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INTRODUCTION |
DNA polymerases (pol) 1 play essential roles in the duplication of genetic material and DNA
repair in both prokaryotes and eukaryotes (1). In Saccharomyces
cerevisiae there exist three essential nuclear DNA polymerases,
pol 
, 
, and 
(2-4). Despite years of study, several perhaps
equally plausible models exist for the function of each polymerase
during DNA replication. Pol plays the role of a primase in the
initiation of DNA replication on both leading and lagging strands in
the simian virus 40 in vitro system (5). Pol and pol are required for the bulk of the replication on the leading and lagging
strands (6-8). The precise location of pol and pol on leading
and lagging strands, however, is not known. Pol , pol , and pol
have also been shown to participate in DNA repair (1).
Polymerase was the first proofreading polymerase purified from
yeast (2). Since then it has been purified from a number of sources
including Schizosaccharomyces pombe, the silkworm
Bombyx mori, and HeLa cells (3-7). The S. cerevisiae pol consists of four subunits, Pol2p, Dpb2p, Dpb3p,
and Dpb4p, with an estimated stoichiometry of 1:1:4:4 (4). These four
proteins are encoded by the POL2, DPB2,
DPB3, and DPB4 genes (4,
8).2 The POL2 gene
encodes the 256-kDa catalytic subunit of pol (8). Mammalian and
S. pombe Pol2p show strong sequence similarity to yeast
Pol2p (7, 9). The DPB2, DPB3, and DPB4
genes encode the remaining 80-, 34-, and 29-kDa subunits of the yeast
pol holoenzyme, respectively (4, 10, 11).2 A functional
and structural human homolog of DPB2, DPE2, has recently been described (12). Dpb2p has also recently been shown to
share amino acid similarity with the B subunits of pol (Pol12p), pol (Pol31p), and many other DNA polymerases (13). DPB3
and DPB4 from other sources have recently been identified
(27). The POL2 and DPB2 genes are essential for
the growth of yeast (8, 10), and the phenotype of temperature-sensitive
pol2 and dpb2-1 mutants suggests a role for each
in DNA replication (10). However, the precise function of
DPB2 is not known. The DPB3 gene is
non-essential, but dpb3 mutants have increased frequency of
spontaneous mutation as expected of a DNA replication defect (11). The
role of the DPB4 gene is currently unknown. Genetic analysis
indicates that DPB11, which is essential for the initiation of DNA replication, may also be important for the function of the pol
complex (14); however, Dpb11p does not seem to copurify with the
holoenzyme. Another protein, encoded by SLD2 (also known as
DRC1), also appears to participate in DNA replication
through interaction with Dpb11p and pol , but does not copurify with the complex (15, 16).
Early studies revealed two main classes of replication-defective
pol2 mutants. One class mapped to the N-terminal catalytic region, pol2-9 and pol2-18. The second class
mapped in the very C-terminal amino acids, pol2-11 and
pol2-12 (17-19). This suggested that the protein might
have at least two essential functions. Supporting this idea, the
C-terminal mutants were defective both in DNA replication and in the
surveillance mechanisms that monitor DNA damage and replication blocks
during S phase, the S/M, and the intra-S checkpoints (19, 20). The
catalytic domain mutants, pol2-9 and pol2-18,
in contrast, were defective only in DNA replication (19). We made
serial 10 amino acid deletions over the C-terminal 120 amino acids to
identify the amino acids specifically involved in the checkpoint
function of the C terminus. A 20-amino acid stretch between two
putative zinc fingers (see Fig. 8) was critical for both DNA
replication and the S/M checkpoint. Given the phenotypes of the
C-terminal mutants, we reasoned that the mutations might affect either
protein/DNA or protein/protein interactions. In fact, the mutations
affected at least two important protein/protein interactions. We
discovered that the Pol2 protein dimerized using two-hybrid assays, and
that the inter-zinc finger mutants failed to do so, suggesting that the
dimerization was not an artifact of the two-hybrid assay (21). Second,
we found that the C terminus of Pol2p interacted strongly with the
Dpb2p subunit by two-hybrid analysis, and that the inter-zinc finger
mutations also destabilized this interaction, although they did not
abolish it (21). These results led to the model that Pol2p C-terminal
amino acids were involved in assembling an active holoenzyme, as also
suggested by Sugino, Araki, and collaborators (22) based on studies of the holoenzyme purified from yeast.
The importance of the Pol2p C terminus at the replication fork was made
clear when it was found that the entire catalytic domain of Pol2p can
be deleted and cells remain viable, as long as the C-terminal 110-kDa
fragment of the protein is intact (23, 24). Thus, it is the C-terminal
half of the protein that provides the only non-redundant essential role
in DNA replication, rather than, as would have been expected, the
catalytic polymerase activity.
The large size of the pol complex currently precludes direct
structural studies. However, a first order picture of the arrangement of subunits that assemble into pol can be obtained by methods similar to those used to analyze the Escherichia coli
replicase, i.e. purification of individual subunits or
mixtures of subunits and analysis of their interactions. We have
analyzed the protein/protein interactions in pol by both
biochemical analysis and two-hybrid assay of pairwise combinations of
subunits. We find that Pol2p and Dpb2p form a stable complex and that
Dpb3p and Dpb4p form a tight complex. It seems logical to propose that
these two subcomplexes may interact to form the holoenzyme observed in
yeast extracts, since Pol2p and Dpb3p interact strongly. We also find
that the Pol2p·Dpb2p complex is dimeric, with dimerization of the
catalytic subunit, Pol2p, being mediated by dimerization of Dpb2p.
Mutations that reduce Pol2p/Dpb2 interaction and dimerization have
severe growth defects. This suggests, but does not prove, that pol is active as a dimeric protein.
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EXPERIMENTAL PROCEDURES |
Materials--
Plasmid PTZ18 was from Bio-Rad. Plitmus39,
M13KO7, restriction enzymes, T4 DNA ligase, and Klenow large fragment
were from New England Biolabs. E. coli CJ236 and mutagenesis
kit were obtained from Bio-Rad. Grace's medium and supplements for
culturing Sf9 insect cells were from Invitrogen. PfastbacI and
pfastbacHTb baculovirus vectors, E. coli DH10bac, Cellfectin
reagent, SFM medium for insect cells, and heat-inactivated fetal bovine
serum were obtained from Life Technologies, Inc. Agarose
Ni2+/NTA beads were from Qiagen. Oligonucleotides were
synthesized by the oligonucleotide facility at California Institute of
Technology. Plasmid miniprep kits were from Qiagen. Western blotting
reagents and nitrocellulose membrane were obtained from Amersham
Pharmacia Biotech. The His6 monoclonal antibody was
from Sigma. Polyclonal antibody for the pol complex was provided by
Dr. Akio Sugino (Osaka University, Osaka, Japan).
Construction of POL2, DPB2, DPB3, and DPB4 Recombinant
Baculoviruses--
The pSEY18-POL2 vector (17) was digested with
SacI, and the purified POL2 fragment was cloned
at the SacI site in the PTZ18 vector to create PTZ18-POL2.
To excise the C terminus of POL2, PTZ18-POL2 was digested
with BsrGI and the vector was ligated with itself. The
ligated vector containing the N terminus of POL2 was
transformed into E. coli CJ236, and cells were infected with M13KO7 phage to make a POL2 phagemid. Uracil containing
single-stranded DNA phagemid template was prepared as described earlier
(21). The single-stranded DNA template was annealed to an
oligonucleotide, which was designed to introduce a SacI
restriction enzyme site near the ATG start of POL2.
Mutagenesis was carried out as described (21). The mutagenic
oligonucleotide used is as follows: P1, 5'-C AAA CAT CAT A
G A GC T C TC CCC TGA G-3'.
The mutation was confirmed by DNA sequencing. This resulted in
PTZ181-POL2. The C-terminal BsrGI fragment of POL2 (21) was subcloned into the PTZ181-POL2 to reconstitute the full-length POL2 gene resulting in PTZ182-POL2. For pol2-E
and pol2-F mutants, plitmus39 containing the mutagenized C
terminus (21) was used to reconstitute the full-length
pol2-E and pol2-F, which resulted in PTZ183-POL2
and PTZ184-POL2, respectively. The PTZ182-POL2, PTZ183-POL2, and
PTZ184-POL2 were digested with SacI, and the purified
fragment was subcloned in the pfastbacI baculovirus vector at
SacI site. Colonies were screened by colony hybridization, and positive clones were confirmed by restriction digestion and DNA sequencing.
The DPB2 gene was excised from the pGal4BD-DPB2 vector
(21) by NcoI/SalI digestion and cloned in the
pfastbacHTb vector. The DPB3 gene (21) was cloned at
BamHI/XhoI sites in pfastbac1 and pfastbacHTb
vectors. The open reading frame of clone YDR121W, which encodes
DPB4,2 was synthesized by PCR using yeast
genomic DNA as template and cloned at BamHI/XhoI
in pfastbac1 and pfastbacHTb vectors. (The Western blot in Fig. 3
confirms that the gene we cloned actually encodes Dpb4p.) The
DPB2, DPB3, and DPB4 genes in
pfastbacHTb have a His6 epitope and 16 additional amino
acids at the N terminus. The oligonucleotides used for PCR cloning of
DPB3 and DPB4 are as follows: DPB31, 5'-C AAC CGT
GTT GGA TCC AAA ATG TCC AAC TTA G-3'; DPB32, 5'-CA ATA ATC TCG AGA TCA
CTA AGG ATC GGT G-3'; DPB41, 5'-GAC CAT ATA TTT GGA TCC ACG ATG CCA
CC-3'; and DPB42, 5'-TAG ACA GTT TCC CTC GAG GGG TTA CGT TTG-3'.
The C terminus of POL2 was prepared by PCR (25) and cloned
at SacI/XhoI sites in pfastHT1 and pfastHA
baculovirus vectors. pfastHT1 and pfastHA were made by inserting
oligonucleotide cassettes shown below into pfastbac1. The pfastHT1 and
pfastHA vectors contain His6 and HA epitopes at
BamHI/EcoRI sites, respectively. The
oligonucleotides used are as follows: HT1, 5'-GA TCC ATG TCG CAT CAT
CAT CAT CAT CAT GGT G-3'; HT2, 5'-AA TTC ACC ATG ATG ATG ATG ATG ATG
CGA CAT G-3'; HA1, 5'-GA TCC ATG TAC CCA TAC GAC GTC CCA GAC TAC GCT
G-3'; HA2, 5'-AA TTC AGC GTA GTC TGG GAC GTC GTA TGG GTA CAT G-3'; K1, 5'-G CCC TCG GAG AGA GCT CTC ATG GAC GAG G-3'; K2, 5'-CA TTG AAC CTC
GAG TTA TAT ACT G-3'.
The preparation of baculoviruses was performed using the Bac to Bac
system from Life Technologies Inc. Briefly, recombinant pfastbacI or
pfastHTb vectors were transformed into E. coli DH10bac cells. Bacmid DNA was prepared according to the manufacturer's instructions and immediately transfected into log phase Sf9
insect cells using Cellfectin reagent. The culture supernatant
containing the baculovirus particles was collected after 3 days,
amplified twice, and stored in darkness at 4 °C. As a control,
pfastbacI vector plasmid was used to prepare wild type baculovirus.
Expression and Purification of the pol Subunits--
For
expression of proteins, 70 × 106 log phase Sf9
insect cells were layered on four 175-cm2 tissue culture
flasks. After cells were attached (1 h), supernatant was removed and
cells were infected with viral supernatant at multiplicity of infection
of 10 for POL2 (or pol2-F mutant), 5 for
DPB2, 5 for DPB3, and 5 for DPB4. The
cells were incubated for 2 h at room temperature with gentle
rocking. Then, 5 ml of additional Grace's complete medium was added
and cells were incubated at 27 °C. After 2 days, cells were
harvested, washed once with cold 1× Tris-buffered saline, flash frozen
in liquid N2, and stored at 
70 °C. For pairwise
coinfections, Sf9 cells were infected with the corresponding
baculoviruses as described above and incubated at 27 °C for 2 days.
To purify various pol subunits, the frozen cells (70-100 × 106 cells) were thawed once on ice and then resuspended in
2 ml of buffer A (20 mM Tris, 100 mM NaCl, 5 mM EGTA, 20% glycerol, pH 7.9) containing leupeptin (20 µg/ml) and pepstatin A (20 mg/ml). After lysis, cells were kept on
ice for 15 min and centrifuged at 12,000 rpm in a microcentrifuge for
10 min and supernatant was recovered. 300 µl of
Ni2+/NTA-agarose beads (50:50 in buffer A) were added to
the supernatant, and the mixture was kept at 4 °C with end to end
rotation for 2 h. All further purification steps were carried out
at 4 °C unless otherwise stated. After binding of the proteins,
beads were centrifuged at 1000 rpm using a swinging bucket centrifuge
and the unbound protein fraction was collected. The beads were
extensively washed (8 × 0.8 ml) with buffer A. To elute proteins,
beads were mixed with 350 ml of buffer B (20 mM Tris, 100 mM NaCl, 20% glycerol, pH 7.9) containing 0.3 M imidazole with rotation for 30 min. The mixture was
centrifuged, and the supernatant containing the eluted proteins was
collected. Samples were stored at 70 °C. The total protein,
unbound protein, and eluted protein fractions were analyzed using 10%
SDS-PAGE and Western blotting. The protein concentration was determined
using Bradford assay using bovine 
-globulin as a standard.
Gel Filtration Analysis--
The SMART system (Amersham
Pharmacia Biotech) and precision column Superdex 200 PC (2.4 ml)
(Amersham Pharmacia Biotech) were used for the analysis. The column was
equilibrated at 4 °C with 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 M KCl, and 10% glycerol. 50 µl
(50 µg) of the protein eluted from the Ni2+/NTA column
was injected on the column. The flow rate was set at 40 µl/min, and
protein fractions (40 µl) were collected. The fractions were analyzed
by 10% SDS-PAGE and assayed for DNA polymerase activity using
poly(dA)/oligo(dT) as a DNA template as described below. Marker
proteins (Amersham Pharmacia Biotech) were chromatographed in a similar
manner. To cleave the N-terminal His6 sequence, the proteins (20-30 µg) were incubated with rTEV protease enzyme as described in the manufacturer's instructions (Life Technologies, Inc.). The cleaved peptide and the rTEV protease were removed by adding
Ni2+/NTA beads to the protein mixture and collecting the
unbound fraction.
Yeast Two-hybrid Protein/Protein Interactions--
The
lexA DNA binding domain (LEX A-BD) plasmid BTM116, provided
by Stanley Fields (University of Washington, Seattle, WA), and
Gal4 activation domain plasmid pACT2 (Matchmaker two-hybrid system; CLONTECH) were used for cloning the
C-terminal fragment of POL2 (aa 1265-2222),
DPB2, DPB3, DPB4, and the N-terminal
fragment of POL2 (aa 1-1265). Construction of the pACT2/C
terminus POL2, DPB2, and DPB3 were
described previously (21). pACT2/N terminus POL2 and
DPB4 were cloned by generating a PCR product using
yeast genomic DNA as a template and primers to give flanking
SmaI/XhoI sites
(5'-CTGCCCGGGCATGTTTGGCAAGAAAAAAAACAACGG-3' and
5'-GGTCGCCTCGAGCTAGTCCATGGAAGGAATCTCCG-3') and
NcoI/BamHI sites
(5'-CATATTTTTTCCATGGCGATGCCACCA-3' and
5'-TACTAGACAGGATCCATAGCGGG-3').
Plasmid pBTM116BD-C terminus POL2 was cloned by generating a
PCR product using pK1 (21) with primers generating flanking SmaI/BamHI sites
(5'-AGATTCCTTCCCCGGGCGAGGACTATGT-3' and
5'-CGTTATATACTGGATCCTCATATGGTCAAATC-3').
pBTM116BD-N-terminus POL2, DPB2,
DPB3, and DPB4 were cloned via the univector
plasmid-fusion system (UPS) (25). UPS uses Cre recombinase to
facilitate a loxP site-specific recombination event.
DPB2, DPB3, and DPB4 were excised from
previously isolated pAS2-1 vectors (21) and ligated into the pUNI15
vector at the NcoI and BamHI sites,
downstream of the loxP site,
5'-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3'. The N terminus
POL2 was cloned into pUNI15 downstream of the
loxP site after PCR with SmaI primers
5'-CTGCCCGGGCATGATGTTTGGCAAGAAAAAAAACAACGG-3' 5'-GGTCGCGCCCGGGTAGTCCATGGAAGGAATCTCCG-3'. A loxP site
was inserted into the polylinker of pBTM116 at
SmaI/PstI, designated pBTM116loxP. The respective
pUNI15 plasmids were recombined with pBTM116loxP, and the
pBTM116BD-N-terminus POL2, DPB2, DPB3,
and DPB4 plasmids were isolated as described (25). P1 Cre
recombinase was either from Novagen or purified from GST-Cre-expressing
pBL21DE3plys/pQL123 (25). (pUNI15 and pQL123 plasmids were supplied by
the Elledge laboratory, Baylor College of Medicine, Houston, TX).
The L40 two-hybrid strain (MATa his
200 trp1-901
leu2-3, 112 ade2 lys2-801am
URA3::(lexAop)8 -lacZ
LYS2::(lexAop)4-HIS3) (26) was
co-transformed with the binding domain and activation domain fusion
constructs using the polyethylene glycol/lithium method. Transformants
were selected on Leu
/Trp/His
synthetic agar plates plus or minus 1 mM
3-amino-1,2,4-triazole incubated at 30 °C for 3-5 days. Colonies
that grew were tested for 
-galactosidase activity using a filter
lift assay and then quantified in a subsequent -galactosidase assay
(both as per instructions from CLONTECH).
-Galactosidase units of activity were calculated as -
galactosidase activity = 1000 × A420/(t × v × A600), where t = time (min)
required for the reaction and v = 0.1 × concentration
factor. All quantitative measurements were done in replicates of
4-11n and are reported as an average ± S.D.
Polymerase Assays--
The polymerase assays were as
described, using poly(dA)/oligo(dT)10 (1:20,
template to primer chains) as DNA template (4, 5). Reaction mixtures
(60 ml) consisted of 50 mM Tris-HCl, pH 7.5, 2 mM dithiothreitol, 10% glycerol, 8 mM
MgCl2, 17 µg/ml poly(dA)/oligo(dT)10, 120 mM KCl, 100 µg/ml bovine serum albumin, and 50 µM [3H] dTTP (100 cpm/pmol). The poly(dA)
/oligo(dT)10 mixture was annealed in 25 mM
Hepes, pH 7.1, 60 mM KCl prior to the reaction. The
reactions were conducted at 37 °C for 15 min. The reactions were
stopped by spotting the reaction mixture on Whatman DE81 filter paper
(2.4 cm diameter). The filters were washed (five times) with 0.5 M Na2HPO4, two times with water,
and then rinsed with 95% ethanol. Filters were dried, 3 ml of
scintillation mixture was added, and radioactivity was measured. One
unit of polymerase activity is defined as 1 nmol of dNMP
incorporated/h.
Mutagenesis and Subcloning--
The point mutations in the
inter-zinc finger domain of POL2 were constructed as before
(20). The oligonucleotides used for the mutagenesis are as follows:
L2146D (5'-G TTT TTC AAT GTC GTG TTC TTG C-3'), L2150D
(5'-GAT ATC AGA ACG GTC TTT TTC AAT C-3'), and
L2146D,L2150D (5'-GAT ATC AGA ACG GTC TTT TTC AAT
GTC GTG TTC TTG CAA C-3').
The mutations were verified by automated DNA sequencing, and the
full-length POL2 gene was reconstituted in pRS314 as
described previously (21).
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RESULTS |
Interactions between the Essential Subunits, Pol2p and
Dpb2p--
If two proteins directly contact each other, one of which
is tagged with a His6 peptide, it is expected that the
protein without the His6 tag will co-purify with the
protein fused to the His6 peptide upon Ni2+/NTA
chromatography. We have used this as the basis of a strategy to
identify putative protein contacts between known subunits of pol
. In order to assess the interaction of Pol2p with Dpb2p subunit, Sf9 cells were infected with
Pol2p·His6-Dpb2p recombinant baculoviruses. After 48 h, cells were harvested, extracts prepared, and proteins purified by
Ni2+/NTA affinity chromatography. As shown in Fig.
1A, Pol2p coeluted from
Ni2+/NTA with His6-Dpb2p (Fig. 1A,
lane 1E). Pol2p could not be detected in
fractions from cells carrying only Pol2p virus without
His6-Dpb2p (Fig. 1A, lane
2E). Thus, Pol2p interacts directly with Dpb2p.
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Fig. 1.
Pol2p/Dpb2p interactions in pol complex.
A, Western blots of affinity-purified proteins. Sf9
insect cells (40 × 106 ) were coinfected with Pol2p
and His6-Dpb2p baculoviruses and expression and
purification of proteins was carried out as described under
"Experimental Procedures." Proteins were analyzed by Western
blotting using pol antibody. Lane 1T, total
cell protein for cells infected with Pol2p and His6-Dpb2p
virus; lane 1E, protein from cells infected with
Pol2p and His6-Dpb2p bound to Ni2+/NTA and
eluted with imidazole; lane 2T, total cell
protein for cells infected with Pol2p virus; lane
2E, protein from cells infected with Pol2p virus only that
bound to Ni2+/NTA and eluted with imidazole. B
and C, interaction of 110-kDa Pol2p fragment with Dpb2p.
B, Western blot of HA-Pol2p (aa 1265-end) eluted from
Ni2+/NTA after coinfection with His6-Dpb2p.
HA-Pol2p (aa 1265-end)·His6-Dpb2p coinfection and the
His6-Dpb2p infection were analyzed by Western blotting
using pol antibody. Lane 1, eluted protein
from HA-Pol2p (aa 1265-end)·His6-Dpb2p coinfection;
lane 2, control showing eluted protein from
His6-Dpb2p infection. C, protein fractions of
cells infected with HA-Pol2p (aa 1265-end) and His6-Dpb2p
were separated on 10% SDS-PAGE and bands were visualized by staining
with Coomassie Brilliant Blue. Lane 1, total cell
protein (150 mg); lane 2, protein not bound to
the Ni2+/NTA column; lane 3, protein
bound to Ni2+/NTA and eluted in the presence of 0.3 M imidazole (8 mg).
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Previous two-hybrid analysis had suggested that the C-terminal fragment
of Pol2p (110 kDa, aa 1265-end) is sufficient for interaction between
Pol2p and Dpb2p (21). We confirmed this biochemically by showing that
when the truncated HA-Pol2p (aa 1265-end) and His6-Dpb2p
were coexpressed, HA-Pol2p (aa 1265-end) coeluted with
His6-Dpb2p from Ni2+/NTA beads (Fig. 1,
B (lane 1) and C (lane 3)).
Two C-terminal Mutations, pol2-E and pol2-F, Which Show Growth
Defects, Weaken Interaction between Pol2p and Dpb2p--
The Pol2-Fp
has a deletion of aa 2153-2162, between the two putative C-terminal
zinc fingers (21). Previously, using two-hybrid analysis, we
demonstrated that pol2-Fp showed reduced affinity for DPB2
(21). Here, we further investigated the pol2-Fp/Dpb2p interactions
in vivo and in vitro. Dosage suppression has been used to argue for protein/protein interaction in the past. For example,
overexpression of SLD2 (also known as DRC1)
suppresses dpb11 growth defects, and the two proteins have
been shown to interact by coimmunoprecipitation from yeast extracts
(15, 16). The ability of the DPB2 gene expressed under the
GAL1,10 promoter in a high copy number plasmid to suppress
the temperature-sensitive phenotype of pol2-F was tested. As
shown in Fig. 2A,
pol2-F was able to grow at 37 °C when DPB2 was
induced by galactose but not when Gal1,10 was repressed by
glucose. This supports the idea that the two proteins may interact and
thus stabilize the thermolabile pol2-Fp.
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Fig. 2.
Interaction of pol2-Fp with Dpb2p.
A, suppression of pol2-F by DPB2. The
pol2-F yeast strain, harboring YEp18-DPB2 or YEp18 only, was
streaked on minus-tryptophan, minus-uracil minimal medium plate
containing raffinose and 2% galactose and incubated at 37 °C for 3 days. B, interaction between pol2-Fp and Dpb2p; Sf9
insect cells (40 × 106) were coinfected with
pol2-Fp·His6-Dpb2p, and proteins were purified by
Ni2+/NTA affinity chromatography as described under
"Experimental Procedures." The various protein fractions as shown
in the top of the figure were separated on 10% SDS-PAGE and
analyzed by Western blotting using pol antibody. As indicated,
lane 1 is from the coinfection and
lane 2 is a control in which pol2-Fp was not
expressed. Lane 1T, total protein loaded on
Ni2+/NTA column; lane 1E, protein
bound to and eluted from Ni2+/NTA column; lane
2T, total protein; lane 2E, bound and
eluted protein.
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To analyze the interactions of pol2-Fp and Dpb2p biochemically,
Sf9 cells were coinfected with pol2-Fp and
His6-Dpb2p recombinant baculoviruses and the ability to
form a complex was assessed using Ni2+/NTA beads as above.
As predicted, the pol antibody showed only a very weak pol2-Fp band
associated with the His6-Dpb2p (Fig. 2B,
lane 1E).
Since assays for catalytic activity can be more sensitive than Western
blotting, the polymerase activity of the affinity-purified fractions
was also used to compare the amount of Pol2p and mutant pol2-F protein
copurifying with His6-Dpb2p. As shown in controls in Table
I, no DNA polymerase activity eluted from
the Ni2+/NTA column when cells were infected with
His6-Dpb2p. Nor was there any detectable DNA polymerase
activity in Ni2+/NTA column fractions when cells were
infected with untagged Pol2p virus (Table I). DNA polymerase activity
was detected in Pol2p·His6-Dpb2p and
pol2-Fp·His6-Dpb2p coinfections, however. As shown in
Table I, the activity of pol2-Fp·His6-Dpb2p was 10-fold
reduced compared with wild-type Pol2p·His6-Dpb2p.
However, the pol2-F complex polymerase activity was still significantly
(50-fold) higher than the control lacking any Pol2p (Table I). We
conclude that there is residual but reduced interaction between pol2-Fp
and Dpb2p. Taken together with the Western blot, we attribute the
reduction in activity to reduction in the amount of pol2-F protein
present, although we cannot rule out that the polymerase activity
itself may also be reduced. Thus, the pol2-F mutation reduces but does
not abolish Pol2p/Dpb2p interaction. A similar reduction in recovery of
Pol2p using the His6-Dpb2 was observed with a second mutant
protein, pol2-Ep, carrying a deletion of aa 2143-2152 that fails to
support growth at any temperature (data not shown) (21).
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Table I
Enzymatic activity of purified wild type and mutant pol complexes
or subcomplexes by Ni2+/NTA affinity chromatography
The polymerase activity of the complexes was measured using
poly(dA)/oligo(dT)10 (1:20) as DNA template. The assay
conditions are described under "Experimental Procedures." The
purifications were done in parallel as were the assays, and 1-2 µg
of protein was used in the polymerase determination. Determinations
were made a minimum of three times, and the average is reported.
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Interactions between Pol2p and the Non-essential Subunits Dpb3p and
Dpb4p--
To determine if the catalytic subunit also interacted with
Dpb3p and/or Dpb4p, Pol2p was expressed in the presence of either His6-Dpb3p or His6-Dpb4p and
Ni2+/NTA chromatography was performed. Western blotting
failed to detect Pol2p copurifying with either His6-Dpb3p
or His6-Dpb4p alone (data not shown). However, the
affinity-purified Pol2p and His6-Dpb3p or Pol2p and
His6-Dpb4p complexes did contain DNA polymerase activity
(Table I). This activity was 50 times higher than the background levels
in His6-Dpb3p or His6-Dpb4p affinity-purified from cells that did not express recombinant yeast Pol2p (Table I).
Thus, there may also be direct interactions between Pol2p and Dpb3p and
Dpb4p (Table I). These interactions with Pol2p are weaker than
Pol2p/Dpb2p interaction, both as measured by Western blotting and DNA
polymerase activity recovered (Table I) (see also two-hybrid analysis,
Fig. 7).
Direct Interactions between Dpb2p, Dpb3p, and Dpb4p in the
Absence of Pol2p--
Sf9 insect cells were coinfected with
pairwise combinations of His6-Dpb2p·Dpb3p,
His6-Dpb2p·Dpb4p, His6-Dpb3p·Dpb4p, and
Dpb3p·His6-Dpb4p baculoviruses. Ni2+/NTA
affinity-purified proteins were monitored by Western blotting using pol
antibody. The mobilities of the respective tagged species are shown
in Fig. 3 (lanes
5-7). Both untagged Dpb3p and Dpb4p, which migrate slightly
faster than the tagged proteins in lanes 5 and
6, coeluted with His6-Dpb2p (Fig. 3,
lanes 1 and 2). Neither untagged Dpb4p
nor untagged Dpb3p bound to the column in the absence of a
His6 tag (data not shown). Thus, Dpb2p interacts independently with Dpb3p or Dpb4p and interaction doesn't require Pol2p. Dpb3p coeluted with His6-Dpb4p and Dpb4p coeluted
with His6-Dpb3p in the coinfections (Fig. 3,
lanes 3 and 4). This indicates that
Dpb3p and Dpb4p form a direct complex without Pol2p or Dpb2p to mediate
their interaction. The results in Figs. 1 and 3 suggest that Pol2p and
Dpb2p form a complex and that Dpb3p and Dpb4p form a complex. We
propose that these two complexes may interact to form the holoenzyme as
purified from yeast (4), although further studies will be necessary to
verify this hypothesis. This organization is consistent with the recent
results obtained with in vitro translation with human pol
subunits (27).
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Fig. 3.
Dpb2p, Dpb3p, and Dpb4p interactions.
40 × 106 Sf9 insect cells were infected with
various pairwise combinations of His6-Dpb2p,
His6-Dpb3p, Dpb3p, His6-Dpb4p, and Dpb4p
baculoviruses, and proteins were purified by Ni2+/NTA
affinity chromatography as described under "Experimental
Procedures." The bound proteins from each infection as indicated were
separated on 10% SDS-PAGE and analyzed by Western blot analysis using
pol polyclonal antibody.
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The C Terminus of POL2 (aa 1265-end) Does Not Form a
Homodimer--
Using yeast two-hybrid analysis, we have previously
shown that the C terminus of POL2 (aa 1265-end), lacking
the entire polymerase domain, interacts with itself, i.e.
dimerizes. We also showed that the C terminus is sufficient for
interaction with DPB2 (Ref. 21, and see Fig. 1). To
investigate dimerization biochemically, we cloned the C-terminal half
of POL2 (aa 1265-end) in pfastHT1 and pfastHA baculovirus
vectors, which fuse POL2 (aa 1265-end) to the
His6 peptide and hemagglutinin (HA) epitope tags,
respectively, and thus produce 110-kDa polypeptides when expressed.
Cells were coinfected with His6-Pol2p (aa 1265-end) and
hemagglutinin epitope-tagged HA-Pol2p (aa 1265-end) recombinant
baculoviruses and Ni2+/NTA-purified proteins were analyzed
by Western blotting using 12CA5 (for HA epitope) and pol polyclonal
antibodies. Although both HA-Pol2p (aa 1265-end) and
His6-Pol2p (aa 1265-end) (Fig. 4, B and C) were
clearly expressed and present in the total cell lysate (Fig.
4A, lane 3T), the Ni2+/NTA
affinity-purified His6-Pol2p fraction did not contain
detectable HA-Pol2p (aa 1265-end) (Fig. 4A, lane
3E). Recovery of His6-Pol2p (aa 1265-end) from
the Ni2+/NTA column was verified by SDS-PAGE and Western
blotting using pol antibody (Fig. 4, B and
C). Control cells singly infected with HA-Pol2p (aa
1265-end) or His6-Pol2p (aa 1265-end) or
His6-Dpb2 showed efficient expression of HA-Pol2p but no
HA-Pol2p in affinity column eluates (Fig. 4A,
lanes 1, 2, and 4), as
expected. The positive control in Fig. 4A (lane
5) shows that HA-Pol2p is capable of binding and copurifying
with His6-Dpb2p and can be detected in a
Ni2+/NTA eluate. The experiment was repeated using various
salt concentrations (50-300 mM) during lysate preparation
and affinity purification, but the HA-Pol2p (aa 1265-end) was not
detected in the eluted protein fraction. Thus, the dimerization of
Pol2p detected by the two-hybrid system either is mediated by another
protein not present in insect cells or is too weak to survive the
isolation procedure used here in the absence of the N-terminal
domain.
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Fig. 4.
Failure of the 110-kDa C terminus of
POL2 (aa 1265-end) to dimerize. A-C,
biochemical test for dimerization of the 110 kDa fragment.
A, Western blot using anti-HA 12CA5 monoclonal antibody.
Sf9 insect cells (40 × 106) were singly
infected or coinfected in pairwise combination with baculoviruses for
His6-Pol2p (aa 1265-end), HA-Pol2p (aa 1265-end), and
His6-Dpb2p as indicated. After cell lysis, the proteins
were purified using Ni2+/NTA affinity chromatography as
described under "Experimental Procedures." Protein from the total
cell lysate (T) and bound protein fractions eluted with
imidazole (E), were separated on 10% SDS-PAGE and subjected
to Western blotting using 12CA5 anti-HA epitope monoclonal antibody.
B, protein from the cells infected with
His6-Pol2p (aa 1265-end) were separated on 10% SDS-PAGE
and the gel was stained with Coomassie Brilliant Blue. Lane
1, total cell protein (150 mg); lane
2, protein that failed to bind to the Ni2+/NTA
beads; lane 3, bound proteins (4 mg) eluted in
the presence of 0.3 M imidazole. C, Western blot
of extract and Ni2+/NTA-bound fractions using pol antibody. Lane 1, unbound protein;
lane 2, bound protein eluted in the presence of
0.3 M imidazole.
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Dpb2p Forms a Homodimer--
Since Pol2p does not itself
appear to dimerize but does interact with Dpb2p, it seemed plausible
that dimerization might occur through Dpb2p. Although we previously
failed to find self interaction for DPB2 in the two-hybrid
system (21), we show below with a more sensitive two-hybrid analysis
that Dpb2 dimerization does occur. To test for dimerization
biochemically, His6-Dpb2p was purified from the Sf9
insect cells and analyzed by gel filtration. To exclude the possible
interference by the N-terminal His6 epitope, we cleaved the
epitope by site-specific rTEV protease and the intact Dpb2p was further
purified. The protein was then injected onto a Superdex 200 PC gel
filtration column. The majority of the protein was eluted at the
apparent molecular mass of 160 kDa, as expected of a Dpb2p dimer (Fig.
5A). This confirms the
two-hybrid results presented below, and we propose that dimerization of
Pol2p may therefore occur through Dpb2p.
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Fig. 5.
Dpb2p forms a homodimeric complex.
A, gel filtration analysis of affinity-purified
His6-Dpb2p. The N-terminal His6 sequence in
His6-Dpb2p was cleaved by rTEV protease as described under
"Experimental Procedures." 10 µg of the purified Dpb2 was
injected on the Superdex 200 PC column, and protein fractions were
separated by 10% SDS-PAGE and silver staining. B, gel
filtration analysis of affinity-purified protein from a
His6-Dpb2p·His6-Dpb3p·Dpb4p coinfection. 50 µg of the protein eluted from Ni2+/NTA resin was injected
on Superdex 200 PC column. The protein fractions were separated on 10%
SDS-PAGE and silver-stained. The polymerase activity was determined
using poly(dA)/oligo(dT) as described under "Experimental
Procedures." Size markers were thyroglobulin (669 kDa), catalase (232 kDa), aldolase (158 kDa), and bovine serum albumin (67 kDa).
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Further evidence for dimerization of Dpb2p was obtained when we
applied a His6-Dpb2p·His6-Dpb3p·Dpb4p
affinity-purified mixture, prepared from coinfections of cells with the
three corresponding viruses, to the gel filtration column. SDS-PAGE and
silver staining of the protein fractions showed that a
His6-Dpb2p·His6-Dpb3p·Dpb4p complex eluted
at 190 kDa (Fig. 5B). The elution position suggests a
2Dpb2p·1Dpb3p·1Dpb4p complex, assuming a globular complex.
Gel Filtration Analysis of Pol2p·Dpb2p Complexes Reveals a
600-kDa Heterotetrameric Form--
To assess whether Dpb2 dimerization
might mediate Pol2p dimerization, we performed gel filtration analysis
of complexes isolated from cells coinfected with Pol2 and
His6-Dpb2. A substantial amount of DNA polymerase activity
eluted in fractions 8-10, corresponding to 600 kDa. Both Pol2p and
Dpb2p were present in these fractions (Fig.
6A). Given that Pol2 is a
256-kDa protein and Dpb2 is an 80-kDa protein, the most likely
composition of the protein in this 600-kDa peak is a heterotetramer
consisting of two Pol2p·Dpb2p heterodimers. Additional polymerase
activity also eluted at 210 kDa. Since this is smaller than Pol2p (256 kDa) itself, this may represent proteolyzed fragments and we have not
analyzed this (these) species in detail. It is worth pointing out that
pol activity has previously been isolated from yeast and shown to contain a 120-kDa form of the Pol2p, and there is a 120-kDa band in
this fraction. Unlike here, that form was not shown to be complexed with Dpb2p, Dpb3p, or Dpb4p (4, 5). This polymerase activity was due to
some form of Pol2p, however, since no polymerase was observed if
His6-Dpb2p alone or
His6-Dpb2p·His6-Dpb3p·Dpb4p was applied to
the column (Fig. 5). In fact, controls showed that no polymerase
activity was observed in any of the gel filtration experiments shown in
this work when Pol2p was omitted.
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Fig. 6.
Gel filtration analysis of Pol2p·Dpb2
complexes reveals a heterotetrameric form. 80 µg of
Pol2p·His6-Dpb2p of the protein eluted from the
Ni2+/NTA was injected onto a Superdex 200 PC column, and
proteins were separated on 10% SDS-PAGE and silver-stained. The
polymerase activity of the protein fractions was determined using
poly(dA)/oligo(dT) as described under "Experimental Procedures."
The size markers were thyroglobulin (669 kDa), catalase (232 kDa),
aldolase (158 kDa), and bovine serum albumin (67 kDa). B,
gel filtration analysis of the
Pol2p·His6-Dpb2p·His6-Dpb3p·Dpb4p
affinity-purified sample. 50 µg of the protein eluted from the
Ni2+/NTA was injected on Superdex 200 PC column, and
proteins were separated on 10% SDS-PAGE and silver-stained. The
polymerase activity of the protein fractions was determined using
poly(dA)/oligo(dT) as described under "Experimental Procedures."
C, stabilization of pol2-Fp·Dpb2p complex by Dpb3p and
Dpb4p. Sf9 cells (70 × 106 ) were coinfected
with pol2-Fp·His6-Dpb2p·His6-Dpb3p·Dpb4p,
and proteins were purified by Ni2+/NTA affinity
chromatography. The various protein fractions were subjected to 10%
SDS-PAGE, and the resulting gel was stained with Coomassie Blue.
Lane 1, total cell protein before addition of
Ni2+/NTA-agarose (200 µg); lane 2,
protein not bound to the beads; lane 3, proteins
eluted in the presence of 0.3 M imidazole (20 µg).
D, Western blotting of
pol2-Fp·His6-Dpb2p·His6-Dpb3p·Dpb4p.
Protein fractions as described in Fig. 6C were analyzed by
Western blotting using pol antibody. E, gel filtration analysis of
pol2-F·His6-Dpb2p·His6-Dpb3p·Dpb4p. 50 µg of the eluted protein from Ni2+/NTA resin was injected
on Superdex 200 PC column. The protein fractions were separated on 10%
SDS-PAGE and silver-stained. The polymerase activity was determined
using poly(dA)/oligo(dT) as described under "Experimental
Procedures."
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It was possible, since these experiments were carried out in the
absence of Dpb3p·Dpb4p complexes, that the putative 600-kDa complex
might be an artifact and might not form in their presence. To test this
possibility, Sf9 insect cells were coinfected with Pol2p,
His6-Dpb2p, His6-Dpb3p, and Dpb4p recombinant
baculoviruses. All four proteins were recovered from the affinity
column, as revealed by Western blots (data not shown). Gel filtration
revealed a DNA polymerase peak at 600 kDa, with the same mobility and
composition (Fig. 6B) as the complex formed in the absence
of Dpb3p and Dpb4p (Fig. 6A). Although there was no
detectable Dpb3 or Dpb4 in the 600-kDa peak, there was more of the
600-kDa form compared with the 210-kDa (presumably) degraded form seen
also in Fig. 6A. Although it is not clear how Dpb3p and
Dpb4p increase the amount of the heterotetrameric peak, these data show
at the least that the heterotetramer form can be a major reproducible
form of this Pol2p·Dpb2p subassembly. We speculate that the apparent
relative stabilization of the 600-kDa form by Dpb3 and Dpb4p may be
related to the fact that coexpression of the latter two subunits with
Pol2p·Dpb2p also results in an increase in recovery of overall DNA
polymerase activity (Table I). It is noteworthy that, if Dpb3 and Dpb4
are not tagged with His6, they do not copurify from
extracts with His6-Pol2p nor with Pol2p·His6-Dpb2p (data not shown).
We next asked if the pol2-Fp mutant could form heterotetramers
with Dpb2p, even though its interaction with Dpb2p was weak compared
with wild-type Pol2p (see Fig. 2A). Sf9 cells were
coinfected with pol2-Fp, His6-Dpb2p,
His6-Dpb3p, and Dpb4p recombinant baculoviruses, and
protein complex was purified using Ni2+/NTA chromatography.
Coexpression of the His6-Dpb3p and Dpb4p proteins appeared
to stabilize the pol2-Fp·Dpb2p complex since pol2-Fp now clearly
coeluted with His6-Dpb2p (compare Fig. 2B (lane 1E) with Fig. 6C
(lane 3)). This effect of Dpb3p and Dpb4p was
reflected in increased recovery of DNA polymerase activity (Table I).
The increased recovery allowed us to analyze the
pol2-Fp·His6-Dpb2p complex by gel filtration. In contrast
to the results with wild type Pol2p·His6-Dpb2p (Fig.
6A), the polymerase activity of pol2-Fp mutant eluted in a
single peak at an apparent molecular mass of 190 kDa (Fig.
6E), suggesting severe degradation. The striking reduction
of the amount of the 600-kDa Pol2p·Dpb2p complex containing pol2-Fp
demonstrates that the inter-zinc finger mutation destabilizes the
Pol2p·Dpb2p complex and reduces formation of the heterotetramer. (We
note parenthetically that a small amount of polymerase may be found in
fraction 6. Multimeric His6-Dpb2p is also observed in
fractions 5 and 6 (Fig. 6E). A small amount of pol2-Fp is
presumably present but below the level of detection by silver staining
in these fractions.)
The experiments in Figs. 5 and 6 have been repeated at least twice,
using at least two different infections; therefore, we believe the
amounts of activity and protein are reproducible. We conclude that the
inter-zinc finger domain stabilizes interaction between Pol2p and Dpb2p
and therefore may be involved in dimerization.
Semi-quantitative Analysis of Subunit Interactions Using the Yeast
Two-hybrid Assay--
Although the fusion of proteins to Gal4 required
for implementation of the two-hybrid assay can interfere with normal
protein/protein interactions, nevertheless the approach has been
successful in many cases in estimating affinities between different
proteins. To compare the strength of the various protein/protein
interactions described above, we used a two-hybrid assay in which the
reporter strain has eight lexA operators in tandem
controlling the lacZ gene and four lexA operators
controlling the HIS3 gene. Strong protein/protein
interactions show high levels of -galactosidase expression, but even
weak interactions are detectable. The POL2, DPB2,
DPB3, and DPB4 genes were fused both to the
lexA DNA binding domain and to the Gal4
activation domain as described under "Experimental Procedures." The
C-terminal and N-terminal portions of POL2 were tested
individually. The strength of the interactions between the various
proteins was quantitated by measuring -galactosidase activity in all
possible pairwise combinations of the proteins. The results are
collated in Fig. 7. Supporting the
biochemical evidence, the C terminus of Pol2p interacted with Dpb2p,
Dpb3p, and Dpb4p (Figs. 1A and 3). The strength of the
interactions was Dpb2>Dpb3
Dpb4. Although we had previously failed
to detect DPB2 self-interaction in a two-hybrid assay with a
less sensitive reporter strain (21), with the current assay the
interaction was easily detectable, as supported by the gel filtration
analysis that showed a homodimer (Fig. 5A). Although we have
shown that the C-terminal half of Pol2p is sufficient for interaction
with Dpb2p, a fusion containing the N-terminal half of Pol2p also
showed weak interactions with Dpb2p, consistent with the fact that high
copy plasmids expressing high levels of DPB2 suppress
N-terminal temperature-sensitive pol2-18 mutant (10).
Dpb3p·Dpb3p and Dpb4p·Dpb4p homodimerization was not observed under
the same conditions, but there was a very strong interaction between
Dpb3p and Dpb4p, confirming that they form a stable heterodimer (Fig.
3). Dpb2p·Dpb3p and Dpb2p·Dpb4p showed very weak but detectable
interaction, again consistent with the recovery of co-complexes from
the Ni2+/NTA column (Fig. 3).
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Fig. 7.
Yeast two-hybrid analysis. The L40
two-hybrid yeast strain was used for the assay. C-term
corresponds to amino acids 1265-2222, and N-term
corresponds to amino acids 1-1265 of Pol2p. The strain was transformed
with the LexA DNA binding domain plasmid and Gal4 activation domain
plasmid, and the colonies were selected on
Leu/Trp/His synthetic agar
plates containing 1 mM 3-amino-1,2,4-triazole. The colonies
that grew were further analyzed by X-gal filter lifts, and interactions
were quantitated by -galactosidase assays. The measurements were
done in replicates of 4-11 samples, and the data are reported as an
average. Numbers in parentheses are standard
deviation.
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An overview of these data is provided in the model shown in Fig. 7. In
summary, the C terminus of Pol2p interacts with Dpb2p, Dpb3p, and
Dpb4p. Dpb3p and Dpb4p directly interact with each other in the absence
of Pol2p and Dpb2p. Dpb2p forms a homodimer and interacts with the N
terminus of Pol2p as well as the C terminus. The dimerization of the
Pol2p C terminus seen in the two-hybrid system is probably indirect and
may occur through interaction of two Pol2p subunits with a Dpb2 dimer.
Point Mutations in the C-terminal Inter-zinc Finger Domain of
POL2--
In our previous study, we showed that the pol2-E
and pol2-F mutants just described showed defects in DNA
replication and the S/M checkpoint (Fig.
8A and Ref. 21). Now we have
provided further evidence that the defect lies in interaction with
other subunits. However, it is not clear whether the specific amino
acid residues between the zinc fingers play an essential role or if the
deletions cause a structural change adjacent to the amino acid
residues. To investigate specific amino acids, we made point mutations
in two conserved leucines (L2146D and L2150D) in the inter-zinc finger domain (Fig. 8, A and B). The mutant proteins
were analyzed for complementation of a pol2 strain using
a plasmid shuffling assay as described previously (21). The
pol2-310 and pol2-311 mutants, with L2146D or
L2150D mutations, respectively, showed no detectable growth defect.
However, the pol2-312 mutant, with an L2146,L2150D double
mutation, was inviable at any temperature (Fig. 8C). These results strengthen the conclusion that specific amino acids covered by
the pol2-E mutation are essential for cell viability,
although it does not prove that there is no structural change.
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Fig. 8.
Characterization of the point mutations
affecting the putative interzinc finger domain of
POL2. A, schematic diagram of putative
zinc finger of POL2. The pol2-E and
pol2-F mutants each containing site-specific 10 consecutive
amino acid deletion is highlighted in a box (21).
B, amino acid alignment showing the conservation of leucine
amino acid residues in pol from various sources. C,
point mutations in the conserved leucine. The mutants were prepared as
described under "Experimental Procedures." The mutant
pol2 genes were subcloned in the pRS314 vector
and introduced into the A1128
pol2-3::LEU2(YEpPOL2) as described
before (21). The transformants were replica plated on agar plates
lacking tryptophan and containing 5-fluoroorotic acid
(5-FOA) at 24 °C and 370 °C for 4 days.
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DISCUSSION |
DNA polymerases are large molecular machines that play an integral
role in chromosomal replication. Polymerase plays a major role in
the elongation stage of the replication of chromosomal DNA. Recently, a
number of studies have indicated an additional role of pol in S/M
checkpoint regulation, recombination, and transcriptional silencing
(16, 19, 21, 28-31). By site-specific deletion analysis in the
putative zinc finger domain of POL2, we showed that the
amino acid residues in the inter-zinc finger domain are essential for
DNA replication, DNA repair, and the S/M checkpoint. We have also shown
that the putative zinc finger participates in protein/protein
interactions. In this study we have extended our analysis of the
inter-zinc finger domain of POL2 and its protein/protein
interactions. By point mutations, we showed that the two conserved
leucines in the inter-zinc finger domain are essential for the cell
viability. The essential nature of the leucine amino acid residues
suggests that protein/protein interactions mediated through the
inter-zinc finger could be mainly hydrophobic in nature, although we
cannot completely rule out ionic and hydrogen bond interactions, given
the sequence shown in Fig. 8B. A recent crystal structure
analysis of a cocrystal containing the phage RB69, "PCNA"-like
sliding clamp and the C-terminal, 11-residue peptide of RB69 DNA
polymerase showed that the "PCNA" is bound in a hydrophobic pocket
through Leu-897, Met-900, and Phe-901 amino acid residues of the
polymerase peptide (32). The C-terminal 11 amino acid residues of the
polymerase have also been shown to be essential for the interaction of
T4 DNA polymerase with the T4 sliding clamp (33). It is possible that
another subunit of pol , Dpb2p, uses a similar mode of interaction
with Pol2p, as suggested by our comprehensive genetic and biochemical analysis of the pairwise protein/protein interactions between the
various subunits of pol complex.
Studies of contacts between four of the subunits of pol gave the
following picture of their organization. Pol2p forms a stable complex
with Dpb2p. The human homolog of Dpb2p also forms a very tight complex
with the mammalian Pol2p catalytic subunit (12), suggesting a
structural conservation between human and yeast pol complexes.
Dpb3p and Dpb4p also form a strong complex, and we propose that the
Pol2p·Dpb2p dimer interacts with the Dpb3p·Dpb4p dimer to form the
holoenzyme isolated from yeast by Sugino and colleagues (4). The
proteins in the Pol2p·Dpb2p heterodimer can make multiple contacts
with proteins in the Dpb3p·Dpb4p dimer. Pol2p interacts independently
with Dpb3p and Dpb4p, but the interactions are weaker than in the
presence of Dpb2p. The Pol2p/Dpb3p and Pol2p/Dpb4p interactions are
also much weaker than the Pol2p/Dpb2p interaction (both biochemically
and by two-hybrid analysis). Dpb2p also interacts independently with
Dpb3p and Dpb4p (Fig. 3), but both purification of complexes from
insect cells infected with baculoviruses carrying all four subunits and
two-hybrid analysis suggests this interaction may be weak (Fig. 7). The
C terminus of Pol2p is sufficient to interact with Dpb2p. A model for
the interaction of the subunits is shown in Fig. 7.
Several studies based on purification of pol activity from yeast
cells support the contacts suggested here. Pol purified from a
dpb2-ts mutant lacked Dpb2p but contained Dpb3p (10). This
is consistent with our observation of a direct Pol2p/Dpb3p contact.
When pol was purified from a dpb3 yeast strain, Dpb2p copurified with Pol2p but Dpb4p was not present (11). This can be
explained if Dpb3p and Dpb4p interact more strongly with each other
than with Pol2p or Dpb2p and if Dpb3p interacts more strongly with
Pol2p than does Dpb4p alone, as our results suggest (Fig. 7). By the
same token, it is possible that a dpb4 mutation would result in the dissociation of Dpb3p from the pol complex, although the interaction between Dpb3p and Pol2p is stronger than between Dpb4p
and Pol2p by two-hybrid analysis (Fig. 7). Recent work on the human pol
is also consistent with our findings.
We also attempted to produce the four-subunit pol complex by
coexpressing baculoviruses containing all four subunit genes (data not
shown). We were not able to recover all four proteins unless we tagged
both Dpb2p and Dpb3p. Since our experiment involved simultaneous
coexpression of all four proteins, it is possible that the four subunit
complex must assemble in a specific order. For instance, since we have
established that Dpb3p and Dpb4p interact with Pol2p, it is possible
that their interaction prevents proper association with Dpb2p. Such
precise order of assembly has been observed in the E. coli
replicase, where addition of - components before the addition of
prevents dimerization (34). Another possibility is that another
protein such as Dpb11p or Sld2p, which interact with pol , might be
essential for proper assembly. Yet another alternative is that Dpb3p
and Dpb4p function by virtue of their histone-fold motifs to allow
entry of pol into nucleosomes, as recently proposed (27). Thus,
Pol2p·Dpb2p efficiently assemble with Dpb3p and Dpb4p only in the
context of chromatin. In regard to a role for Dpb3p in chromatin
structure, it is intriguing that dpb3 mutations were
identified in a screen for genes involved in rDNA silencing. The
structure of silenced chromatin in the rDNA locus may somehow be
altered in dpb3 mutants to allow transcription of the DNA
(31). All four subunits of the human enzyme have been shown to
coimmunoprecipitate when using an antibody to the Dpb4p homolog, p17,
tagged with a FLAG epitope (27).
Our biochemical analysis indicates that Pol2p dimerizes. The
dimerization of the Pol2p is most likely not direct (Fig. 4), but
instead occurs through homodimerization of the Dpb2p subunit (Figs.
5-7). This situation is somewhat reminiscent of the pol III' form of
the E. coli replicase described many years ago by McHenry and O'Donnell laboratories (34), in which the pol III core containing polymerase and proofreading nuclease subunits is dimerized by interaction with a dimeric subunit. The pol inter-zinc finger region is crucial for dimerization, as we proposed previously (21),
since gel filtration of the pol2-Fp mutant complex did not show a
heterotetrameric polymerase complex, demonstrating that the mutation
destabilizes Pol2p interactions with Dpb2p. This was more directly
verified by showing that little pol2-Fp coeluted with
His6-Dpb2p from the Ni2+/NTA column. The
overexpression of Dpb2p under the GAL1,10 promoter suppressed the growth defect of the C-terminal pol2-F
temperature-sensitive mutant at the nonpermissive temperature (Fig. 2),
suggesting that we have identified a physiologically significant
interaction domain. It will be of interest to isolate and examine the
phenotype of dpb2 mutants that fail to dimerize.
The dimerization of pol is interestingly different from the
dimerization of pol . Pol31p, the B subunit of pol which is
homologous to Dpb2p, does form a heterodimer with the catalytic subunit, Pol3p. However, Pol31p does not form a homodimer. Instead Pol31p interacts with PCNA and with the third pol subunit, Pol32p. It is the latter subunit, Pol32p, that homodimerizes and then leads to
dimerization of the Pol3p·Pol31p heterodimer (35, 36). Recently, pol
from S. pombe has also been shown to be a dimer of the
heterotetramer containing Pol3p, Cdc1p, Cdc27p, and Cdm1p and
dimerization was mediated by the essential Cdc27 subunit (37). Mutants
of pol that fail to dimerize have not been described to date.
Although it is thought that leading and lagging strand elongation are
coordinated through the use of two different gene products, pol and
pol , one on each strand, obviating the need for dimeric polymerases
as are found in prokaryotes, a possible role for dimeric polymerases in
eukaryotes could be coordination of the two forks emanating from a
single origin. The idea that the two branch points of a bidirectional
replication fork are connected into a binary replisome was put forward
as a mechanism for preventing rotation of the two forks with respect to
one another in order to reduce tangling of the sister chromatids (38).
Dimerization of the polymerases could both link the forks physically
and ensure synchronous activity at both forks. It has previously been
proposed that DNA helicases may also have an organizational role in
tethering forks to each other (39).
(pol 
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Braun Laboratories
147-75, California Institute of Technology, Pasadena, CA 91125. Tel.:
626-395-6053; Fax: 626-405-9452; E-mail:
jcampbel@cco.caltech.edu.
