(Received for publication, October 24, 1994)
From the
The catalytic polypeptide of human DNA polymerase was
overexpressed in BSC-40 cells (African green monkey kidney cell line)
using the vaccinia virus/pTM1 system. The recombinant human DNA
polymerase
was purified to homogeneity in two steps using an
immunoaffinity column and a single-stranded DNA-cellulose column.
Levels of expression were about 1% of soluble cytosolic protein. The
recombinant catalytic subunit was fully active and exhibited enzymatic
properties similar to that of the native two-subunit enzyme including
the possession of an associated 3` to 5` exonuclease activity.
Recombinant pol
was stimulated by proliferating cell nuclear
antigen (PCNA); however, the degree of stimulation was lower than that
of the native human enzyme. Analysis of a double mutant of the
catalytic subunit, H142R/F144S, showed that it had a greatly reduced
sensitivity to PCNA, suggesting that the PCNA binding site of pol
may be located in this region of the N terminus.
DNA polymerase (pol
) (
)was first isolated
from the rabbit reticulocyte (Byrnes et al., 1976). Calf
thymus (Lee et al., 1984) and human (Lee et al.,
1991) pol
have been isolated to near homogeneity and consist of
two subunits of 125 and 50 kDa. The 125-kDa polypeptide was
demonstrated to be the catalytic polypeptide by activity staining (Lee et al., 1991). A central role of this enzyme activity in
mammalian DNA replication has been established through the
investigation of the in vitro replication of the SV40 genome.
A complex array of accessory proteins are also involved. The functional
insertion of pol
at the replication fork requires the actions of
replication factor C, which leads to the recruitment of PCNA to the
primer template terminus followed by binding of pol
. Replication
protein A, a single-stranded DNA binding protein, further potentiates
the activity of the pol
complex. The replication of SV40 has now
been reconstituted in vitro using isolated components (Waga et al., 1994).
Because of the importance of the DNA
polymerases, they have been intensively studied, and there is now a
great deal of information on the primary structures of these enzymes
from a wide range of organisms that span a broad evolutionary spectrum.
Cloning of the human pol cDNA showed that it is a member of a
family of polymerases that include T4 DNA polymerase (Wong et
al., 1988; Wang, 1991). cDNAs for the calf thymus (Zhang et
al., 1991) and human (Chung et al., 1991; Yang et
al., 1992) pol
catalytic subunit have been cloned, as have
the genes for the yeast homologues of pol
(Boulet et
al., 1989; Pignede et al., 1991). Mammalian and yeast pol
are more highly conserved with each other than with their
homologous pol
enzymes (Yang et al., 1992). Multiple
sequence alignments have shown that there is a highly conserved central
core region in the pol
family that is thought to contain most of
the catalytic site (Wang, 1991). There is less conservation in the
C-terminal regions, which, however, do contain two zinc finger regions
that are thought to be involved in DNA binding. Analysis of the
N-terminal regions show that there is some conservation among the
members of the pol
group of enzymes, and that these regions are
conserved to some extent in herpes simplex virus type I DNA polymerase
(Yang et al., 1992). The regions involved in the 3` to 5`
exonuclease activity of yeast pol
have been identified by
site-directed mutagenesis (Simon et al., 1991). Little is
known of the regions of pol
that are involved in its functional
interactions with other proteins. We have attempted to develop a system
for the overexpression of pol
that will provide a ready means for
the preparation of the enzyme and for study of its structure-function
relationships. In this report we describe the successful expression of
the active catalytic subunit of pol
using a vaccinia virus/pTM1
expression system in BSC-40 cells.
The human DNA pol
cDNA clone (Yang et al., 1992) was digested with SacI and BamHI. After purification by agarose gel
electrophoresis, the 2.7-kilobase pair fraction from SacI to
termination codon was selected and then was ligated into pTM1-
-5`,
which had been previously digested with SacI and BamHI. After transformation of E. coli DH5
competent cells, the single colony harboring the full-length pol
coding sequence in pTM1 (pTM1-
) was selected. The DNA sequence of
pTM1-
was again confirmed by DNA sequencing (Sanger et
al., 1977).
The pTM1 plasmid allows the insertion of a coding sequence for a given protein under the control of the T7 promoter and also contains the 5`-untranslated sequence of the encephalomyocarditis virus untranslated leader region. This plasmid was designed to allow expression of foreign messages in mammalian cells in the presence of T7 RNA polymerase following transfection with the plasmid. Eukaryotic expression is based on simultaneous infection with a recombinant vaccinia virus (vvTF7-3) that synthesizes the bacteriophage T7 RNA polymerase. Efficient translation of the uncapped mRNAs in mammalian cells is due to the presence of the encephalomyocarditis virus untranslated leader region (Nakano et al. 1991). For transient expression in mammalian cells, an infection/transfection protocol is performed with the plasmid and the recombinant vaccinia virus expressing T7 RNA polymerase (Nakano et al., 1991).
Figure 1:
SDS-PAGE and Western blot analysis of
BSC-40 cells for the expression of the catalytic subunit of human pol
. BSC-40 cells were infected/transfected with vvTF7-3 and
pTM1 or pTM1-
(see ``Experimental Procedures''). Cell
extracts were then prepared and subjected to SDS-PAGE and Western
blotted with a monoclonal antibody directed toward the C terminus of
human pol
. Leftpanel shows the Coomassie Blue
staining of SDS-PAGE gels. LaneS, protein standards
(
-macroglobulin, 180 kDa;
-galactosidase, 116
kDa; fructose 6-phosphate kinase, 84 kDa; pyruvate kinase, 58 kDa;
fumarase, 48 kDa; lactate dehydrogenase, 36 kDa; triosephosphate
isomerase, 26 kDa). pTM1-
and pTM1 refer to
cytosolic cell extracts from cells transfected with either the
pTM1-
or the pTM1 control plasmid. Centerpanel,
Western blots of the cytosolic cell extracts (50 µl) from cells
transfected with the pTM1-
and the pTM1 plasmids. Rightpanel, Western blots (50 µl) of the nuclear and
cytosolic extracts from cells transfected with the pTM1-
plasmid.
When the nuclear extract was concentrated 20-fold and Western blotted,
a weak reaction was detectable (not shown).
Figure 2:
Purification of recombinant pol by
immunoaffinity chromatography. A cell extract from BSC-40 cells that
had been transfected with the pTM1-
plasmid was chromatographed on
an immunoaffinity support (see ``Experimental Procedures'').
The diagram shows the assay of the fractions from the column that were
eluted with 30% ethylene glycol, 50 mM Tris-HCl, 0.7 M NaCl, 0.1 mM EDTA, pH 7.9. Samples (5 µl) of the
fractions were assayed for DNA polymerase activity using poly(dA-dT) as
the template (solidtriangles) or with
poly(dA)/oligo(dT) in the presence of PCNA (solidcircles), and for 3` to 5` exonuclease activity using
3`-end labeled poly(dT) (solidsquares).
Figure 3:
Western blot analysis of the fractions
obtained by immunoaffinity chromatography. Samples (50 µl) from the
column fractions shown in Fig. 2were subjected to SDS-PAGE and
stained for protein with Coomassie Blue (lowerpanel)
and Western blotted using a C-terminal antibody against pol (upperpanel; only the 125 kDa band is
shown).
The peak
fractions of the immunoaffinity column were then passed through a
single-stranded DNA cellulose column. After this step, the recombinant
protein was near homogeneous and was Western blotted by a monoclonal
antibody to the C-terminal region of human pol (Fig. 4).
The summary of the purification achieved is shown in Table 1.
Approximately a 100-fold purification was required, i.e. the
pol
polypeptide is expressed at about 1% of the soluble cytosolic
protein. The levels of enzyme activity and protein far exceed those
present in the BSC-40 cytosol, so that contamination of the expressed
protein with endogenous BSC-40 pol
, or for that matter, with
vaccinia virus DNA polymerase, is unlikely, particularly since it was
purified by a specific immunoaffinity chromatography procedure.
Figure 4:
SDS-PAGE and Western blot analysis of
recombinant pol after single-stranded DNA cellulose
chromatography. The diagram shows the SDS-PAGE analysis of pol
obtained after single-stranded DNA-cellulose chromatography (see
``Experimental Procedures''). Samples of the peak fractions
of activity (50 µl) were subjected to SDS-PAGE and stained with
Coomassie Blue (A) and Western blotted using anti-pol
monoclonal antibody (B). S refers to protein
standards as in Fig. 1.
Figure 5:
Sensitivity of native and recombinant pol
to aphidicolin. Purified human placental pol
(Lee et
al. 1991) and purified recombinant pol
-p125 (5 µl each)
were assayed in the presence of increasing amounts of aphidicolin. The
results were expressed as relative activities (solidcircles, recombinant pol
; opencircles, native pol
).
Figure 6:
Inhibition of recombinant pol by
KCl. Purified human placental pol
(Lee et al. 1991) and
purified recombinant pol
were assayed using poly(dA)/oligo(dT) as
the template in the presence of PCNA and in the presence of increasing
amounts of KCl. The results were expressed as relative activities (squares, recombinant pol
; circles, native pol
.).
The response of recombinant pol
to PCNA was examined. The recombinant enzyme was sensitive to
PCNA and was stimulated 4.5-fold by 300 ng of PCNA. A highly purified
human placental pol
preparation (Lee et al., 1991)
purified by conventional means was stimulated 10-fold by the same
amount of PCNA (Fig. 7). In the same experiments, examination of
the reaction products showed that the stimulation of recombinant pol
by PCNA is also due to an effect on processivity.
Figure 7:
Response of recombinant pol to PCNA.
Purified human placental pol
(Lee et al. 1991) and
purified recombinant pol
-p125 (5 µl each) were assayed using
poly(dA)/oligo(dT) as the template in the presence of increasing
amounts of PCNA. The results were expressed as relative activities (squares, recombinant pol
; circles, native pol
.) The rightpanel shows an analysis of the
reaction products to determine effects of PCNA on the processivity of
pol
(see ``Experimental Procedures''). Lanes1 and 5, human placental pol
in the
absence and presence of PCNA; lanes2 and 6,
recombinant pol
in the absence and presence of PCNA; lane3, PCNA alone; lane4, template
alone.
Figure 8:
Lack
of PCNA response by a double mutant of pol . The H142R/F144S
mutant (squares) and the wild type recombinant mutant pol
(circles) were assayed in the presence of increasing
amounts of PCNA using poly(dA)/oligo(dT) as the template (see
``Experimental Procedures'').
We report here the first successful overexpression of the
catalytic subunit of human pol as a soluble active protein using
a vaccinia virus/plasmid vector system. The amounts of protein
expressed were estimated to be 1% of the soluble protein. In practical
terms, about 22 µg of p125 could be prepared to near homogeneity
using a simple 2-step procedure involving immunoaffinity chromatography
and single-stranded DNAcellulose chromatography. Isolation of the
mammalian pol
is extremely tedious, and only microgram amounts of
purified enzyme can be obtained (Lee et al., 1984, 1991). Only
a handful of laboratories have ever reported preparations of
demonstrable purity owing to the difficulties of its isolation, and
even highly purified pol
preparations commonly consist of
multiple polypeptide components (Goulian et al., 1990; Feher
and Mishra, 1994). There are no previous reports of the expression of
mammalian pol
, and we also have encountered extreme difficulty in
expression of this cDNA in E. coli. Yeast DNA pol
has
been overexpressed in E. coli (Brown and Campbell, 1993) as an
insoluble and inactive aggregate, and the recombinant enzyme was
obtained after solubilization with urea and renaturation.
These
studies show that the vaccinia virus (vvTF7-3)/pTM1 system is
effective for the expression of the human pol catalytic subunit.
The use of the vaccinia virus/pTM1 infection/transfection system has
been shown to allow for very efficient overexpression of genes in
mammalian cells (Fuerst and Moss, 1989; Moss et al., 1990) and
also allows for rapid screening of the mutants. Levels of the foreign
transcripts have been estimated to be as high as 30% of the total
steady-state RNA in the cytoplasm of the infected cells after 24 h as
shown for T7-lac Z transcripts (Fuerst and Moss, 1989). The recombinant
vv/T7 hybrid expression system offers certain advantages over the
baculovirus system. For example, site-directed mutants of the target
gene can be constructed rapidly in the expression vector pTM1, and
their biological activities can be assayed by the
infection/transfection protocol (Moss et al., 1990). In
addition, protein synthesized in insect cells are likely to be
different from those synthesized in mammalian cells with respect to
some of post-translational modifications (Miller, 1988). Thus, the
vaccinia virus system allows for overexpression in a mammalian cell
context that provides some assurance that any post-translational
modifications would be those normally encountered.
Our studies show
that the recombinant p-125 is almost exclusively found in the cytoplasm
of the BSC-40 cells. Our findings differ from those of the adenovirus
DNA polymerase, which was transiently expressed using the same vv/pTM1
expression system (Nakano et al., 1991) in that the
recombinant adenovirus polymerase was distributed both in the cytoplasm
and nucleus. It may be possible that translocation of pol -p125 to
the nucleus is a relatively slow process, or alternatively, it may be a
regulated process.
Recombinant pol was characterized and
exhibited essentially similar catalytic properties as the native
enzyme. However, we observed that its PCNA response was consistently
lower than that of enzyme isolated from human placenta. It has been
previously suggested that the catalytic polypeptide is not responsive
to PCNA and that this property is conferred by the p50 subunit. The
evidence is inferential and is based on isolation of enzyme
preparations that consist only of the 125-kDa subunit and do not
respond to PCNA. Mouse cells have been suggested to possess of two
forms of pol
, one consisting of only a 125 kDa, and the second
having two subunits of 125 and 50 kDa. The first is not stimulated by
PCNA, suggesting that the 50-kDa subunit is required for the
interaction of polymerase
with PCNA (Goulian et al.,
1990). Rabbit reticulocyte pol
was reported to have a single
subunit of 122 kDa (Goscin and Byrnes, 1982). This form was later
reported by Lu and Byrnes(1992) to be a PCNA-independent form. The same
authors have also purified a PCNA-dependent DNA polymerase
from
rabbit bone marrow that is similar to the calf thymus DNA polymerase
in consisting of two subunits and being responsive to PCNA.
Chiang et al.(1993) purified a pol
from Drosophila to near homogeneity, which consisted of a single 120-kDa
polypeptide and is not stimulated by PCNA. Our results show that
unequivocally the human p125 subunit does respond to PCNA. Our data
agree with that of Brown and Campbell(1993), who showed that
overexpressed yeast pol
in E. coli is stimulated about
2.5-3-fold by PCNA. Nevertheless, our findings do indicate that
the p50 subunit may be important in the response of the catalytic
subunit to PCNA, since the degree of stimulation by PCNA was
consistently lower than that of the native two subunit enzyme
consisting of 125- and 50-kDa subunits. In other preliminary studies,
we have shown by reconstitution experiments that the addition of the
p50 subunit also potentiate the response of the recombinant p125
subunit to replication factor C and replication protein A in the
presence of PCNA.
The vv/pTM1 system thus provides a useful means
for preparation of the catalytic subunit as well as a means for
structure-function studies of pol by mutagenesis. Examination of
an adventitious double mutant in which 2 residues in the N terminus,
His-142 and Phe-144, were mutated to Arg and Ser, respectively, showed
that its response to PCNA was severely attenuated. This strongly
suggests that this region is involved in PCNA binding. This is the
first report of a site-directed mutant that is affected in its PCNA
binding and is consistent with other studies using synthetic peptides
that suggest that the PCNA binding region of pol
is localized to
a region between residues 129 and 149 (Zhang et al., 1995).