From the Department of Biochemistry and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
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ABSTRACT |
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Two forms of DNA polymerase II () of
Saccharomyces cerevisiae, Pol II* and Pol II, were purified
to near homogeneity from yeast cells. Pol II* is a four-subunit complex
containing a 256-kDa catalytic polypeptide, whereas Pol II consists
solely of a 145-kDa polypeptide derived from the N-terminal half of the
256-kDa polypeptide of Pol II*. We show that Pol II* and Pol II are
indistinguishable with respect to the processivity and rate of
DNA-chain elongation. The equilibrium dissociation constants of the
complexes of Pol II* and Pol II with the DNA template showed that the
stability of these complexes is almost the same. However, when the
rates of dissociation of the Pol II* and Pol II from the DNA template were measured using single-stranded DNA as a trap for the dissociated polymerase, Pol II* dissociated 75-fold faster than Pol II.
Furthermore, the rate of dissociation of Pol II* from the DNA template
became faster as the concentration of the single-stranded DNA was
increased. These results indicate that the rapid dissociation of Pol
II* from the DNA template is actively promoted by single-stranded DNA.
The dissociation of Pol II from the DNA template was also shown to be
promoted by single-stranded DNA, although at a much slower rate. These
results suggest that the site for sensing single-stranded DNA resides
within the 145-kDa N-terminal portion of the catalytic subunit and that
the efficiency for sensing single-stranded DNA by this site is
positively modulated by either the C-terminal half of the catalytic
subunit and/or the other subunits.
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INTRODUCTION |
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In Saccharomyces cerevisiae, DNA polymerase II (),
as well as DNA polymerases I (
) and III (
), are required for DNA
replication (1, 2). Deletion of the POL2 gene, which codes
for the catalytic subunit of DNA polymerase II, causes cell death with
a terminal dumbbell morphology, the hallmark of a defect in DNA
replication (3). Direct measurements of in vivo DNA
synthesis in temperature-sensitive pol2 mutants revealed
that chromosomal DNA replication ceases at the restricted temperature
(4, 5). The observation that a pol2 mutant defective in
3'
5' exonuclease activity exhibits a mutator phenotype at a variety
of genetic markers throughout the genome supports the idea that
participation of the polymerase in chromosomal DNA replication is not
restricted to certain sites on the genome (6). Recent work by Aparicio
et al. (7) revealed that DNA polymerase II is recruited to
the origin at the time of initiation of DNA replication and proceeds
along the DNA with the replication fork. This finding, together with
others, strongly supports the idea that DNA polymerase II is a
component of the replication apparatus and that it is responsible for
DNA synthesis on either the leading or lagging strand. However, whereas
DNA polymerase I is responsible for laying down RNA-DNA primers,
specific roles for DNA polymerases II and III at the fork have not been determined (8, 9).
DNA repair is another cellular process for which the function of DNA polymerase II might be required. Among the DNA polymerase mutants of S. cerevisiae, pol2 mutants were exclusively deficient in the repair synthesis of base-damaged DNA (10). For nucleotide excision repair, DNA polymerase II and III are potentially responsible, as pol2 and pol3 double mutants showed accumulation of single-strand breaks in their chromosomes after UV irradiation (11).
Besides DNA replication and repair, DNA polymerase II has recently been assigned a new role in the S-phase checkpoint. Certain pol2 mutants are defective in their transcriptional response to DNA damage, specifically in S-phase, and are unable to prevent entry into mitosis when DNA replication is blocked by hydroxyurea (12, 13). The Dpb11 protein, which has been suggested to be an additional component of the DNA polymerase II complex on the basis of genetic studies, is also implicated in both DNA replication and checkpoint control (14). A temperature-sensitive dpb11 mutant showed a defect in S-phase progression at the restricted temperature with accompanying loss of viability owing to abnormal nuclear segregation. Furthermore, an involvement of Rfc5 and Rfc2, which are components of replication factor C (RF-C)1, in the S-phase checkpoint was also suggested (15, 16).2 Therefore, the view has emerged that the replication apparatus acts as a sensor for aberrant DNA replication as it progresses along the template with the replication fork. DNA polymerase II may play a central role in this process.
A high degree of conservation in the primary structure of the catalytic
subunit of DNA polymerase between yeast and human, as well as a
similarity in their biochemical properties, suggests that the role of
DNA polymerase II in yeast may apply to eukaryotic cells in general
(17, 18). The finding that expression of human DNA polymerase
depends on cell proliferation is in support of its involvement in
chromosomal DNA replication (19). However, experiments by Zlotkin
et al. (20) designed to capture DNA polymerases in contact
with the nascent DNA by the photolabeling method detected only DNA
polymerases
and
on SV40 DNA, consistent with their requirement
in in vitro SV40 DNA replication (21-23). DNA polymerase
, in addition to polymerases
and
, was photolabeled by
nascent cellular DNA; however, unlike DNA polymerases
and
, the
signal responded poorly to mitogenic stimulation, which increases the proportion of replicative DNA synthesis relative to repair synthesis. From these results, Zlotkin et al. (20) proposed that the
major replicative polymerases, not only in SV40 but also in nuclear DNA
synthesis, are DNA polymerases
and
. DNA polymerase
was proposed to be required for cellular DNA replication, such as that
observed during post-replicational repair coupled to fork movement, and
in checkpoint activities, where it would assure accurate and
coordinated DNA synthesis during the cell cycle.
In any case, little is known about the mechanism of action of DNA
polymerase II () in specific replicative reactions except for its
ability to catalyze DNA synthesis. This is largely the result of a lack
of biochemical characterization of the polymerase, especially of the
intact polymerase associated with all of its subunits. In accordance
with the complexity of the biological functions of DNA polymerase II in
S. cerevisiae, the enzyme possesses a rather complex
structure. In its most intact form, DNA polymerase II purified from
S. cerevisiae consists of four subunits (24). These comprise
a catalytic 256-kDa subunit and auxiliary subunits of 80 and 34 kDa,
which are encoded by the POL2, DPB2, and
DPB3 genes, respectively (3, 25, 26). The gene for the
smallest subunit of 29 kDa has been identified
recently.3 The large
catalytic subunit is composed of two domains; the N-terminal half
encodes the catalytic domain, which is conserved among
aphidicolin-sensitive DNA polymerases, and the C-terminal half is a
region unique to DNA polymerase II (2, 3, 27). Although the N-terminal domain is sufficient for polymerase and 3'
5' exonuclease activities in vitro, mutational analyses suggest that the C-terminal
domain is also involved in DNA replication (5, 12). In addition, the
C-terminal domain is involved in the functioning of the S-phase checkpoint and the response mechanism to DNA damage, as mutations in
this domain selectively cause defects in these processes (12). In as
much as the C-terminal domain is known to be required for holding the
auxiliary subunits together as a complex (3), the defects caused by
mutations may be either the direct effect of impaired functioning of
the C-terminal domain or the result of loss of auxiliary subunits from
the complex. The functions of the auxiliary subunits are mostly
unknown.
Elucidation of the biochemical functions of the C-terminal domain of
the Pol2 catalytic subunit and the auxiliary subunits of DNA polymerase
II is critical for our understanding of the specific roles of the
polymerase in vivo. As a first step, we have taken advantage
of the fact that different forms of DNA polymerase II can be purified
from cells of S. cerevisiae. Besides the four-subunit complex described above, a single polypeptide of 145 kDa, a proteolytic product of Pol2, has been purified to near homogeneity (24). Because
the polypeptide retains both polymerase and 3'5' exonuclease activities, it probably contains most, if not all, of the N-terminal domain but lacks the C-terminal domain. Therefore, we compared the
biochemical activities of the two forms of DNA polymerase II. The
intact complex consisting of four subunits is called Pol II*, whereas
the 145-kDa single polypeptide is called Pol II, following the proposal
of the previous study (24). Here, we show that Pol II* is readily
displaced from a DNA template by single-stranded DNA, whereas, in
comparison, the sensitivity of Pol II to displacement by
single-stranded DNA is greatly reduced. This difference in sensitivity
to single-stranded DNA defines for the first time a biochemical
activity specific for the C-terminal domain of Pol2 and/or the
auxiliary subunits.
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EXPERIMENTAL PROCEDURES |
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Materials--
Ultrapure dNTPs (minimal diphosphate, sodium
salt), bovine serum albumin (DNase-Free), Ready-To-Go T4 polynucleotide
kinase, poly(dA)300, and oligo(dT)10 were all
from Amersham Pharmacia Biotech; [-32P]dTTP (800 Ci/mmol) and [
-32P]dATP (3000 Ci/mmol) were from
Amersham; 70% glutaraldehyde was from WAKO; the restriction enzyme,
Alw44I, was from TOYOBO; the protein assay kit and bovine
gamma globulin were from Bio-Rad; the DNA molecular weight marker and
-EcoT14I digest were from TAKARA Biochemicals. DNA
Polymerase III (
) (3500 units/mg, about 50% pure) and replication
factor A (RF-A) (>90% pure) were purified from S. cerevisiae as described previously (28).
X174 viral DNA
was from New England Biolabs, and the 18-mer (5'-CTTCTGCGTCATGGAAGC-3') and 20-mer (5'-GCATAAAGTGCACCGCATGG-3') oligonucleotides were synthesized by Sawady Technology and purified by high pressure liquid
chromatography. The 60-mer
(5'TCGAGGTCGACGAATTCTAGTGATGGTGATGGTGATGCAGCAGGTCCGAGATGACGTACT-3') and
15-mer (5'-AGTACGTCATCTCGG-3') oligonucleotides (29) were synthesized and PAGE purified by Pharmacia Biotech OligoExpress.
Nucleic Acids--
The homopolymer poly(dA)300 and
oligo(dT)10 were mixed at a weight ratio of either 20:1 or
5:1 in 20 mM Tris-HCl (pH 8.0) containing 20 mM
KCl and 1 mM EDTA, heated at 65 °C for 5 min, and then
slowly cooled at room temperature. X174 ssDNA was singly primed with
an 18-mer (map positions 11-28) synthetic oligonucleotide (30). The
primer DNA and the
X DNA were mixed at a molar ratio of 3:1 in 10 mM Tris-HCl (pH 8.0) containing 270 mM NaCl and
1 mM EDTA. The mixture was heated at 80 °C for 10 min,
transferred to 56 °C for 15 min, and slowly cooled at room
temperature. A linear form of
X ssDNA was made as follows. The
20-mer DNA (map positions 4772-4791) (30) was labeled at its 5'-end by
T4 polynucleotide kinase and hybridized to
X ssDNA under the
conditions described above for primed
X DNA. The hybridized region
contains the unique Alw44I site of
X DNA. After diluting
the reaction mixture 8-fold, the
X DNA was digested completely with
Alw44I and extracted with phenol-chloroform. The sample was
further diluted 8-fold with TE (pH 7.5), heated at 65 °C for 15 min,
and chilled quickly on ice. The denatured oligonucleotides were removed
by centrifugation of the sample in a Centricon-100 (Amicon).
Concentrated sample was diluted again with TE (pH 7.5) to the initial
volume, and the procedures were repeated again to remove denatured
oligonucleotides. The removal of the oligonucleotides was complete as
monitored by counting radioactivity in the sample. The 60-mer (d60) and 15-mer (d15) synthetic oligonucleotides were labeled at their 5'-ends
by T4 polynucleotide kinase. The 15-mer DNA was hybridized to the
60-mer DNA (d60:d15) (14,000 cpm/pmol) by mixing 5'-labeled 15-mer and
unlabeled 60-mer (or unlabeled 15-mer and 5'-labeled 60-mer) at a molar
ratio of 1:1 in 20 mM Tris-HCl (pH 8.0) containing 20 mM KCl and 1 mM EDTA. The mixture was heated at
90 °C for 3 min, incubated at 65 °C for 2 h, and cooled
slowly at room temperature. Hook-structure DNA was a gift of Dr. H. Maki, Nara Institute of Science and Technology (31).
Purification of DNA Polymerase II (Pol II* and Pol II)-- Pol II* and Pol II were purified from CB001 yeast cells (24). Cells (2 kg) were grown, and extracts (fraction I) were subjected to ammonium sulfate precipitation (fraction II) and SP-Sepharose Fast Flow (Pharmacia Biotech) column chromatography (fraction III), as described previously (24). Fraction III was dialyzed against buffer A (50 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 10 mM NaHSO3, 1 mM PMSF, 10 mM 2-mercaptoethanol) until the conductivity was equivalent to buffer A + 0.1 M NaCl. After removal of insoluble material by centrifugation (12,000 × g, 4 °C, 15 min), the sample was loaded onto a POROS Q column (5 × 10 cm) (PerSeptive Biosystems) equilibrated with buffer A + 50 mM NaCl. The column was washed with 2 column volumes of the equilibration buffer, and polymerase activity was eluted by 11 column volumes of a linear gradient from 50 to 500 mM NaCl in buffer A at a flow rate of 3 ml/min. Four major peaks of the polymerase activity were obtained. Immunological detection with antiserum against Pol II* revealed that the fourth peak eluting at 300 mM NaCl corresponded to that of Pol II* (four-subunit complex of 256, 80, 34, and 29 kDa polypeptides), whereas the third peak eluting at 250 mM NaCl contained several different subassemblies of DNA polymerase II. The major constituents of the third peak seemed to be complexes of 145- and 34-kDa polypeptides and of 256- and 80-kDa polypeptides. Pol II* and Pol II were purified from the peaks eluting at 300 and 250 mM NaCl, respectively.
Pol II* Purification--
Fractions containing the peak eluting
at 300 mM NaCl were pooled, proteins were precipitated with
ammonium sulfate (0.4 g/ml sample), and the precipitate was collected
by centrifugation (39,000 × g, 4 °C, 30 min). The
suspension of the pellet in buffer C (50 mM HEPES, pH 7.4, 10% glycerol, 1 mM EDTA, 10 mM
NaHSO3, 1 mM PMSF, 10 mM
2-mercaptoethanol) was dialyzed against the same buffer until the
conductivity reached that of buffer C + 50 mM NaCl
(fraction IV) and loaded onto a Mono S HR 10/10 column (Pharmacia
Biotech) equilibrated with buffer C + 50 mM NaCl. After
washing the column with 3 column volumes of the equilibration buffer,
the activity was eluted by a linear gradient of 20 column volumes of
50-500 mM NaCl in buffer C at a flow rate of
0.25 ml/min. The activity was recovered in a peak eluting at 280 mM NaCl with a shoulder at 290 mM NaCl. Four
polypeptides of 256, 80, 34, and 29 kDa, which were cross-reactive with
the Pol II* antiserum, coincided with the activity in the peak at 280 mM NaCl, whereas the activity in the shoulder peak
coincided with another set of four bands of 256, 80, 30, and 29 kDa.
Because the 34- and 30-kDa polypeptides are known to be encoded by the
same gene, DPB3 (26), the complex containing the 30-kDa
polypeptide was probably the result of degradation of the 34-kDa
polypeptide and was separated from the Pol II* complex at this step.
The fractions in the peak at 280 mM NaCl were pooled (fraction V) and applied directly to a HiTrap heparin column (5 ml)
(Pharmacia Biotech) equilibrated with buffer D (10 mM
sodium phosphate, pH 7.0, 10% glycerol, 1 mM EDTA, 10 mM NaHSO3, 1 mM PMSF, 10 mM 2-mercaptoethanol) containing 300 mM NaCl.
The column was washed with 3 column volumes of the equilibration
buffer, and the activity was eluted by 20 column volumes of a linear
gradient of 300 mM to 1 M NaCl in buffer D. The
activity was eluted in a single peak at 640 mM NaCl and
coincided with the presence of four bands of 256-, 80-, 34-, and 29-kDa
polypeptides, as detected by SDS-PAGE. As the activity in this fraction
was unstable at 4 °C and sensitive to freezing and thawing,
presumably as a result of the low protein concentration, eluates were
collected into tubes containing BSA and Triton X-100R at final
concentrations of 0.5 mg/ml and 0.01%, respectively. This treatment
prevented enzyme inactivation. The overall recovery of the activity was 24%, assuming that the Pol II* activity recovered from the POROS Q
column was 100%. The specific activity of the final sample of Pol II*
was 4,400 units/mg protein (without BSA) (total 6,200 units), and the
purity was approximately 70% (without BSA) as estimated by SDS-PAGE.
The pool of the peak fractions of the HiTrap heparin column was
dialyzed against 50 mM Tris-HCl, pH 7.5, 50% glycerol, 1 mM EDTA, 50 mM NaCl, 10 mM
2-mercaptoethanol, and 5 mM DTT, and aliquots were stored
at 80 °C (fraction VI).
Pol II Purification--
Fractions that had eluted as a peak at
250 mM NaCl from the POROS Q column were pooled, and
proteins were precipitated with ammonium sulfate (fraction IV') and
chromatographed on the Mono S HR 10/10 column as described above. The
activity was recovered in three major peaks eluting at 200, 240, and
310 mM NaCl. The activity in the peak at 200 mM
NaCl was sensitive to high salt (120 mM KCl), and the
proteins in the peak showed no cross-reactivity with Pol II* antiserum.
However, they were cross-reactive with Pol I antiserum. The activities
in the peaks at 240 and 310 mM NaCl were resistant to 120 mM KCl, which is a characteristic of DNA polymerase II.
Immunological detection with the Pol II* antiserum revealed that the
peak at 240 mM NaCl contained two polypeptides of 145 and
34 kDa, whereas the peak at 310 mM NaCl contained
polypeptides of 256 and 80 kDa. The activity in the former peak was
further purified. The peak fractions were pooled and dialyzed against buffer D (fraction V') and loaded onto a HiTrap heparin (5 ml) equilibrated with buffer D + 100 mM NaCl. After washing the
column with 3 column volumes of the same buffer, the activity was
eluted with linear gradient of 10 column volumes of 100 mM
to 1 M NaCl in buffer D. The activity recovered in a single
peak at 730 mM NaCl coincided with the presence of only the
145-kDa polypeptide, as detected by immunoblot analysis using the Pol
II* antiserum. The 34-kDa polypeptide appeared in slightly earlier
fractions, suggesting that the association between the two
polypeptides, if any, is not maintained during purification. The peak
fractions were pooled, dialyzed against buffer B (10 mM
potassium phosphate, pH 7.0, 10% glycerol, 10 mM
NaHSO3, 1 mM PMSF, 10 mM
2-mercaptoethanol) (fraction VI'), and loaded onto a Macro-Prep ceramic
hydroxyapatite, type I (CHT-I) column (1 ml) (Bio-Rad) equilibrated
with buffer B. After washing the column with 3 column volumes of the
same buffer, the activity was eluted with a linear gradient of 15 column volumes of 10-500 mM potassium phosphate at a flow
rate of 0.1 ml/min and recovered in one major peak at 200 mM potassium phosphate. The activity coincided with the
presence of the 145-kDa polypeptide, namely Pol II. There was also a
very small peak at 280 mM NaCl, which co-eluted with the
145- and 34-kDa polypeptides. This peak probably represents the complex
of the two polypeptides that contaminated the previous fraction. The
specific activity of the final sample of Pol II was 58,000 units/mg
(total 1600 units), and the purity of the sample was estimated to be
approximately 90% as judged by SDS-PAGE and silver staining of the
gel. To prevent enzyme inactivation, BSA, EDTA, DTT, and Triton X-100R
were added to the final sample at final concentrations of 0.3 mg/ml, 1 mM, 5 mM, and 0.01%, respectively, and
aliquots were stored at 80 °C (fraction VII').
Amino Acid Sequence Analysis of Pol II-- The amino acid sequences of peptides generated by digesting Pol II with lysylendopeptidase were analyzed using a PSQ-10 protein sequencer (Shimadzu). Among the sequences determined, the ones most proximal to the N terminus and the C terminus of the catalytic subunit (total 2222 amino acids) were NH2-LSFVNSNQLFEARK (amino acids 191-204) and NH2-RNQLTNEEDPLVLPSEIPSMDEDYV (amino acids 1246-1270), respectively (3). As the molecular mass of the polypeptide (amino acids 191-1270) was calculated to be 126 kDa, Pol II contains most, if not all, of the N-terminal half of the catalytic subunit but lacks most of the C-terminal region.
Assay for Polymerase Activity during Purification--
The assay
measures incorporation of [-32P]dTTP into
trichloroacetic acid-insoluble material using
poly(dA)300oligo(dT)10 (20:1) as the DNA
template. The assay conditions were as described previously (24),
except the reaction was carried out in a volume of 20 µl at 30 °C
for 30 min. One unit of polymerase activity corresponds to the
incorporation of 1 nmol of dTTP per h.
Assay with Singly Primed X ssDNA--
The reaction mixture
(320 µl) contained 35 mM Bis Tris-HCl, pH 6.3, 8 mM MgCl2, 10% glycerol, 100 µg/ml BSA, 2 mM DTT, 100 µM each of dATP, dCTP, and dGTP,
50 µM [
-32P]dTTP (1000-2000 cpm/pmol),
2.7 µg of singly primed
X174 ssDNA, 58 µg of RF-A, and the
polymerase to be assayed. After preincubation of the components above,
except for the [
-32P]dTTP, at 30 °C for 5 min, the
reaction was initiated by the addition of [
-32P]dTTP.
After incubation at 30 °C for the times specified, 40-µl samples
were withdrawn, quenched by adding 40 µl of 50 mM EDTA, and divided into two halves. One was used for counting the radioactive dTTP incorporated into acid-insoluble material as described previously (24). The other was used for an analysis of the DNA products of the
reaction. The DNA was precipitated by ethanol precipitation, the
resulting pellet was dissolved in 10 µl of alkaline loading buffer
(30 mM NaOH, 30 mM EDTA, 10% w/v sucrose,
0.04% bromcresol green, 0.2% SDS), and the suspension was separated
by electrophoresis through a 1% alkaline agarose gel (15 × 13.5 × 0.3-cm gel, 40 V, 11 h). An EcoT14I digest
of
DNA was labeled at its 5' ends by T4 polynucleotide kinase and
used as a size standard. After neutralizing the gel in 7%
trichloroacetic acid, the gel was dried and visualized using a
Bio-Imaging Analyzer BAS-1500 (Fuji film).
Measurement of the Rate of Dissociation of the Pol:DNA
Complex--
DNA polymerase (Pol II* or Pol II) and
poly(dA)300oligo(dT)10 (5:1) were mixed as
indicated in the figure legends in a reaction mixture containing 35 mM Bis Tris-HCl, pH 6.3, 5 mM
MgCl2, 10% glycerol, 100 µg/ml BSA, and 2 mM
DTT and pre-incubated at 30 °C for 2 min to allow the Pol:DNA
complex to reach equilibrium. X ssDNA circle was then added to the
reaction at the concentrations specified in the figure legends. At
different time points after the chase with the
X DNA, samples were
withdrawn, and [
-32P]dTTP (20 µM) was
added to initiate DNA synthesis. The reaction was terminated after 2 min at 30 °C and the radioactivity incorporated into acid-insoluble
material was counted. When the rate of dissociation of Pol:DNA complex
was measured by a gel mobility shift assay, 5'-labeled d60:d15 was used
for complex formation. The Pol:DNA complex was formed and chased with
the
X DNA as described above. After the times indicated, samples
were removed, fixed with glutaraldehyde (0.8%) for 5 min at 30 °C,
and subjected to non-denaturing gel electrophoresis as described
below.
Gel Mobility Shift Assays-- Samples (6 µl) containing the Pol:DNA complex cross-linked with glutaraldehyde as described above were mixed with 1 µl of loading buffer (20% w/v sucrose, 1 mg/ml bromophenol blue) and subjected to non-denaturing gel electrophoresis at 4 °C in a 4% polyacrylamide gel (15 × 15 × 0.1 cm) for 3.5 h at 100 V in Tris-glycine buffer (50 mM Tris, pH 8.5, 0.38 M glycine, and 2 mM EDTA). The gel was fixed with 12% (v/v) methanol and 10% (v/v) acetic acid, washed with distilled water, dried at 70 °C, and exposed for quantification using a Bio-Imaging Analyzer BAS-1500 (Fuji film).
Other Methods-- Protein concentration was determined by Bio-Rad protein assay system based on the method of Bradford using bovine gamma globulin as a standard (32). DNA concentration was determined spectrophotometrically.
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RESULTS |
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Both Pol II* and Pol II Catalyze Highly Processive DNA Synthesis at
the Same Rate--
First, we compared the polymerase activities of Pol
II* and Pol II by measuring their processivity and rate of DNA-chain
elongation. In these assays, Pol II* or Pol II was pre-incubated with
the singly primed circular X174 ssDNA coated with RF-A in the
presence of dATP, dGTP, and dCTP, and then DNA synthesis was initiated by adding 32P-labeled dTTP. The products of elongation were
analyzed by electrophoresis on an alkaline agarose gel. During the
first 3 min, a burst in DNA synthesis was observed in both reactions
with Pol II* and Pol II (Fig.
1A). The product DNA at these
early time points ran as rather distinct bands, and the size increased
to reach the full length within the time period of the initial burst in
DNA synthesis (Fig. 1B). Because the levels of incorporation
during the burst DNA synthesis were less than 3% of the total
incorporation obtained when all the input template primers were
utilized and fully elongated, these results indicated that both Pol II*
and Pol II started elongation uniformly and that the polymerases have a
capacity to elongate DNA all the way around the viral DNA circle without dissociating from it. However, it was also evident that a
considerable proportion of the polymerase molecules paused at, or
dissociated from, the DNA template before completing the elongation of
the viral DNA. The broad distribution of the elongation products on the
gel suggested that polymerases paused at (or dissociated from) many
positions on the viral DNA, although some specific stop sites were
evident. The rates of DNA elongation by Pol II* and Pol II at 30 °C,
calculated from the maximum size of the elongation products at the
first three time points, were 30 and 36 nucleotides/s, respectively.
After 3 min in the time course, the overall rate of DNA synthesis
decreased owing to the contribution of recycling events by the
polymerases. The recycling of Pol II seemed to occur at a slightly
higher rate than that of Pol II*. DNA synthesis with low processivity,
carried out by DNA polymerase III (
) (Pol III) of S. cerevisiae in the absence of PCNA and RF-C, showed no biphasic
kinetics. Accordingly, an accumulation of short elongation products was
observed, even after 30 min when the level of incorporation was
equivalent to that of Pol II* or Pol II. From these results, we
concluded that the intrinsic capacity of Pol II* for highly processive
DNA synthesis is fully retained by the N-terminal half of the catalytic
subunit, namely Pol II. Also, the rates of DNA elongation by Pol II*
and Pol II were almost indistinguishable.
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Pol II* Dissociates from the DNA Template Much Faster than Pol
II--
In search for a specific function for the auxiliary subunits
of Pol II* and/or the C-terminal half of the catalytic subunit, we
compared the ability of Pol II* and Pol II to interact with the DNA
template and found an unexpectedly large difference in their rates of
dissociation from the template. The assay system was based on the one
reported by Maga and Hübscher (29). DNA polymerase has been
shown to have an affinity for single-stranded DNA, and this can be used
to trap the polymerase if it dissociates from the DNA template (29,
33). In our assay, the polymerase (Pol II* or Pol II) was incubated
first with the DNA template, poly(dA)300oligo(dT)10 (5:1), to allow the
Pol:DNA complex to reach equilibrium. The reaction was then chased with
circular single-stranded
X174 DNA, samples were removed at different
time points, and the level of the remaining Pol:DNA replication
complexes was quantified by measuring DNA synthesis.
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Dissociation of Pol II* from the DNA Template Is Actively Promoted
by Single-stranded X174 DNA--
DNA polymerases with high
processivity tend to have slow dissociation rates from the DNA
template. Because both Pol II* and Pol II are highly processive enzymes
and are indistinguishable from each other in this respect (Fig. 1), the
rapid dissociation of the Pol II* from the DNA template suggested that
the observed dissociation rate might not be a true reflection of the
intrinsic stability of the Pol II*:DNA complex. The equilibrium
dissociation constant (KD) of a given protein-DNA
interaction is a measure of the affinity of the protein for the DNA,
and it represents the same quantity
koff/kon. If we assume
that the rates of association (kon) do not
differ so much from one complex to another because the "on" rate is
usually diffusion limited for DNA binding proteins, the
KD value of a given complex gives us a good estimate of its rate of dissociation that is intrinsic to the complex. Therefore, we measured the equilibrium dissociation constants (KD) of Pol II*:DNA and Pol II:DNA complexes.
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Dissociation of Pol II Is Also Promoted by Single-stranded X174
DNA, Although at a Much Slower Rate--
The rate of dissociation of
the Pol II:DNA complex observed in Fig. 3 was very slow compared with
that of Pol II*. This implies that the Pol II:DNA complex is not
sensitive to the displacement promoted by
X DNA. However, the fact
that two different values for the half-life of the complex were
obtained from two different experiments (Figs. 3 and 4) suggested the
possibility that this complex is also subject to second-order
displacement.
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Deoxyoligonucleotides Also Promote the Dissociation of Both Pol II*
and Pol II from the DNA Template--
We then tested a 60-mer (d60)
synthetic deoxyoligonucleotide for its ability to displace the
polymerases from complexes with the DNA template. The assay system was
the same as described in Fig. 3, except d60 was used as a trap for the
dissociated polymerase. The concentration of d60 sufficient to trap
dissociated polymerases was determined to be 6.4 µM as
DNA molecules (380 µM as nucleotides). Under these
conditions, the half-lives of the Pol II*:DNA and Pol II:DNA complexes
were 10 and 40 s, respectively (Fig.
8), indicating that oligomer DNA can also
promote dissociation of both Pol II*:DNA and Pol II:DNA complexes.
These results, taken together with those obtained using X DNA,
strongly suggest that it is single-stranded DNA that is sensed by the
DNA polymerase when it is displaced from the DNA template. Consistent
with the previous data (Figs. 3 and 7), the semi-logarithmic
presentation of the dissociation curve for the Pol II:DNA complex
produced a straight line, whereas that of the Pol II*:DNA complex did
not.
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DISCUSSION |
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Among the DNA polymerases in S. cerevisiae, DNA
polymerase II () is unique in that it inherently exhibits highly
processive DNA synthesis. In an assay using poly(dA)oligo(dT) as a
template, it has been shown that not only Pol II* but also Pol II
possess the capacity to polymerize at least 100 nucleotides per binding event to the primer template (24). In the present study, we confirmed
the results in an assay using a singly primed
X174 DNA (5.4 kilobases) coated with RF-A. Under these conditions, both Pol II* and
Pol II were capable of elongating DNA all the way around the
X DNA
circle without dissociating from it. However, the polymerases also
showed a tendency to pause or dissociate at many sites on the template,
as pointed out previously (34). The observation that DNA polymerase II
becomes responsive in the presence of salt to the processivity factors,
PCNA and RF-C, seems to have resulted in a neglect of the importance of
the high intrinsic processivity of the polymerase (18, 35).
Nevertheless, the structural basis for this highly processive DNA
synthesis, which must reside within the N-terminal portion of the
catalytic subunit, as well as its biological importance, certainly
warrants further investigation. Our estimation for the rate of DNA
elongation was 30 to 40 nucleotides/s at 30 °C by either Pol II* or
Pol II. This value is comparable with that of Burgers (34), which was
estimated in the presence of PCNA and RF-C (at least 50 nucleotides/s
at 30 °C). The rate may actually be somewhat stimulated by PCNA and RF-C, as in the case of mammalian Pol
and PCNA (29). It was impossible to distinguish Pol II* and Pol II on the basis of their polymerase activities both in terms of processivity and rates of DNA
replication.
We obtained a clue to the functions of the C-terminal half of the
catalytic subunit and/or the auxiliary subunits by characterizing the
DNA complexes of Pol II* or Pol II. Although the intrinsic stability of
the two complexes, as indicated by their equilibrium dissociation
constants, was almost the same, the rates of complex dissociation
measured in the presence of single-stranded DNA, which served as a trap
for free DNA polymerase, were 75-fold faster for the Pol II*:DNA
complex than for the Pol II:DNA complex. The rapid dissociation of Pol
II*:DNA complex was shown to be a second-order process whose rate
depended on the concentration of single-stranded DNA. Thus, Pol II*
bound to a DNA template is displaced through its ability to sense
single-stranded DNA. We further showed that the rate of dissociation of
Pol II:DNA complex increased at higher concentrations of
single-stranded DNA, although the rates were still much slower than
those of the Pol II*:DNA complex. Therefore, Pol II can also sense
single-stranded DNA but, unlike Pol II*, is displaced from the DNA
template with low efficiency. These results suggest that there is at
least one site in the N-terminal half of the catalytic subunit, besides
the one responsible for interaction with the DNA template, that
recognizes single-stranded DNA and promotes dissociation of the Pol:DNA
complex. An additional site(s) that stimulates the single-stranded
DNA-directed displacement of the polymerase seems to be present in the
C-terminal half of the catalytic subunit and/or in the auxiliary
subunits of Pol II*. One of the candidates for this site could be the
active center for 3'5' exonuclease activity in the N-terminal half
of the catalytic subunit. The polymerase displacement seen in the
present study may occur as a result of conformational changes in the
primary DNA binding site of the polymerase provoked by an interaction between single-stranded DNA and the exonuclease domain. However, considering the fact that the active site for the exonuclease has a
greater preference for the free 3'-end of the DNA, this possibility
seems unlikely as the displacement of Pol II was promoted with equal
efficiency by circular and linear
X DNA or by oligomer DNA and hook
DNA whose 3'-ends are sequestered.
The 75-fold difference in the rates of dissociation between Pol II*:DNA
and Pol II:DNA complexes was reduced to 4-fold when 60-mer
single-stranded DNA was used instead of X DNA at equivalent amounts
in terms of nucleotide concentration. The 60-mer DNA selectively increased the dissociation rate of the Pol II:DNA complex to a level
that was comparable with that of the Pol II*:DNA complex. The
observation suggests that the small size and high molar concentration of the 60-mer DNA made it accessible to the putative sensor site for
single-stranded DNA on Pol II. The site(s) on the C-terminal half of
the catalytic subunit and/or on the auxiliary subunits may modulate the
affinity for single-stranded DNA of the site that resides in the
N-terminal half of the catalytic subunit. Identification of the sites
responsible for polymerase displacement and clarification of the
mechanism awaits further investigation. In addition, it will be
interesting to ascertain if Pol:DNA complexes show active displacement
when they are in the elongation or the idling states of DNA synthesis.
In as much as the rapid displacement of Pol II* promoted by the
single-stranded DNA requires the C-terminal domain of the catalytic
subunit and/or other subunits that are structurally unique to this
polymerase, it is suggested that the displacement process is a reaction
specific to DNA polymerase II (
). However, our trial to set up the
dissociation assays with other essential DNA polymerases of S. cerevisiae, Pol I (
) and Pol III (
), failed because of a
difficulty in detecting the DNA complexes of these polymerases owing to
their inherent instability. It would be necessary and of interest to
compare Pol II* and Pol III in this displacement process in the
presence of PCNA and RF-C.
A major restriction in characterizing the displacement process is that the single-stranded DNA in our assay plays two roles; one is that it serves as a trap for the dissociated polymerase, and the other is that it promotes displacement of the polymerase from the DNA template. A search for single-stranded DNA species that can act only as a trap but not as a promoter of polymerase displacement has so far been unsuccessful. Double-stranded DNA (blunt-ended linear double-stranded DNA of pUC119) does not act inhibitory to DNA synthesis by Pol II* and Pol II, so it cannot be used as a trap (data not shown). Because we cannot separate the two phenomena, it is difficult to test different DNA species, such as double-stranded DNA and RF-A-coated single-stranded DNA, for their ability to promote polymerase displacement. However, we observed no difference in amounts of Pol II*:DNA complex formed at given concentrations of the polymerase and the template DNA in the presence or absence of double-stranded DNA, showing that the double-stranded DNA does not affect the equilibrium state of Pol II*:DNA complex formation (data not shown). The observation suggests that the double-stranded DNA does not have an ability to promote polymerase displacement.
Maga and Hübscher (29) have made kinetic analyses of the
interaction of DNA polymerase (Pol
) from fetal calf thymus with
DNA. In their study, they determined the equilibrium dissociation constant of the complex between the Pol
and a synthetic
oligodeoxynucleotide (61-mer) hybridized to a 15-mer primer (d61:d15)
to be 6 nM. They also observed that the Pol
:DNA complex
dissociated with a half-life of 7 min at 22 °C using single-stranded
M13 DNA as a trap. The slow dissociation may be due to a lack of the
stimulatory function that we described for the second-order
displacement of the polymerase by single-stranded DNA because their
preparation of Pol
consisted only of the catalytic subunit of 145 kDa and an auxiliary subunit of 45 kDa. However, a direct comparison of
their results with ours is made difficult because of a crucial
discrepancy. They observed a higher affinity of Pol
for d61:d15
than that for d61. On the contrary, we saw no difference in the
affinity of Pol II* or Pol II for d60:d15 and d60 DNA (data not shown).
Therefore, whereas Pol
in the complex described by Maga and
Hübscher (29) is exclusively at the site of the primer, the
interaction of Pol II* (or Pol II) with the DNA template is the result
of nonspecific binding of the polymerase to the single-stranded DNA.
This discrepancy may be attributed to a difference in the properties of
Pol
from fetal calf thymus and Pol II* (Pol II) from S. cerevisiae. Titration of Pol II* or Pol II in the gel mobility
shift assay showed that one molecule of d60:d15 can be bound by at
least two molecules of either polymerase. When we analyzed the Pol:DNA
complexes, the conditions were chosen as such that the polymerase and
the DNA template formed a complex with 1:1 stoichiometry.
Because Pol II* and Pol II show no preference for binding to the primer
terminus, the competitive inhibition (Fig. 4A) of complex
formation between the polymerase and DNA templates by single-stranded
DNA is as if diluting the specific activity of the radioactive DNA
template. In the case of Pol II, complex formation, as detected by the
radioactivity in the shifted band, was almost completely inhibited when
X DNA was present at a 60-fold higher concentration in terms of
nucleotides over that of 32P-labeled d60:d15, which is
consistent with this notion. In contrast, an equimolar concentration of
X DNA was sufficient to completely inhibit complex formation between
Pol II* and the labeled d60:d15. The binding of Pol II* to the
X DNA
predominates over that to d60:d15, probably because active displacement
of the polymerase is taking place in the reaction. The Pol II*
initially bound on d60:d15 is displaced by the
X DNA. Also, the Pol
II* bound on the
X DNA stays on the DNA perhaps because
intramolecular displacement is favored. The preference for the
intramolecular displacement of Pol II* is also suggested from the time
course of DNA synthesis by Pol II* in the presence of
X DNA, as
shown in Fig. 2A. The dTTP incorporation on the template
poly(dA)oligo(dT) reached a plateau after 2 min, irrespective of
the concentration of the
X DNA added to the reaction. These results
suggest that Pol II* is unable to re-associate with poly(dA)oligo(dT)
once the polymerase is trapped by the
X DNA. This peculiar nature of
X DNA on the inhibition of DNA synthesis by Pol II* may also be
attributed to the intramolecular displacement of Pol II* on long
single-stranded DNA.
There are several cellular processes in which the sensitivity of DNA polymerase II to single-stranded DNA might play an important role. First, it may be involved in the sensing mechanism for detecting a replication fork block in the initial stage of the S-phase checkpoint pathway (12). A block in the progression of the replication fork by DNA damage or nucleotide deprivation may generate single-stranded DNA at or near the fork (36). DNA polymerase II, which has been shown to be present at the replication fork (7), may sense single-stranded DNA as a signal of aberrant DNA replication. The resulting displacement of the polymerase could be a part of the biochemical process involved in signal transmission to the downstream components in the S-phase checkpoint mechanism (37). In the SOS response, the checkpoint mechanism of Escherichia coli, the single-stranded DNA created by blocking DNA replication acts as a signal for the sensor protein, RecA, which results in activation of the protein and the induction of downstream reactions (38). The pol2 mutants carrying non-sense mutations in the C-proximal region of the catalytic subunit are defective in their response to DNA damage or replication defects, such as induction of damage-inducible genes and prevention of entry into mitosis (12). This is consistent with the fact that Pol II, lacking the C-terminal half of the catalytic subunit, does not have an ability to efficiently sense single-stranded DNA. Second, the activity may efficiently translocate the polymerase to the single-stranded DNA region where DNA synthesis is required. Single-stranded gaps generated as intermediates in the nucleotide excision repair may attract polymerases. Another possible site is the intermediate structure for DNA recombination. It has recently been reported that Holliday junctions accumulate when DNA elongation is blocked and that DNA polymerase II might be required for the formation of the recombination intermediates, probably through its capacity to stabilize such structures (39). A role for recombination in the restoration of collapsed or blocked replication forks during DNA replication has been proposed (40). Pol II may take a part in this process by efficiently translocating onto the cross-stranded structure and extending the length of the invading strand. One other possible site of action is the single-stranded regions generated on the lagging strand. If the polymerase is involved in lagging strand DNA synthesis, this activity may provide a means for efficient retargeting of the polymerase to the next primer after the completion of the synthesis of an Okazaki fragment. The activity of Pol II* described in this report should further our understanding of the biological role of DNA polymerase II in S. cerevisiae.
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports, and Culture of Japan.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.
Present address: Graduate School of Biological Sciences, Nara
Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara
630-01, Japan.
§ To whom correspondence should be addressed: Dr. Akio Sugino, Dept. of Biochemistry and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-879-8331; Fax: 81-6-877-3584; E-mail: asugino{at}biken.osaka-u.ac.jp.
The abbreviations used are:
RF-C, replication
factor C; RF-A, replication factor A; PCNA, proliferating cell nuclear
antigen; Pol II*, four-subunit complex form of DNA polymerase II ()
of S. cerevisiaePol II, degradative form of catalytic
subunit of DNA polymerase II (
) of S. cerevisiaePol
III, DNA polymerase III (
) of S. cerevisiaePol
, DNA
polymerase
ssDNA, single-stranded DNAPAGE, polyacrylamide gel
electrophoresisPMSF, phenylmethylsulfonyl fluorideBSA, bovine serum
albuminDTT, dithiothreitol.
2 Noskov, V., Araki, H., and Sugino, A. (1998) Mol. Cell. Biol. 18, in press.
3 T. Ohya, T. Ohara, S. Maki, K. Hashimoto, Y. Kawasaki, and A. Sugino, unpublished results.
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REFERENCES |
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