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
) has three
subunits of 125, 58, and 55 kDa. The gene for the 125-kDa catalytic
subunit (POL3) has been known for several years. Here we
describe the cloning of the genes for the 58- and 55-kDa subunits using
peptide sequence analysis and searching of the yeast genome data base.
The 58-kDa subunit, encoded by the POL31 gene, shows
23-28% sequence similarity to the 48-kDa subunit of human Pol
and
to S. pombe Cdc1. POL31 is allelic to
HYS2 and SDP5. The 55-kDa subunit is encoded by the POL32 gene (ORF YJR043c in the yeast data base). Very
limited sequence similarity was observed between Pol32p and
Schizosaccharomyces pombe Cdc27, the functionally analogous
subunit in S. pombe Pol
. The POL32 gene is
not essential, but a deletion mutant shows cold sensitivity for growth
and is sensitive to hydroxyurea and DNA damaging agents. In addition,
lethality was observed when the POL32 deletion mutation was
combined with conditional mutations in either the POL3 or
POL31 gene. Pol32
strains are weak
antimutators and are defective for damage-induced mutagenesis. The
POL32 gene product binds proliferating cell nuclear
antigen. A gel filtration analysis showed that Pol32p is a dimer in
solution. When POL31 and POL32 were
co-expressed in Escherichia coli, a tetrameric (Pol31p·Pol32p)2 species was detected by gel filtration,
indicating that the two subunits form a complex.
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INTRODUCTION |
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DNA polymerase (Pol
)1 is the major
replicative DNA polymerase in the eukaryotic cell. This insight is
based on extensive in vitro studies using the simian 40 virus as a model system, and on genetic and biochemical studies in the
yeast Saccharomyces cerevisiae and Schizosaccharomyces
pombe (reviewed in Refs. 1 and 2). These genetic studies have not
only shown a role for Pol
in bulk DNA replication but also for
maintaining genome fidelity via the proofreading exonuclease activity
of this enzyme (3, 4). A similar, but perhaps less defined role has
also been identified for DNA polymerase
, whereas the synthetic
function of DNA polymerase
-primase appears to be limited to that of
initiator RNA-DNA synthesis for priming Okazaki fragments on the
lagging strand of the DNA replication fork (5-7).
The best characterized Pol from mammalian cells is the enzyme
purified from fetal calf thymus tissue, a heterodimer with a catalytic
subunit of 125 kDa and a second subunit of 48 kDa (8). The small
subunit is required for efficient stimulation of the polymerase
processivity by the proliferating cell nuclear antigen (PCNA) (9,
10).
The forms of Pol isolated from bakers' and fission yeast are more
complex. Our previous studies of S. cerevisiae Pol
indicated that the enzyme might consist of three or more subunits (11). Very recently, Pol
has been isolated from S. pombe as an
enzyme with five distinct subunits, four of which also appear to be
subunits based on genetic arguments (12).
In this paper we describe an improved purification of S. cerevisiae Pol as a three-subunit enzyme, the cloning of the
two small subunits of Pol
, and a genetic and biochemical analysis of
those subunits. Surprisingly, deletion of the gene for the smallest
subunit, POl32, was not a lethal mutation. Nevertheless, it
resulted in a phenotype consistent with its involvement in DNA
replication. We also show that this subunit interacts with PCNA. In the
accompanying article (55), the properties of a two-subunit form of
Pol
, which is analogous to mammalian Pol
, is compared with the
three-subunit form.
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MATERIALS AND METHODS |
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Strains--
The Escherichia coli strains used were
DH5, BL21 (DE3), and ABLE-C, lac(lacZ
)[KanR McrA- McrCB- McrF-
Mrr- HsdR(rK- mK-)][F' proAB lacIqZDM15 Tn10 (TetR)]. Some of the
plasmids containing the POL32 gene were toxic to E. coli. They were successfully propagated in ABLE-C cells
(Stratagene, La Jolla, CA) which maintained these plasmids at a reduced
copy number. The yeast strains are listed in Table
I. Most strains were constructed using
standard genetic methods. To obtain a "START to STOP"
POL32 deletion strain, the HIS3 gene was PCR
amplified with two-hybrid POL32-HIS3 primers as described
(13). The PCR product was used to transform diploid strain YM4590 to
His+. Correct integration of HIS3 in the
POL32 locus was confirmed by PCR with appropriate primers
and by Southern analysis. The resulting diploid was sporulated to give
PY74a. To obtain additional POL32 deletion mutants, genomic
DNA from PY74 was PCR amplified with two primers which were located
about 330 nucleotides upstream and 250 nucleotides downstream of the
HIS3 integration site, respectively, and the appropriate
his3 strains, e.g. PY116, transformed with the
PCR product to His+, PY117 (Table I). Proper integration
and loss of the POL32 gene were confirmed by PCR and by
Southern analysis.
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Media-- Standard media were used as described (14). YPDA medium is yeast extract/peptone/dextrose with 20 µg/ml adenine. Hydroxyurea (75 mM final concentration) was added to YPDA after autoclaving.
Plasmids--
With the exception of pBL385, all plasmids in this
study are derived from standard ColE1 based AmpR vectors.
Gene coordinates are based on those given in the
Saccharomyces genome data base
(http://genome-www.stanford.edu/Saccharomyces/) with the first
nucleotide of the translation initiation codon defined as +1. In the
genome data base the POL31 (HYS2,SDP5)
gene is YJR006w and the POL32 gene is YJR043c. Plasmid
pBL331 contains a genomic XbaI-SalI
POL31 fragment (coordinates 139 to +2044) cloned into the
SpeI-SalI sites of pRS314 (Bluescript ARS
CEN TRP1) (15). The POL31 gene was PCR amplified with
an amino-terminal primer which introduces an NcoI site at
the initiation codon position and a carboxyl-terminal primer which
extends beyond the genomic PvuII site (coordinate +1559).
The NcoI-PvuII fragment was cloned into the
NcoI-HindIII (filled) sites of pPY55, thereby
putting the POL31 gene under control of the phage T7
promoter (16). This is plasmid pBL361. The accuracy of the amplified
DNA was confirmed by DNA sequence analysis. pBL384 contains a
2.2-kilobase yeast genomic SpeI-PstI fragment
(coordinates
542 to +1656) cloned into the
SpeI-PstI sites of pRS314 (Bluescript ARS
CEN TRP1). pBL389 was derived from pBL384 by recloning
POL32 into pRS316 (Bluescript ARS CEN URA3) using
appropriate polylinker restriction sites. The POL32 gene in
pBL384 was PCR amplified with a primer which introduces a
BclI site at nucleotides 3-8 and a carboxyl-terminal primer
which extends beyond the HincII site (nucleotides +1113). The BclI (filled)-HincII fragment was cloned into
the NcoI (filled)-SalI (filled) sites of pMON5839
(pACYCori KanR) putting the gene under control of the
Ptac-G10L promoter-leader. This is plasmid pBL385.
Purification of Pol--
The purification is largely based on
the one published previously (11). All purification steps were carried
out at 0-4 °C. Unless otherwise indicated, all buffers contained
10% glycerol, 1 mM EDTA, 0.2 mM EGTA, 3 mM dithiothreitol, 0.02% Nonidet P-40, 10 mM
NaHSO3, 2 µM pepstatin A, and 4 µM leupeptin. The NaCl concentration in millimolar in the
buffer is indicated with a subscript. Strain BJ405 was grown, broken
open, and fractionated with ammonium sulfate as described before
(11).
Peptide Sequence Analysis--
Pol MonoQ fraction (30 µg)
was concentrated by acetone precipitation and separated by 10%
SDS-PAGE. The gel was stained with Coomassie Brilliant Blue,
appropriate gel slices were excised and treated with lysyl
endopeptidase as described (17). The peptides were recovered from the
gel slices by centrifugation through a nylon mesh (18). They were
separated by reverse phase HPLC and sequenced by the protein chemistry
facility at Washington University.
Overexpression of POL31 and POL32 in E. coli--
Strain BL21
(DE3) contained pBL361 (POL31), pBL385 (POL32),
or both. A single colony was grown overnight in 10 ml of LB medium with
100 µg/ml ampicillin, 50 µg/ml kanamycin, or both, and inoculated into 1 liter of the same medium at 37 °C. When the OD595
reached 0.6, isopropyl -D-thiogalactopyranoside was
added to the culture to a final concentration of 1 mM, and
the culture shaken for another 3 h at 37 °C. The culture was
then harvested and the cells suspended in 5 ml of 50 mM
Tris-HCl, pH 8.1, 10% sucrose, and an equal volume of 2 × lysis
buffer was added (lysis buffer is 50 M Tris-Cl, pH 8.1, 2 mM EDTA, 0.2 mM EGTA, 2 µM
leupeptin, 2 µM pepstatin A, 5 mM sodium
bisulfite, and 3 mM dithiothreitol). All further steps were
carried out at 0-4 °C. Lysozyme was added to 0.6 mg/ml and the
mixture was stored on ice for 30 min with occasional mixing. Nonidet
P-40 and phenylmethylsulfonyl fluoride were then added to 0.05% and 1 mM, respectively, and, after another 10 min on ice, the
mixture was sonicated to reduce the viscosity. After a spin at
27,000 × g for 20 min, the supernatant was discarded and the precipitate was washed with 10 ml of wash buffer (lysis buffer,
except that the pH of the Tris-HCl was reduced to 7.5 and NaCl was
added to 2 M final). After repeating the wash procedure, the precipitate was homogenized with 10 ml of denaturation buffer (50 mM Tris-HCl, pH 7.5, 6 M urea, 2 mM
EDTA, 0.2 mM EGTA, 10 mM sodium bisulfite, 2 µM leupeptin, 2 µM pepstatin A, 0.5 mM phenylmethylsulfonyl fluoride). The suspension was
shaken gently for 1 h and spun for 30 min at 27,000 × g. The supernatant was diluted to a protein concentration of
1 mg/ml with denaturation buffer and dialyzed for 2 × 3 h
against 2 × 200 ml of dialysis buffer (40 mM Hepes pH
7.4, 20% glycerol, 1 mM EDTA, 0.1 mM EGTA, 3 mM dithiothreitol, 2 µM pepstatin A, 10 mM NaHSO3, 2 µM leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, and 200 mM
ammonium sulfate). The solution was cleared by centrifugation and
stored at
70 °C. Protein concentrations were determined according
to Bradford (19).
Gel Filtration Analysis-- 200-µl samples were injected onto a 20-ml Superose 12 column in 40 mM Hepes-NaOH, pH 7.5, 10% ethylene glycol, 1 mM EDTA, 0.02% Nonidet P-40, 0.2 M NaCl, 1 mM dithiothreitol, 5 mM NaHSO3, and 2 µM each of leupeptin and pepstatin A. The column was run at 25 °C at 0.4 ml/min. Fractions of 300 µl were collected and analyzed by 10% SDS-PAGE.
Protein Interaction Blots--
Protein-protein interaction blots
were carried out with a PCNA derivative containing an amino-terminal
phosphorylatable tag, Ph-PCNA, MRRASVGS-PCNA. Ph-PCNA was overproduced
in E. coli from plasmid pMM83 (a gift of Michael McAlear,
Wesleyan University) and purified as wild-type PCNA (20). The
replication properties of Ph-PCNA were indistinguishable from wild-type
(data not shown). Phosphorylation of 1 µg of Ph-PCNA was carried out
in a 50-µl reaction containing 20 mM Hepes-NaOH, pH 7.0, 12 mM MgCl2, 1 mM dithiothreitol,
100 mM NaCl, 50 µCi of [-32P]ATP, and 3 units of bovine heart cAMP-dependent protein kinase (catalytic subunit, Sigma) at 30 °C for 30 min. The labeled protein was passed through a Sephadex G-50 column, equilibrated in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, to remove unincorporated radioactivity.
DNA Polymerase Assay-- The 50-µl reaction contained 20 mM Tris-HCl, pH 7.8, 8 mM MgAc2, 0.2 mg/ml bovine serum albumin, 4% glycerol, 1 mM dithiothreitol, 80 µM each dATP, dGTP, and dCTP, 20 µM [3H]dTTP (400 cpm/pmol), 200 µg/ml activated salmon sperm DNA, 1 mM spermidine, and enzyme. Assays were assembled on ice and incubated at 37 °C for 30 min. They were stopped by addition of 100 µl of 25 mM EDTA, 25 mM sodium pyrophosphate, and 50 µg/ml salmon sperm DNA, followed by 1 ml of 10% trichloroacetic acid. After 10 min on ice, the mixture was filtered over a GF/C filter. The filter was washed with 2 × 2 ml of 1 M HCl, 0.05 M sodium pyrophosphate, rinsed with ethanol, dried, and counted in a counting fluid in a liquid scintillation counter. One unit of enzyme incorporates 1 pmol/min of nucleotide into acid-insoluble radioactivity. When inhibition by BuPhdGTP was measured, the concentration of dGTP was lowered to 10 µM.
Measurements of Spontaneous Mutation Rates and Damage-induced Mutagenesis-- To measure forward mutation rates to canavanine resistance, PY82 or PY83 cells were grown to saturation in YPDA broth, diluted to approximately 100 cells/ml in 20 separate cultures for each strain, and again grown to saturation in YPD. Cells were then plated on complete synthetic media without arginine and with 80 µg/ml canavanine (14). Colonies appearing after 4 days of growth at 30 °C were counted. Reversion or suppression of the lys2-1 ochre mutation was measured similarly in strains PY71 and PY125. Plating was on synthetic complete media lacking lysine.
20-ml cultures of PY82 and PY83 each were grown to saturation at 30 °C. The cells were collected by centrifugation, washed with water, resuspended to 5 × 107 cells/ml in 50 mM KH2PO4, pH 7.2, briefly sonicated to disperse clumped cells, and treated with 0.75% MMS at room temperature. Aliquots were quenched in an equal volume of 10% cold Na2S2O3. The cultures were either further diluted and plated on complete synthetic medium lacking arginine to determine survival, or concentrated 4-fold and plated on complete synthetic medium without arginine and with 80 µg/ml canavanine to determine mutation frequencies. Several canavanine plates were used to obtain mutation frequency data points when survival was low. To obtain UV-survival curves and UV-induced mutagenesis frequencies, the sonicated cells in phosphate buffer were plated on the two media and immediately irradiated with the indicated doses of UV light. Colonies appearing after 4 days of growth at 30 °C were counted. ![]() |
RESULTS |
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Purification of Pol--
Our attempts to obtain acceptable
quantities of Pol
based on the original purification scheme were
hampered by several problems relating to enzyme instability, enzyme
loss, and proteolysis (11). In particular, use of a mildly hydrophobic
HPLC column (propyl silica gel) resulted in large losses in yield and
activity. Yet this matrix appeared very desirable as it greatly
separated Pol
from bulk protein. We found that inclusion of
broad-range ampholytes (3.5-9) in the buffers substantially increased
both yield and activity of Pol
, in particular when silica gel
columns were used. Therefore, ampholytes were included in all columns
starting at the propyl silica gel step. The purification is summarized
in Table II.
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Isolation of the POL31 Gene--
Amino acid sequence analysis
resulted in only one short peptide sequence for the 58-kDa subunit
(Fig. 2). A BLAST search of the complete
yeast data base revealed only one perfect match to this sequence, but
there were several other genes with only one mismatch. The perfect
match was with HYS2. This gene was initially isolated in a
screen for yeast mutants which confer hydroxyurea-sensitive cell growth
(24). Hydroxyurea sensitivity can result from defects in nucleotide
metabolism, DNA replication, DNA repair, or cell-cycle checkpoints.
HYS2 is an essential gene and the hys2-1 mutant
isolated by Sugimoto et al. (24) showed a
temperature-sensitive growth defect with a terminal phenotype
consistent with a role for the HYS2 gene product in DNA
replication. Recently, the same gene has been identified as an
extragenic suppressor, sdp5-1, of the temperature-sensitive
growth of a POL3 mutant (25). The HYS2 (SDP5) gene shows strong sequence similarity with the small
subunit gene of human Pol and the second largest subunit of S. pombe Pol
(Fig. 2). This strongly indicates that the
HYS2(SDP5) gene encodes one of the small subunits
of Pol
. We have designated this gene as POL31 to indicate
that it is the second subunit of Pol
. Positive confirmation that
POL31 encodes the 58-kDa subunit of Pol
came from two
additional experiments: (i) expression of POL31 in E. coli yielded a polypeptide with the exact same electrophoretic mobility as the 58-kDa subunit (data not shown). (ii) Pol31p was overproduced in E. coli and the protein bound to
nitrocellulose. The filter was incubated with a rabbit serum raised
against the entire Pol
complex. The bound antibodies were eluted
from the filter with an alkaline buffer and used in a Western analysis of yeast Pol
; they recognized the 58-kDa subunit specifically (Fig.
3).
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Isolation of the POL32 Gene--
The amino acid sequence analysis
of the 55-kDa subunit yielded four short sequences (Fig. 2). A BLAST
search of the yeast genome data base yielded the YJR043c open reading
frame as the only possible candidate gene. Three of the four peptide
sequences did not exactly correspond to the amino acid sequence
predicted by the YJR043c gene in the genome data base. Therefore, the
cloned gene was sequenced again. The sequence was identical to that
present in the data base. We conclude that the amino acid changes, 4 out of a total of 27 informative positions, originate from peptide sequence misassignments (Fig. 2). We have designated this gene as
POL32 to indicate that it is the third subunit of Pol.
Like many other genes involved in DNA replication, the POL32
gene has a putative MluI cell cycle box located 141 nucleotides upstream of the translational start site. The
MluI cell cycle box confers periodic expression in the cell
cycle with an increased expression at the G1/S phase
(26).
POL32 Deletion Mutants Are Viable and Cold-sensitive for
Growth--
The entire POL32 gene was deleted in diploid
strain YM4590 while tagging the deletion with the HIS3
auxotrophic marker as described under "Materials and Methods."
Sporulation of the heterozygous diploid, i.e.
POL32/pol32::HIS3, and tetrad analysis showed a 2:2 segregation for His+, indicating that the
POL32 gene is not essential for yeast cell growth. Southern
analysis confirmed the deletion of the POL32 gene in the
His+ spores (data not shown).
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The pol32 Mutation Exhibits Synthetic Lethality with Mutations
in Other Pol
Genes--
Our studies strongly suggest that Pol32p is
part of the Pol
complex. In order to determine further genetic
interactions between POL32 and other components of Pol
holoenzyme, we attempted to isolate double mutants by strain crossing.
In S. cerevisiae, the catalytic subunit of Pol
is encoded
by the CDC2 gene, which is now commonly referred to as
POL3 (30, 31). The cdc2-1 mutation confers
temperature-sensitive growth upon yeast (32). When crosses were carried
out between a cdc2-1 strain and a pol32
strain, no progeny were obtained which were cdc2-1 pol32
,
suggesting synthetic lethality between the two mutations (data not
shown). To confirm synthetic lethality, the cdc2-1 pol32
double mutant was made in the presence of a complementing centromere
plasmid carrying the wild-type POL32 gene and the
URA3 gene as a selectable marker. The strain was grown on
non-selective media for 10 generations, and plasmid loss was determined
in the double mutant by plating on 5-fluoroorotic acid-containing media
which allows growth of those cells which have lost the plasmid (Table
III). No growth was observed indicating
that loss of the POL32 complementing plasmid was not
tolerated and that, therefore, a cdc2-1 pol32
double mutation is lethal. In comparison, the single mutant control strains showed 55-80% loss of the POL32 plasmid. Loss of the
complementing plasmid with the URA3 marker in the double
mutant was also tolerated if the strain contained an additional
POL32 plasmid, e.g. pBL384 (POL32
TRP1) (Table III, entry 2). Similarly, a double mutant carrying the temperature-sensitive hys2-1 allele of POL31
together with pol32
failed to lose a complementing
centromere plasmid carrying the wild-type POL32 gene and the
URA3 maker, indicating synthetic lethality between
pol31 and pol32 mutations. Again, the single mutant control strain KSH542-1 readily lost the POL32
plasmid (Table III).
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Pol32 Mutants Are Damage-sensitive and Deficient for Induced
Mutagenesis--
Pol
participates in various forms of DNA repair
including repair of methylation damage and UV damage (25, 34-36). The
pol32
strain was more sensitive to UV irradiation than
wild-type. Also, exposure to MMS had a profound effect on cell
viability (Fig. 5).
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Defective Pol from a pol32
Strain--
Lack of the 55-kDa
subunit of Pol
has serious consequences in vivo. To
assess its effect in vitro, the POL32 deletion
was introduced in a protease-deficient strain used in our laboratory for biochemical studies. The protease-deficient strain and its pol32
derivative were grown up in gram quantities,
extracts were made and fractionated by phosphocellulose chromatography
and MonoQ HPLC. Fractionation of extracts from wild-type cells
separated, in order of elution, Pol
from Pol
and Pol
(Fig.
6A). In contrast, fractionation of extracts from the isogenic pol32
strain
lacked Pol
, but showed a novel peak of polymerase activity eluting
prior to that Pol
. We have designated this new activity Pol
*. A
Western analysis showed that the Pol
* peak from pol32
extracts contained both the catalytic subunit and the 58-kDa subunit of
Pol
, but not the 55-kDa subunit, whereas the Pol
peak from
wild-type extracts contained all three subunits (Fig.
6B).
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Interactions between Pol Subunits and PCNA--
A comprehensive
two-hybrid analysis was carried out to determine protein-protein
interactions between the three subunits of Pol
and of PCNA with each
of these three subunits (51). The POL3 gene was fused to the
acidic activation domain of GAL4, and the POL31,
POL32, and POL30 (PCNA) genes to both the
GAL4 activation domain and the bacterial lexA DNA-binding
domain. Pairwise combinations of binding domain and activation domain
plasmids were tested for interaction by measuring
-galactosidase
activity in strain L40 which places this marker gene under
transcriptional control of the lex operator (52). The results are given
in Table IV. Strong interaction signals
were obtained for Pol3p-Pol31p, Pol3p-Pol32p, and Pol31p-Pol32p
combinations. A weak, but statistically significant interaction signal
was obtained for Pol32p-Pol32p, indicating that this subunit may form a
homodimer, whereas the Pol31p-Pol31p combination yielded background
levels of
-galactosidase activity. When interactions with PCNA were
measured, all interaction signals were low, like observed previously
with other two-hybrid experiments involving PCNA (53, 54). For
instance, even though PCNA is known to form a stable trimer, only 1.9 units of
-galactosidase activity was measured for the PCNA-PCNA pair
(Table IV). In comparison, the interaction signal between PCNA and
Pol32p was high, especially in the orientation with POL32
fused to the LexA DNA-binding domain and POL30 to the
GAL4 activation domain. The analogous orientation for the
Pol31p-PCNA pair gave a very low but statistically significant signal,
whereas background levels of
-galactosidase activity were measured
for the pair in the opposite orientation. In conclusion, this analysis
supports a model in which Pol3p interacts with Pol31p and with Pol32p,
Pol31p interacts with Pol32p, Pol32p interacts with itself,
i.e. forms a dimer, and PCNA interacts with Pol32p, and
perhaps also with Pol31p. However, because the two-hybrid method may
detect both direct and indirect interactions, we turned to biochemical
methods to investigate a possible interaction between these
polypeptides.
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Pol31p and Pol32p Form a Heterotetramer--
In order to test
whether the interactions between the small subunits suggested from the
two-hybrid analysis could be reproduced in vitro, the small
subunits were overproduced in E .coli. Both Pol31p and
Pol32p when overproduced in E. coli were found in inclusion bodies. This was also observed when Pol31p and Pol32p were overproduced simultaneously (data not shown). The three different precipitates were
dissolved in a buffer containing 6 M urea and the urea was removed by stepwise dialysis. The preparations were analyzed by gel
filtration. Analysis of the Pol31p preparation was uninformative. The
subunit was largely aggregated and the bulk of the protein eluted in
the void volume, while the rest streaked across the entire elution
profile (Fig. 7B). In
contrast, very little Pol32p was aggregated and the majority eluted as
a single peak with an apparent Mr of 105,000, consistent with a dimeric form for this subunit (Fig. 7A).
When the preparation resulting from the simultaneous overexpression of
POL31 and POL32 was analyzed, most of Pol31p was
still aggregated and most of Pol32p eluted as a dimer. However, a peak
of Pol31p and a shoulder of Pol32p was observed at fractions 31 and 32, corresponding to a molecular weight of 230,000 (Fig. 7C).
Quantitation of the bands in the Coomassie-stained gels showed an
approximate 1:1 ratio of Pol31p and Pol32p in these fractions (Fig.
7E). To confirm that fractions 31 and 32 contained a
distinct complex, these fractions were combined and reinjected onto
the column. Coelution of the bulk of both subunits at fraction 31 and
32 confirmed the existence of a complex with an apparent molecular weight of 230,000 (Fig. 7D). However, because the subunits
were visualized by silver staining, quantitation of the data could not
be carried out. This analysis shows that Pol31p and Pol32p form a
complex in the absence of the catalytic subunit of Pol and indicate
that this complex may be that of a dimer of a
heterodimer:(Pol31p·Pol32p)2.
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Pol32p Interacts with PCNA--
The subunits of Pol were
separated by SDS-PAGE, transferred to nitrocellulose, and probed with a
32P-labeled form of PCNA. Under the experimental conditions
used, binding of PCNA was only observed to the Pol32p subunit, but not to Pol3p nor to Pol31p (Fig. 8). These
data largely agree with the two-hybrid results (Table IV). Extracts
from E. coli cells overexpressing POL32, but not
extracts from cells overexpressing POL31, showed binding of
32P-labeled PCNA confirming that the binding was specific
for this subunit. The signal was abolished by inclusion of an excess of cold PCNA (Fig. 8) or Pol32p (data not shown) in the blotting solution.
Regardless of the different experimental conditions used in this assay
(renaturation and probing were carried out at pH 6.8; 0.05% Triton
X-100 was included during renaturation and probing; the guanidinium
hydrochloride renaturation was omitted; probing was carried out in
different buffers, e.g. with EDTA instead of Mg or in 30 mM NaCl instead of 150 mM NaCl) no interaction signal between PCNA and Pol31p could be detected (data not shown).
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DISCUSSION |
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Subunit Structure of Pol--
The enormous advantage of the
availability of the Saccharomyces genome data base is
exemplified in the project described in this paper. Peptide sequences
derived from the two small subunits of Pol
were obtained in
1992-1993. However, these were too small for any attempts at gene
isolation. Periodic BLAST searches of the growing yeast sequence data
base gave positive hits in 1995. Further biochemical studies allowed an
unambiguous assignment of the genes for the 58-kDa as YRJ006w and the
55-kDa subunit as YJR043c, respectively. These genes have been renamed
POL31 and POL32, respectively, to indicate that
they are subunits of Pol
.
Repair Defects in Pol--
A large number of mutations have
been isolated in the POL3 gene, three mutations in the
POL31 gene and the single deletion mutation of the
POL32 gene described here (24, 25, 36, 40-42). All
conditional mutants tested are sensitive to hydroxyurea, indicating defects in DNA replication. Similarly, sensitivity to alkylating agents
is greatly enhanced in mutants containing mutations in one of the three
Pol
genes (e.g. see Fig. 6). However, Pol
mutations appear to fall in two classes with regard to the effect of UV irradiation. Most POL3 mutants are not sensitive to UV.
These include the cdc2-1, cdc2-2,
hpr6-1, mut7-1, pol3-01, and pol3-14 alleles of POL3 (25, 36, 40-42). When mapped, the mutations fall in the exonuclease or polymerase domains of this subunit. A
POL31 mutant (hys2-1) is also insensitive to UV
irradiation (24). Only two POL3 mutants, pol3-11
and pol3-13, with mutations in the carboxyl-terminal
cysteine-rich domain of this subunit are sensitive to UV irradiation
(25). The pol32 deletion mutant is also UV-sensitive. These
UV-sensitive mutants are also deficient for damage-induced mutagenesis
(Fig. 6) (25). In addition, pol32
strains are weak
antimutators, which may indicate that their primary repair defect is in
error-prone repair. The Rev3p DNA polymerase is required for
error-prone DNA repair. A rev3 deletion mutant is deficient
for error-prone repair and is a weak antimutator (43). The experiments
described here and by Giot et al. (25) show that Pol
also
functions in the mutagenic repair of UV lesions, presumably in
cooperation with Rev3p. Recent genetic studies have also implicated
Rev3p and Pol
in the repair of MMS-induced lesions, perhaps via a
mutagenic pathway as well (35).
Biochemical Interactions between Pol32p and PCNA-- Both Pol32p and Cdc27 show a box of homology (338QGTLESFF345 for Pol32p) which has previously been proposed to bind PCNA (Fig. 2). This sequence is also present in FEN-1 and in mammalian p21 (28, 29). X-ray structure analysis of the complex between human PCNA and a p21 peptide shows that the amino acids in the PCNA-binding box make contacts with the interdomain connector loop region of PCNA (44). Particularly, Met147, Phe150, and Tyr151 in p21 make contacts within and near a hydrophobic pocket in PCNA formed mostly by the interdomain connector loop of PCNA. The amino acids in Pol32p analogous to p21 which are predicted to be essential to PCNA binding are Leu341, Phe344, and Phe345 (Fig. 2). The hydrophobic pocket on PCNA includes Leu126 and Ile128, which are universally conserved amino acids in this otherwise poorly conserved loop (21). In a recent mutational study of PCNA we showed that a mutant PCNA, pcna-79, in which Leu126 and Ile128 were changed to alanine, fails to interact with Pol32p (21). These data support the proposal that proteins containing this consensus PCNA-binding domain target their interaction to the interdomain connector loop. Despite these clear similarities in protein interaction domains between yeast and human cells, considerable divergence of these interaction domains has still taken place as exemplified by the very weak interaction between human p21 and S. cerevisiae PCNA (45).
Subunit Structure of Pol--
Subunit interaction studies of
S. pombe Pol
have revealed interactions between the
catalytic subunit and the second subunit, Pol3-Cdc1, and between the
second and third subunit, Cdc1-Cdc27 (38). Our two-hybrid analysis
confirms these interactions in S. cerevisiae Pol
and
extends it to indicate possible interactions between the catalytic and
the third subunit, Pol3p-Pol32p (Table IV). Interestingly, the
two-hybrid analysis indicates that Pol32p, but not Pol31p, may form a
homodimer. Size fractionation studies with subunits overproduced in
E. coli confirmed the suggestion from the two-hybrid data
that Pol32p is a homodimer and, moreover, that Pol31p and Pol32p
interact to form a tetramer (Fig. 7). Because Pol31p overproduced in
E. coli did not renature efficiently, we could not determine
whether this subunit is a monomer or a dimer. However, the human p48
subunit expressed in E. coli as a soluble protein is a
monomer (10). If the dimeric state of Pol32p persists in larger
complexes containing the catalytic subunit of Pol
, the interesting
possibility is presented that Pol
may consist of two catalytic
cores. These studies will be reported in the second paper.
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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, Xavier Gomes for donation of the POL31 two-hybrid constructs, and Alan Hinnebusch for information on GCD14 prior to publication.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM32431.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.
Current address: Dept. of Biochemistry, University of Washington
SJ-70, Seattle, WA 98195.
§ 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; PCNA, proliferating
cell nuclear antigen; RF-C, DNA replication factor C; PCR, polymerase
chain reaction; MMS, methylmethane sulfonate; BuPhdGTP,
N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate;
PCR, polymerase chain reaction; PAGE, polyacrylamide gel
electrophoresis; Tricine; N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
HPLC, high performance liquid chromatography.
2
Synthetic lethality was observed between
pol32 and pol30-52 in one strain (20). In
another strain, however, the double mutant is viable albeit extremely
slow-growing (A. Pautz and P. Burgers, unpublished observations).
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REFERENCES |
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