32 × 108 cells were analyzed in two lanes of a 12%
SDS-polyacrylamide gel, followed by transfer to nitrocellulose. Western
blot analysis was then performed using rabbit polyclonal antibody
directed against E. coli (which was purified against
A. aeolicus as described earlier). Known amounts of pure
recombinant A. aeolicus were analyzed in the same gel
for use as a standard curve to quantitate the in A. aeolicus cells observed in the Western blot. The protein concentration of A. aeolicus was calculated from its
absorbance at 280 nm in 6 M guanidine hydrochloride using
the molar extinction coefficient determined from its 2 Trp, 14 Tyr, and
20 Phe content (280 = 5,690 × 2 + 1,280 × 14 + 2 × 20 = 29,340 M
1
cm1).
Assays of , , ±--
Titration of into
replication reactions were performed using circular M13mp18 ssDNA
primed with a 700-fold molar excess of synthetic DNA 30-mer
oligonucleotide. Reactions contained 138 fmol of primed M13mp18 ssDNA,
100 pmol of DNA 30-mer, 3.6 µg of SSB (when present), 150 ng of
', 0.4 µg of (when present), and the indicated amount of
in 50 µl of 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM
(NH4)2SO4, 2 mM
MgSO4, 0.1% Triton X-100, 4 mM CaCl2, 0.5 mM ATP, and 60 µM each
dATP and dGTP. Reactions were mixed on ice, and then the mixture was
brought to 65 °C for 2 min before initiating synthesis upon addition
of 2 µl of dCTP and [-32P]dTTP (final
concentrations, 60 and 40 µM, respectively). Aliquots were removed and quenched at the times indicated upon adding EDTA and
SDS to final concentrations of 20 mM and 0.5%,
respectively. Quenched reactions were then analyzed in a 0.8%
alkaline-agarose gel. Size standards were also included in the same
gel. Gels were dried followed by analysis using a PhosphorImager.
Replication time course reactions contained 70 ng (25 fmol) of M13mp18
ssDNA, 100 pmol of 30-mer oligonucleotide, 2 µg of SSB (when
present), 100 ng of , 200 ng of ' complex, and 40 ng of in 25 µl of 20 mM Tris-HCl (pH 8.8), 10 mM
KCl, 10 mM (NH4)2SO4,
10 mM MgSO4, 0.1% Triton X-100, 1 mM ATP, 4 mM CaCl2, 10% glycerol,
and 60 µM each dGTP and dATP. Replication time courses were performed at different temperatures as described above except that
SSB was omitted where indicated, and the mixture was brought to the
indicated temperature for 2 min before initiating synthesis by the
addition of 2 µl of dCTP and [-32P]dTTP (final
concentrations, 60 and 40 µM, respectively). Aliquots were removed and quenched at the times indicated upon adding EDTA and
SDS to 20 mM and 0.5%, respectively. Quenched reactions
were then analyzed in 0.8% alkaline-agarose gels. Size standards were included in the same gel. Gels were dried and exposed to film.
Detection of Interaction with and by Elisa--
The
interaction was analyzed as follows: E. coli or
A. aeolicus (2 µg in 100 µl) were placed in wells of
a 96-well vinyl assay plate (Costar) and incubated for 12 h at
4 °C in 20 mM Tris-HCl (pH 7.5), 1 mM EDTA,
2 mM DTT, 5 mM MgCl2, 100 mM NaCl, 20% glycerol. Solutions were removed, and wells
were washed 4 times with 100 µl of TBS. Wells were then blocked with
100 µl of TBS containing 5% non-fat milk for 3 h at 23 °C.
Following the blocking step, wells were washed 4 times with TBS and
then incubated with 4 µg of either E. coli or A. aeolicus for 2 h at 23 °C. Solutions were then
removed, and wells were washed as above and then incubated overnight at
4 °C with 100 µl of 1:500 dilution of polyclonal rabbit antibody
raised against E. coli and purified against A. aeolicus . Wells were washed three times with TBS (5 min each
wash) and then incubated with anti-rabbit horseradish
peroxidase-conjugated antibody (Sigma) for 30 min and developed with an
ECL detection kit (Amersham Biosciences) as described by the manufacturer.
Detection of and interaction was performed as described above
with the following modifications. One hundred microliters of either
E. coli or A. aeolicus (50 µg/ml) were
incubated in wells for 12 h at 4 °C. Following the block step
and the washes with TBS, 100 µl of either E. coli or
A. aeolicus (100 µg/ml) were placed into the wells and
incubated for 2 h at 23 °C. Solutions were then removed, and
wells were washed with TBS as above and then incubated overnight at
4 °C with 100 µl of 1:500 dilution of polyclonal rabbit antisera
directed against E. coli . Wells were then washed with
TBS (3 times for 5 min) and further incubated with a 1:10,000 dilution
of anti-rabbit horseradish peroxidase-conjugated antibody (Sigma) for
30 min and then developed with an ECL detection kit (Amersham
Biosciences) and exposed to film.
Detection of - Interaction by DNA Synthesis--
DNA
synthetic reactions contained 70 ng of M13mp18 ssDNA uniquely primed
with a synthetic DNA 60-mer, 1 µg of E. coli SSB, 100 ng
of polymerase subunit (either S. pyogenes pol C or E. coli ), and when present 400 ng of (S. pyogenes
or E. coli ), in 25 µl of 20 mM
Tris-HCl (pH 7.5), 4% glycerol, 0.1 mM EDTA, 5 mM DTT, 2 mM ATP, 8 mM
MgCl2, 40 µg/ml BSA, and 60 µM each of dGTP
and dCTP. Reactions were preincubated at 37 °C for 2 min, and then
synthesis was initiated upon addition of 1.5 µl of dATP and
[-32P]TTP (final concentrations of 60 and 20 µM, respectively). Reactions were allowed to proceed for
5 min prior to being quenched with an equal volume (25 µl) of 1%
SDS, 40 µM EDTA. One-half of the reaction was analyzed
for total DNA synthesis using DE81 filter paper as described (43).
Assays of A. aeolicus and utilized a different
temperature and conditions as follows. Reactions contained 70 ng (25 fmol) of M13mp18 ssDNA, 100 pmol of DNA 30-mer, 2 µg of A. aeolicus SSB, 50 ng of A. aeolicus , and 400 ng of
A. aeolicus in 25 µl of 20 mM Tris-HCl (pH
8.8), 10 mM KCl, 10 mM
(NH4)2SO4, 10 mM
MgSO4, 0.1% Triton X-100, 1 mM ATP, 4 mM CaCl2, 10% glycerol, 60 µM
each dGTP and dATP. The reaction mixture was incubated at 60 °C for
2 min before initiating synthesis by the addition of 1.5 µl of dCTP
and [-32P]TTP (final concentrations, 60 and 40 µM, respectively). Reactions were allowed to proceed for
5 min before being quenched with an equal volume (25 µl) of 1% SDS,
40 µM EDTA. One-half of the quenched reaction was
analyzed for total DNA synthesis using DE81 filter paper (43).
Gel Filtration Analysis of , , and --
The
mixtures contained 1 mg of and 200 µg of or 0.5 mg of in
a volume of 200 µl of buffer A containing 300 mM NaCl. Proteins were incubated at 24 °C for 15 min before injecting the mixture onto an HR 10/30 Superose 12 column equilibrated with buffer A
containing 300 mM NaCl. In a further experiment, 4 mg (150 µM) and 1 mg (92 µM) were mixed in
a volume of 200 µl of buffer A containing 300 mM NaCl and
analyzed by gel filtration as described above. The controls included
alone (1 mg, 37.5 µM), alone (0.5 mg, 46 µM), and alone (200 µg) in 200 µl of buffer A
containing 300 mM NaCl.
Thermostability Assays--
A. aeolicus , ,
', SSB, and + ' were tested for stability at
different temperatures by incubating the proteins at the temperatures
indicated and then testing them for activity in the M13mp18 replication
assay. Heat treatment was performed in 0.4-ml tubes under mineral oil
in 5 µl of 20 mM Tris-HCl (pH 7.5), 5 mM DTT,
5 mM EDTA, and either (a) 0.352 µg of ;
(b) 0.2 µg of ; (c) 0.125 µg of
'; (d) 0.32 µg of SSB and 0.042 µg of primed
M13mp18 ssDNA; or (e) 0.352 µg and 0.125 µg of
'. Heat treatment was for 2 min at either 70, 80, 85, or 90 °C in the presence of either (a) 0.1%
Triton X-100; 0.05% Tween 20 and 0.01% Nonidet P-40; (b) 4 mM CaCl2; (c) 40% glycerol;
(d) 0.01% Triton X-100, 0.05% Tween 20, 0.01% Nonidet
P-40, 4 mM CaCl2; (e) 40% glycerol,
0.1% Triton X-100; (f) 40% glycerol, 0.05% Tween 20, 0.01% Nonidet P-40; (g) 40% glycerol, 4 mM
CaCl2; (h) 40% glycerol, 0.01% Triton X-100,
0.05% Tween 20, 0.01% Nonidet P-40, 4 mM
CaCl2. After heating, reactions were shifted to ice, and 20 µl of replication assay buffer was added followed by incubation for
1.5 min at 70 °C; 15 µl was then spotted onto a DE81 filter and
DNA synthesis was quantitated (43). The replication assay buffer
contained the following: 60 mM Tris-HCl (pH 9.1 at
25 °C), 8 mM MgCl2, 10 mM
(NH4)2SO4, 2 mM ATP, 60 µM each of dATP, dCTP, dGTP, and 20 µM
[32P]TTP (specific activity 10,000 cpm/pmol), 2 µM DNA 30-mer, and 0.264 µg of primed M13mp18 ssDNA. To
assay , 170 ng of and 100 ng of ' was added to the
reaction. To assay ', 0.9 ng of and 170 ng of were
added to the reaction. To assay '2b ', 90 ng of was
added to the reaction. To assay , 90 ng of and 100 ng of
' was added to the reaction. To assay SSB, 90 ng of E. coli and 100 ng of E. coli ' was added
to the reaction followed by incubation for 1.5 min at 37 °C.
Exonuclease Assay--
Exonuclease assays contained 100 fmol of
a 5'-labeled 50-mer DNA oligonucleotide and 50 ng of either E. coli subunit or A. aeolicus in 20 µl of 20 mM Tris-HCl (pH 7.5), 4% glycerol, 0.1 mM
EDTA, 5 mM DTT, 40 µg/ml BSA, 8 mM
MgCl2. Reactions were incubated at 37 °C for the
indicated times and were quenched upon the addition of 20 µl of 95%
formamide, 40 mM EDTA. Ten microliters of each reaction
were analyzed in a 15% denaturing (6 M urea) polyacrylamide gel.
Calf Thymus DNA Synthesis Assay--
Assays contained 2.5 µg
of activated calf thymus DNA and 0.2 of either E. coli subunit or A. aeolicus in a final volume of 25 µl of
20 mM Tris-HCl (pH 7.5), 4% glycerol, 0.5 mM
EDTA, 5 mM DTT, 40 µg/ml BSA, 8 mM
MgCl2, 1 mM ATP, 60 µM each dCTP, dGTP, dATP, and 20 µM [32P]dTTP.
Reactions were incubated at the indicated temperatures, and aliquots
were removed at the times indicated. DNA synthesis was quantitated
using DE81 paper as described (43).
Clamp Loading Assay--
A C-terminal kinase site was introduced
into A. aeolicus by PCR using a primer with a sequence
encoding a 6-amino acid (RRASVP) recognition motif for
cAMP-dependent protein kinase (44). The gene encoding
A. aeolicus pk was placed into
NdeI/BamHI sites of pET16b, and the A. aeolicus pk was expressed and purified. The
pk was radiolabeled using cAMP-dependent
protein kinase and [-32P]ATP as described for E. coli pk (45). Clamp loading reactions contained
0.125 pmol of singly nicked M13mp18 plasmid DNA (using gpII protein),
0.7 nmol (60 ng) of 32P-, 100 ng of 'complex
(when present) and 2.4 µg of A. aeolicus SSB (when
present) in 50 µl of reaction buffer (20 mM Tris-HCl (pH
8.8, at 25 °C), 10 mM KCl, 10 mM
(NH4)2SO4, 10 mM
MgSO4, 0.1% Triton X-100, and 1 mM ATP).
Reactions were incubated for 10 min at 70 °C and then applied to 5 ml Bio-Gel A15m columns (Bio-Rad) pre-equilibrated with 20 mM Tris-HCl (pH 7.5), 8 mM MgCl2,
5% glycerol, 2 mM DTT, 40 µg/ml BSA, and 50 mM NaCl. Gel filtration was performed at room temperature
(24 °C). Fractions of 200 µl were collected, and 150 µl of each
fraction was analyzed by liquid scintillation counting.
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RESULTS |
The A. aeolicus Clamp Loader--
Studies in the E. coli system demonstrated the need for five proteins to form a
processive polymerase III: , (or ), , ', and (9,
13, 46). A sixth protein, the 3'-5'-exonuclease, further
enhanced the speed and processivity of the holoenzyme constituted from
these five proteins (9). The report on the A. aeolicus
genome sequence (41) documented the presence of A. aeolicus
dnaE (), dnaX (/), dnaN (), and
dnaQ (), but holA and holB genes
encoding and ' were not identified. As we have reported
previously, is a very poorly conserved subunit of the pol III
replicase (38). We identified S. pyogenes holA by isolating
a gene encoding a weak homologue to E. coli and then
showing that the putative formed an active clamp loader complex
with other S. pyogenes subunits (38). In the experiments below, we follow a similar strategy to identify genes encoding both
A. aeolicus and '.
A search of the A. aeolicus genome for the A. aeolicus
holA gene encoding , using E. coli subunit as
the query, produced very weak matches, the best having about 18%
identity to E. coli (see Fig.
2A). Likewise, a search for
the A. aeolicus holB gene encoding ', using E. coli ' as query, produced a similarly weak match (Fig.
2B). To determine whether the putative A. aeolicus holA and holB genes truly encode the and '
subunits of the replicase, we expressed and purified the encoded
proteins and then tested them for clamp loading activity with the
A. aeolicus dnaX product. The genes encoding the putative
A. aeolicus and ' subunits were cloned into the
T7-based pET11 expression plasmid. Purification of A. aeolicus and ' were performed as described under
"Experimental Procedures," and a sample of each preparation is
shown in the SDS-polyacrylamide gel of Fig. 1A.
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Fig. 2.
The sequences of A. aeolicus
and ' subunits. The
amino acid sequences of A. aeolicus and E. coli
(A) and ' (B) are aligned using ClustalX.
The asterisks below the sequence indicate amino
acids that are identical between the two sequences.
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To test ability of the putative A. aeolicus and '
subunits to form a clamp loader complex with A. aeolicus
(), encoded by dnaX, we needed to express and purify
the product of the A. aeolicus dnaX gene. A. aeolicus
dnaX lacks a ribosomal frameshift sequence, and it also lacks the
six or more contiguous A residues that are needed to produce in
T. thermophilus by a transcriptional slippage mechanism
(37). Consistent with the lack of sites for synthesis of the truncated
subunit, expression of the A. aeolicus dnaX gene in
E. coli showed production of only one protein that was the
approximate size of the predicted full-length product (54.3 kDa). As
will be shown later in this report, examination of A. aeolicus cell extracts revealed the presence of only full-length subunit, a truncated version (i.e. ) was not
detected. The A. aeolicus subunit was purified as
described under "Experimental Procedures," and a sample of the
protein preparation is shown in Fig. 1A.
In the E. coli system, (or ) forms a heterotrimer
with and ' (13). Even though the crystal structure of E. coli 3' shows that contacts both and
' directly, mixtures of (or ) with either or ' do not
yield either heterodimer (i.e. or ') complex
(13). To examine the A. aeolicus , , and ' subunits
for protein interactions, combinations of these subunits were mixed and
then analyzed by gel filtration on a Superose 12 column. The analysis
in Fig. 3A demonstrates that a
mixture of + ' does not yield a heterodimer complex, instead the
two proteins elute at distinct positions. This result is similar to
observations in the E. coli system (13). Analysis of the + mixture, in Fig. 3B, shows that the bulk of the and does not comigrate, although a slight amount of appears in
the vicinity of indicating a weak interaction between them. The
elution position of the free and ' subunits in Fig. 3,
A and B, compared with molecular mass standards,
indicates that and ' are each monomeric, similar to and '
of E. coli and S. pyogenes (13, 38). The subunit elutes as an oligomer, possibly a trimer or tetramer, like
E. coli and S. pyogenes (38, 47).
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Fig. 3.
Constitution of the A. aeolicus
' clamp loader. Combinations
of the , , and ' subunits were incubated together and then
analyzed for complex formation by gel filtration analysis on a Superose
12 column. Column fractions were analyzed in 10% SDS-polyacrylamide
gels stained with Coomassie Blue. A, + ';
B, + ; C, + + '; D,
+ '. Subunit positions in the gels are indicated to the
right. The elution positions of protein standards are
indicated at the bottom: 670 kDa, thyroglobulin; 440 kDa,
horse spleen apoferritin; 158 kDa, bovine globulin; 44 kDa, chicken
ovalbumin; and 17 kDa, horse myoglobin.
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A mix of all three subunits results in their comigration as a
' complex that elutes earlier than either , , or '
subunits alone (Fig. 3C). We presume on the basis of
E. coli structural studies of 3' that
the stoichiometry of A. aeolicus ' is 3 1 '1 (235.8 kDa) (19,
47). Consistent with this, densitometric scans of SDS-polyacrylamide
gels of Superose 12 column fractions of ', constituted using
excess ', give a stoichiometry of 3.3
1.1 '1.0.
Previous study of the E. coli and S. pyogenes pol
III systems demonstrated that the and ' subunits form a 1:1
' complex (13, 38). Fig. 3D shows that A. aeolicus and ' also form a ' complex. Analysis of a
mixture of and ' demonstrates that the two subunits coelute in
earlier fractions than the elution position of either or ' alone
(compare D with A and B) and thus form
a :' complex. Densitometric scans of the gel in Fig. 3D yield a stoichiometry of 1.1
'1.0 (a similar stoichiometry is obtained from an
analysis using a 2-fold molar excess of 'over and scanning the
fractions containing ' complex).
Is the A. aeolicus ' complex active as a clamp
loader? To test this, a six-residue kinase recognition site was placed
onto the C terminus of , allowing it to be phosphorylated using
[-32P]ATP and cAMP-dependent protein
kinase. The 32P- was then incubated with ',
ATP, and a circular plasmid having a single nick. After incubation at
70 °C for 10 min, the reaction was applied to a 5-ml Bio-Gel A15m
column. Bio-Gel A15m is a large pore resin that includes most proteins
but excludes the large circular DNA molecule. Therefore, if '
is active in assembly of 32P- onto DNA, some of the
32P- should comigrate with the DNA in the excluded
volume and resolve from the free 32P- in the included
fractions. The results of this 32P- clamp loading
experiment are shown in Fig. 4; about
half of the 32P- is observed to comigrate with DNA in
the excluded fractions (fractions 10-15). As a control, a similar
experiment was performed in the absence of ' complex. The
result demonstrates that essentially no 32P- is
assembled onto the DNA in the absence of '. Hence, the A. aeolicus ' is capable of assembling A. aeolicus 32P- clamps onto DNA. The temperature
resistance of the A. aeolicus ' complex and clamp will be demonstrated later in this report, along with the DNA
polymerase.
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Fig. 4.
Demonstration of clamp loading by A. aeolicus '. The scheme
at the top illustrates the assay. A C-terminal six-residue
tag was cloned onto A. aeolicus that serves as an
efficient substrate for phosphorylation. A. aeolicus
' and 32P- are incubated with a circular
plasmid containing a single nick. Clamp loading by |
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TOP
ABSTRACT
INTRODUCTION
