From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Yeast DNA polymerase (Pol
) consists of
three subunits encoded by the POL3, POL31, and
POL32 genes. Each of these genes was cloned under control
of the galactose-inducible GAL1-10 promoter and
overexpressed in various combinations. Overexpression of all three
genes resulted in a 30-fold overproduction of Pol
, which was
identical in enzymatic properties to Pol
isolated from a wild-type
yeast strain. Whereas overproduction of POL3 together with
POL32 did not lead to an identifiable Pol3p·Pol32p
complex, a chromatographically distinct and novel complex was
identified upon overproduction of POL3 and
POL31. This two-subunit complex, designated Pol
*, is
structurally and functionally analogous to mammalian Pol
. The
properties of Pol
* and Pol
were compared. A gel filtration
analysis showed that Pol
* is a heterodimer (Pol3p·Pol31p) and
Pol
a dimer of a heterotrimer,
(Pol3p·Pol31p·Pol32p)2. In the absence of proliferating
cell nuclear antigen (PCNA), Pol
* showed a processivity of 2-3 on
poly(dA)·oligo(dT) compared with 5-10 for Pol
. In the presence of
PCNA, both enzymes were fully processive on this template. DNA
replication by Pol
* on a natural DNA template was dependent on PCNA
and on replication factor C. However, Pol
*-mediated DNA synthesis
proceeded inefficiently and was characterized by frequent pause sites.
Reconstitution of Pol
was achieved upon addition of Pol32p to
Pol
*.
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INTRODUCTION |
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The subunit structure of eukaryotic DNA polymerase (Pol
)1 remains ambiguous
(for a review, see Ref. 1). The most thoroughly characterized form of
mammalian Pol
is that isolated from calf thymus, a two-subunit
enzyme with a 125-kDa catalytic subunit and a 48-kDa accessory subunit
(2). The accessory subunit is required for efficient stimulation of
Pol
by the proliferating cell nuclear antigen (PCNA) (3, 4).
Likewise, mouse Pol
can be purified in two forms, the single
catalytic subunit form which is not stimulated by PCNA and the
two-subunit enzyme which is stimulated by PCNA (5). The subunit
composition of the two-subunit enzymes is that of a heterodimer (2, 5).
In contrast, Pol
isolated from the two yeasts is more complex with
the enzyme from Saccharomyces cerevisiae having three
subunits and that from Sshizosaccharomyces pombe at least
four, and perhaps five subunits (6, 7).
The three subunits of S. cerevisiae Pol have apparent
sizes by SDS-PAGE of 125, 58, and 55 kDa and are encoded by the
POL3, POL31, and POL32 genes,
respectively (7). The 125-kDa catalytic subunit encoded by the
POL3 (CDC2) gene is very highly conserved in all
eukaryotes (1). The 58-kDa second subunit is encoded by the
POL31 (HYS2, SDP5) gene (7). Mutations in this
essential gene can cause sensitivity to the replication inhibitor
hydroxyurea, for the hys2-1 allele, or suppress the
temperature sensitivity of mutations in the catalytic subunit, for the
sdp5-1 allele (8, 9). Pol31p shows 23-28% sequence
similarity to the 48-kDa subunit of human Pol
and to S. pombe Cdc1. The essential cdc1+ gene
encodes the second subunit of S. pombe Pol
(6). The 55-kDa subunit is encoded by the POL32 gene. Mutants deleted
for POL32 are viable, but show both replication and repair
defects (7). Although the sequence similarity between Pol32p and Cdc27, which is the essential third subunit of S. pombe Pol
, is
very low, other considerations including the presence of a PCNA-binding motif in both subunits denote these two as functional homologues (discussed in Ref. 7).
In the previous paper (7) we have described the cloning of the two
small subunit genes of Pol and their characterization. Here we
describe the overproduction in yeast of the three-subunit form of
Pol
, and of a two-subunit form, called Pol
*, which is analogous
to mammalian Pol
.
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MATERIALS AND METHODS |
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Strains and Plasmids
The yeast strains used in this work are the protease-deficient
galactose-inducible strains BJ2168 (MATa,
ura3-52, trp1-289, leu2-3, 112,
prb1-1122, prc1-407, pep4-3), PY116
(MATa ura3-52 trp1- his3-11, 15 leu2-3, 112 pep4-3
prb1-1122 nuc1::LEU2) and its pol32
derivative PY117 (MATa ura3-52 trp1-
his3-11, 15 leu2-3, 112 pep4-3 prb1-1122 nuc1::LEU2
pol32
::HIS3) (7).
The overproduction plasmids used in this study are based upon the
pRS420 series plasmids into which the GAL1-10 upstream
activating sequence (GAL1-10 upstream activating sequence) including
the transcriptional start sites for the GAL1 and
GAL10 genes, as a 678-nt BamHI-EcoRI
fragment, was inserted into the corresponding plasmid polylinker sites,
resulting into vectors pRS424-GAL (TRP1), pRS425-GAL
(LEU2), and pRS426-GAL (URA3) (10). All vectors
have in addition the yeast 2 µM origin for high copy
maintenance in yeast and the Bluescript SKII+ backbone for
propagation in E. coli. The transcriptional start site of
the GAL1 gene is 60 nt upstream of the BamHI
cloning site and the transcriptional start site of the GAL10
gene is 10 nt upstream of the EcoRI cloning site. Both
promoters are of similar strength. Coordinates are with reference to
the translational start sites. pBL336 (TRP1 GAL1-POL3) has a
3.6-kb HgiAI (trimmed)-HindIII fragment
(coordinates: 45 to 3543) cloned into the BamHI
(filled)-HindIII sites of pRS424-GAL. pBL338 (LEU2
GAL1-POL31) has a 1.6-kb NcoI (filled)-ClaI
(filled) fragment (coordinates: 2 to 1567) from pBL361 cloned into the
SacII site of pRS425-GAL (7). pBL340 (URA3
GAL10-POL32) has a 1.7-kb HpaI-SalI fragment
(coordinates:
20 to 1688) from pBL384 cloned into the
EcoRI (filled)-SalI sites of pRS426-GAL (7).
Cell Growth
A single colony of a plasmid-containing strain from a selective SCGL plate was grown in an air shaker at 30 °C in 100 ml of selective SCGL medium. SCGL medium contains per liter: 1.7 g of yeast nitrogen base without amino acids and ammonium sulfate, 5 g of ammonium sulfate, 30 ml of glycerol, 20 ml of lactic acid, 1 g of glucose, 20 g of agar for solid media, 20 mg each of adenine, uracil, histidine, tryptophan, proline, arginine, and methionine, 30 mg each of isoleucine, tyrosine, and lysine, 50 mg of phenylalanine, and 100 mg each of leucine, glutamic acid, aspartic acid, valine, threonine, and serine. Uracil, tryptophan, and/or leucine were omitted when appropriate to ensure the selective maintenance of plasmids. Prior to autoclaving, the pH of the media was adjusted to 5-6 with concentrated sodium hydroxide. After 2-3 days when the OD660 had reached 0.8-1, the culture was used to inoculate 1200 ml of SCGL media. After overnight growth, when the OD660 was about 1, 1200 ml of YPGL were added. YPGL contains per liter: 10 g of yeast extract, 20 g of peptone, 30 ml of glycerol, 20 ml of lactic acid, 2 g of glucose, and 20 mg of adenine. Prior to autoclaving, the pH of the media was adjusted to 5-6 with concentrated sodium hydroxide. The culture was equally divided over two 4-liter flasks and grown at 30 °C for 3 h. Solid galactose (2% final concentration) was then added to each flask and after 4 h of continuous shaking the cells were harvested.
Enzyme Purification
All steps were carried out at 0-4 °C. The following buffers were used: buffer A: 0.1 M Tris-HCl, pH 7.8, 5% (v/v) glycerol, 175 mM ammonium sulfate, 2 mM EDTA, 1 mM EGTA, 3 mM DTT, 0.025% Nonidet P-40, 5 µM pepstatin A, 5 µM leupeptin, 2 µg/ml chymostatin, 0.5 mM p-methylphenylsulfonyl fluoride, 5 mM benzamidine,10 mM NaHSO3. Buffer B consisted of 25 mM KH2PO4, pH 7.5, 10% glycerol, 2 mM EDTA, 1 mM EGTA, 3 mM DTT, 0.01% Nonidet P-40, 5 µM pepstatin A, 5 µM leupeptin, 2 µg/ml chymostatin, 0.5 mM p-phenylsulfonyl fluoride. Buffer C was 30 mM triethanolamine-HCl, pH 7.3, 1 mM EDTA, 0.5 mM EGTA, 10% glycerol, 0.01% Nonidet P-40, 3 mM DTT, 5 µM pepstatin A, 5 µM leupeptin, 5 mM NaHSO3. Buffer D was 30 mM HEPES-NaOH, pH 7.4, 1 mM EDTA, 0.5 mM EGTA, 10% glycerol, 0.01% Nonidet P-40, 5 mM DTT, 2 µM pepstatin A, 2 µM leupeptin, 0.1% (v/v) ampholytes 3.5-9. Salt concentrations (as NaCl) are indicated by a suffix, e.g. Buffer A500 = Buffer A + 500 mM NaCl. Buffers were precooled on ice water.
Bead beating was carried out in a 350-ml chamber containing 175 ml of glass beads (0.4-0.5 mm diameter) and 80-100 g, wet weight, of cells, resuspended in an equal volume of 2 × buffer A. The chamber was cooled in ice water and the beater turned on for 45 s, followed by a cooling period of 2 min, for a total beating time of 5 min. The lysate was poured in a cold graduated cylinder and the beads were washed with 50 ml of extraction buffer. An aliquot was centrifuged at 45,000 × g for 20 min (cleared lysate, see Table I). The volume of the crude lysate was measured and 40 µl of 10% Polymin P were added per ml of lysate. After 5 min of mixing, the lysate was spun for 40 min at 13,000 rpm in a GSA rotor. Solid ammonium sulfate (0.28 g/ml) was added to the supernatant and dissolved by stirring. The precipitate was collected at 13,000 rpm for 45 min. The pellet was resuspended in 20 ml of Buffer B. After dialysis against 2 × 500 ml of buffer B for 8 h each, the dialysate was cleared by centrifugation at 18,000 rpm for 20 min.
The cleared ammonium sulfate fraction was loaded on a 20-ml phosphocellulose column, equilibrated in buffer B. The column was washed with 40 ml of B25 and eluted with B750. The protein-containing fractions were combined and dialyzed for 2 × 3 h against 150 ml each of buffer C until the conductivity of the dialysate was equal to that of C25.
The dialyzed fraction was injected onto a 8-ml MonoQ column,
equilibrated in buffer C25, washed with 10 ml of
C25, and eluted with a 120-ml linear gradient from
C25 to C500. Fractions of 2.5 ml were
collected, Pol* eluted at ~C150 and Pol
at
~C200.
Individual Mono Q fractions were diluted with 2 volumes of buffer D and
injected onto a 1-ml MonoS column, equilibrated in buffer
D50. The column was washed with 2 ml of D50 and
eluted with a 15-ml linear gradient from D50 to
D500. Pol* eluted at ~D250 and Pol
at
~D400.
Samples of 200 µl were injected onto a 20-ml Superose 6 column in 40 mM Hepes-NaOH, pH 7.5, 10% ethylene glycol, 1 mM EDTA, 0.02% Nonidet P-40, 0.2 M NaCl, 5 mM DTT, 5 mM NaHSO3, and 2 µM each of leupeptin and pepstatin A. The column was run at 0 °C at 0.2 ml/min. Fractions of 300 µl were collected and analyzed by 10% SDS-PAGE and for DNA polymerase activity.
Enzyme Assays
DNA Polymerase-- The DNA polymerase assay on activated DNA is described in the previous paper (7).
Stimulation of Pol by PCNA on Poly(dA)-Oligo(dT)--
The
10-µl reaction contained 20 mM Tris-HCl, pH 7.8, 8 mM MgAc2, 0.2 mg/ml bovine serum albumin, 1 mM dithiothreitol, 25 µM [
-32P]dTTP, 100 ng of poly(dA)-(dT)22
(40:1, nucleotide ratio, 0.4 pmol of primer termini), 0.02 pmol of
Pol
or 0.03 pmol of Pol
*, and PCNA as indicated. Incubations were
for 5 min at 37 °C. The assays were stopped with 7 µl of 95%
formamide, 20 mM EDTA and electrophoresed on a 12%
denaturing polyacrylamide gel.
Pol Holoenzyme Assay on mp18 DNA--
The standard 30-µl
reaction contained 40 mM Tris-HCl, pH 7.8, 8 mM
MgAc2, 0.2 mg/ml bovine serum albumin, 1 mM
dithiothreitol, 100 µM each of dATP, dCTP, and dGTP, and
25 µM [3H]dTTP (100 cpm/pmol dNTP), 0.5 mM ATP, 40 fmol (100 ng) of singly primed SS mp18 DNA (the
36-mer primer is complementary to nt 6330-6295), 850 ng of
Escherichia coli single stranded-binding protein, 75 mM NaCl, 100 fmol of RFC, and Pol
, Pol
*, and PCNA as
indicated in the legend to the figures. Incubations were at 37 °C
for the times indicated. The reactions were stopped and acid insoluble radioactivity was determined as described above. When an
electrophoretic analysis of the replication products was carried out,
[
-32P]dTTP replaced [3H]dTTP, and the
assays were stopped with 10 µl of 60% glycerol, 50 mM
EDTA, 1% SDS, and electrophoresed on a 1 or 1.5% alkaline-agarose gel.
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RESULTS |
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Overexpression of the Subunits of Pol--
An inducible system
for the overexpression of the Pol
genes allows normal cell growth
without possible deleterious effect on cells due to constitutive high
levels of the Pol
subunits. The strain used in this study is the
protease-deficient strain BJ2168. In this strain, the expression of
genes placed under control of the GAL1-10 upstream
activating sequence is appropriately induced by addition of galactose
to the media, but the strain grows very poorly on galactose as sole
carbon source. Satisfactory cell growth was obtained on media
containing as carbon source 3% glycerol, 2% lactate, and a
non-repressing concentration of glucose (0.1%). Galactose was added to
this media to induce expression. The three Pol
genes were cloned
under control of the bi-directional GAL1-10 upstream
activating sequence as described under "Materials and Methods."
Constitutive overproduction of Pol
is inhibitory to yeast cell
growth as a strain carrying the three overexpression plasmids grew less
well on galactose medium than on raffinose, a non-inducing carbon
source (data not shown).
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Subunit Structure of Pol and Pol
*--
Enzyme from the MonoS
step was injected on a Superose 6 column. Pol
eluted at a position
consistent with that of a 520-kDa complex (Fig.
4). This is in agreement with our initial
studies of Pol
in which we noted that the apparent size of Pol
was >300 kDa (11). In contrast, the elution position of Pol
*
indicated a size of 180 kDa for that complex (Fig. 4). The data shown
in Fig. 4 were obtained when concentrated enzyme at 0.4 mg/ml was injected onto the column. Dilution of the injected enzyme to 0.05 mg/ml
did not change the respective elution positions of Pol
and Pol
*
(data not shown).
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Enzymatic Activities of Pol and Pol
*--
The specific
activity of Pol
and Pol
* was measured on activated DNA (data not
shown). Calculated on a weight basis Pol
had a 1.3-fold higher
specific activity than Pol
*. This equates to a 3.2-fold higher
specific activity for Pol
if calculated on a molar basis, or
1.6-fold higher if both catalytic cores in the hexameric Pol
are
active.
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Pol* Poorly Replicates Natural DNA Templates--
DNA synthesis
by Pol
* or Pol
on extended SS DNA templates is virtually
completely inhibited by the presence of 75 mM NaCl in the
assay. On the other hand, these conditions are optimal for replication
by a complex of Pol
with PCNA (13). PCNA was loaded onto singly
primed single stranded-binding protein-coated SS mp18 DNA by RFC and
ATP. Addition of Pol
resulted in extremely fast and efficient DNA
synthesis (Fig. 7A). Only
marginal differences in replication efficiency were observed between
Pol
purified from a wild-type strain of yeast through a six-column
procedure and Pol
purified from the overproducing strain through a
three-column procedure. The fastest complexes complete replication of
the 7,250-nt mp18 circle within 1.5 min at 37 °C, a rate of more
than 80 nt/s. In comparison, Pol
* is a very inefficient enzyme,
replicating only ~2 kb of DNA during the 20-min assay (Fig.
7A). Replication is still dependent on PCNA as no synthesis
was observed in its absence. Pause sites with Pol
* are much more
pronounced than with Pol
, indicating that sites of secondary
structure form major replication barriers for this two-subunit enzyme
(Fig. 7).
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Reconstitution of Pol from Pol
* with Pol32p--
Pol32p
overproduced in E. coli is found in inclusion bodies. The
protein was efficiently renatured from a 6 M urea extract. Its properties indicate that Pol32p is a homodimer (7). Pol
* was
incubated with renatured E. coli expressed Pol32p, and
assayed in a holoenzyme assay on single-stranded mp18 DNA, in order to determine whether in vitro reconstitution of the
three-subunit Pol
could be performed. The result in Fig.
8A shows that reconstitution of Pol
as measured by the formation of a processive holoenzyme proceeded quite efficiently. Controls, a dialyzed urea extract from
E. coli cells or from cells overproducing Pol31p, show that reconstitution is specific for Pol32p.
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DISCUSSION |
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Overproduction Studies in Yeast--
Overproduction of Pol in
yeast was easily accomplished by cloning the genes for its three
subunits under control of the galactose-inducible GAL1-10
promoter (Table I). The enzyme isolated and purified from such an
overproduction strain did not show marked differences with the enzyme
isolated from a wild-type strain (Fig. 7). The slightly lower activity
of Pol
isolated from a non-overproducing strain could be caused by
partial inactivation of the enzyme during the laborious multistep
purification procedure. (7). The availability of overproducing plasmid
also allowed us to investigate the occurrence of partial Pol
complexes and their activities. A yeast two-hybrid analysis with Pol
subunits indicated a strong interaction between Pol3p and Pol31p and
between Pol31p and Pol32p, and also a weak but significant interaction
between Pol3p and Pol32p (7). Yet, we found no biochemical evidence for
a stable complex between Pol3p and Pol32p. When both subunits were
overproduced together and the extracts fractionated on a MonoQ column,
the same results were obtained as when only Pol3p was overproduced:
only one peak of Pol3p derived activity was identified, and this peak
corresponded to the normal elution position of Pol
(Fig. 2A).
Although these biochemical data do not exclude the possibility of a
Pol3p·Pol32p complex as suggested by the two-hybrid analysis, they do
indicate that such a complex would be as unstable as the catalytic
subunit alone. An alternative explanation of the two-hybrid results is that the Pol3p·Pol32p signal was the result of an indirect
interaction with Pol31p serving as a bridge, i.e.
Pol3p·Pol31p·Pol32p. Pol3p forms a strong complex with Pol31p
to give Pol
*, and Pol31p also forms a strong complex with Pol32p, as
do the analogous subunits in S. pombe (6, 7, 14).
Properties of Pol* and Pol
--
Most interestingly, a novel
complex, Pol
*, was isolated from the simultaneous overproduction of
Pol3p and Pol31p. The differences between Pol
* and Pol
are both
structural and enzymatic. (i) Pol
* is a heterodimer, whereas Pol
is a dimer of a heterotrimer (Figs. 4 and 5). With the knowledge that
Pol32p by itself forms a homodimer, it follows that dimerization of the
catalytic core must be the result of Pol32p dimerization (7). In fact,
addition of Pol32p to Pol
* restored the dimeric form of Pol
(Fig.
8, B and C). (ii) The processivity of Pol
alone on poly(dA)·oligo(dT) is higher than that of Pol
* (Fig. 6).
(iii) In the presence of PCNA both DNA polymerases are fully processive
on poly(dA)·oligo(dT). However, much more PCNA is required to
make processive complexes with Pol
* than with Pol
(Fig. 6). PCNA
rapidly dissociates from linear DNA and is only stabilized onto the DNA
by interaction with the polymerase (15-17). Therefore, PCNA trimers
loaded by diffusion onto poly(dA)·oligo(dT) rapidly slide off the DNA
as they fail to be anchored much less efficiently by Pol
* than by Pol
. However, those few PCNA clamps which do form a complex with Pol
*, replicate processively. (iv) Pol
* holoenzyme
(i.e. the complex of polymerase, PCNA, and RFC) is much less
efficient than Pol
holoenzyme in the replication of natural DNA
templates (Fig. 7). Whereas Pol
holoenzyme replicated SS mp18 DNA
with high processivity, replication by Pol
* holoenzyme showed
frequent pausing. Those replication defects were partially suppressed
by a large molar excess of PCNA or Pol
*, suggesting that pausing led
to frequent holoenzyme disassembly, and subsequent reassembly was
stimulated by excess PCNA and Pol
* (Fig. 7B, data not
shown). As the PCNA·Pol
* complex is processive on
poly(dA)·oligo(dT) which lacks secondary structure, it is likely that
the replication defects of Pol
* holoenzyme on SS mp18 DNA are due to
the extensive secondary structure of this template. In conclusion,
although the interactions between PCNA and the Pol3p and/or Pol31p
subunits are essential for establishing a productive PCNA-polymerase
complex, additional interactions between PCNA and Pol32p may stabilize
this complex, particularly during replication of secondary structures
in the DNA template. Alternatively, or in addition, the presence of the
third subunit itself may stabilize the holoenzyme complex.
Yeast Pol* Is Comparable to Human Pol
--
The two-subunit
yeast Pol
* is structurally and functionally analogous to mammalian
Pol
(1, 18). Like Pol
*, mammalian Pol
is purified as a
heterodimer. Depending on the PCNA levels, the synthetic rate of
Pol
* holoenzyme varies from 1.5 to 15 nt/s, much less than the rate
of the three-subunit Pol
at ~100 nt/s (Fig. 7) (19). The latter
rates are comparable to in vivo rates of fork movement in
yeast (20, 21). The range of synthetic rates of human or bovine Pol
holoenzyme in analogous replication reactions is 2-10 nt/s
(e.g. see Refs. 22 and 23). As with yeast Pol
*
holoenzyme, replication by mammalian Pol
holoenzyme is also prone to
pausing. It appears that the large difference in replication efficiency
between yeast and mammalian Pol
is largely or completely accounted
for by the presence of the third subunit in the yeast enzyme. Is this
subunit also present in mammals? As Pol32p and Cdc27, the functional
S. pombe homologue of Pol32p, show only minimal sequence
similarity, it may not be possible to clone or identify this putative
mammalian subunit based on sequence comparison considerations. The
prediction would be that human Pol
containing the third subunit
would be more processive. SV40 might be an attractive assay system for
this third subunit as the assay can be carried out in crude extracts
(24). Unfortunately, as the synthetic rate of the SV40 fork is limited
at 3 nt/s by the rate of the T antigen helicase, it may not be possible
to functionally detect the presence of the third subunit, based on rate
considerations only (22).
Is Pol a Dimer at the Fork?--
The observation that Pol
has a dimeric catalytic core immediately suggests the notion that this
enzyme is also a functional dimer at the replication fork. Replication
of both the leading and the lagging strand by Pol
is the favored
model for SV40 (25, 26). However, in yeast there are several
indications that Pol
plays a major role at the replication fork.
First, Pol
is essential for yeast cell growth and the phenotypes of
temperature-sensitive Pol
mutants indicate that the enzyme is
required for the elongation phase of DNA replication (27, 28). Second,
in vivo cross-linking studies place Pol
at or near the
fork (29). Third, the mutator phenotype of a proofreading-deficient
mutant of Pol
suggests a replication function for Pol
. In
particular, the multiplicative relationship of spontaneous mutation
rates between Pol
exonuclease-deficient mutants and mismatch repair
mutants indicates that these pathways act sequentially, i.e.
that the proofreading function of Pol
is required during DNA
replication and prior to mismatch repair (30). Furthermore, the
observed spectrum of 6-N-hydroxylaminopurine-induced mutations in strains defective for the proofreading exonuclease of
either Pol
or Pol
indicate that the respective exonuclease functions of these DNA polymerases correct the analog induced DNA
replication errors on opposite DNA strands (31). By extension, if we
assume that the DNA synthetic and proofreading functions of a DNA
polymerase are tightly coupled, these latter data indicate that Pol
and Pol
replicate opposite strands of the fork (32). Taken in total,
the various data pointing to the respective replication functions of
these two DNA polymerases remain inconclusive and perhaps even
contradictory.
Function of Pol32p--
The Pol32p subunit of Pol has at least
three functional domains, a basic structural domain for interaction
with Pol31p, an organizational domain for homodimer formation thereby
promoting dimerization of the catalytic core, and a domain which
interacts with PCNA (7). None of these functions is essential for yeast cell growth. Recently, we described the properties of a PCNA mutant, pcna-79, with mutations in conserved residues in the interdomain connector loop (I126A, L128A) (33). The mutant PCNA fails to interact
with the Pol32p subunit and the in vitro replication properties of a pcna-79 containing Pol
holoenzyme are quite similar to those of the Pol
* holoenzyme described here, suggesting that the
observed in vitro phenotype of Pol
* holoenzyme may be due to the loss of a PCNA interaction site. However, mutant yeast cells
containing the pol30-79 mutation differ in phenotype from mutants deleted for the POL32 gene. Whereas both mutants are
sensitive to hydroxyurea indicative of replication defects,
pol32
mutants are cold-sensitive for growth but
pol30-79 mutants are not. Furthermore, pol30-79
is a mutator and pol32
an antimutator with a defect in
damage-induced mutagenesis (7, 33). Loss of Pol32p function shows a
much more severe growth defect than loss of the PCNA-Pol32p interaction. Possibly, the loss of dimerization function of Pol32p rather than PCNA interaction leads to the observed conditional lethal
phenotype in pol32
mutants. If the function of Pol
as a dimeric enzyme is important for yeast replication, it appears that
other factors contribute to the stabilization of a dimeric replisome,
and, therefore, loss of Pol32p-Pol32p interactions can be tolerated. A
comprehensive mutational analysis of the POL32 gene is
required to address these questions appropriately.
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ACKNOWLEDGEMENTS |
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We thank John Majors, Tim Lohman, and members of the Burgers laboratory for helpful discussions during the course of this work.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM32431 (to P. B.).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.
To whom correspondence should be addressed. Tel.: 314-362-3872;
Fax: 314-362-7183; E-mail: burgers{at}biochem.wustl.edu.
1
The abbreviations used are: Pol, DNA
polymerase
; Pol
*, Pol
lacking Pol32p; SS, single-stranded;
PCNA, proliferating cell nuclear antigen; RFC, DNA replication factor
C; DTT, dithiothreitol; BuPhdGTP,
N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate;
PAGE, polyacrylamide gel electrophoresis; HPLC, high performance
liquid chromatography; nt, nucleotide; kb, kilobase(s).
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REFERENCES |
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