(Received for publication, September 16, 1994; and in revised form, January 25, 1995)
From the
DNA polymerase (pol
) from HeLa cells was
purified to near homogeneity, utilizing Mono S fast protein liquid
chromatography for complete separation from pol
. The purified pol
preparation showed two polypeptides of >200 and 55 kDa and a
small amount of active 122-kDa proteolysis product on denaturing
polyacrylamide gels. Pol
(as well as pols
and
) is
optimally active in 100-150 mM potassium glutamate and
15 mM MgCl
. Replication factors RF-A and RF-C,
proliferating cell nuclear antigen, and Escherichia coli single-stranded DNA binding protein showed no significant effect
on this preparation's pol
activity, processivity, or
substrate specificity. The size of the pol
transcript for the
catalytic subunit (>200 kDa) was investigated in both normal human
fibroblasts and HeLa cells. A 7.7-kilobase transcript was detected
which was 5-16-fold more prevalent in proliferating than in
quiescent HeLa cells. No significant difference in the level of pol
transcript in HeLa cells or fibroblasts was seen after
ultraviolet irradiation. Mouse polyclonal antiserum was produced to a
144-amino acid fragment of pol
fused to staphylococcal protein A.
This non-neutralizing polyclonal antiserum specifically recognized the
catalytic subunit of pol
by immunoblotting, but not that of pol
,
, or
. In addition, mouse polyclonal antiserum raised
against column-purified pol
was able to recognize and to
neutralize pol
, and a mouse monoclonal antibody was raised which
was able to recognize specifically the catalytic subunit of pol
.
DNA polymerase (pol
) (
)from HeLa cells,
unlike yeast pol
(1) , contains two apparent subunits of
>200 and 55 kDa (2) and appears to be required for long
patch DNA repair in permeabilized cells(3) . Unlike pol
,
pol
is highly processive in the absence of proliferating cell
nuclear antigen (PCNA). Studies with primed, single-stranded DNA
observed that human single-stranded DNA binding protein (HSSB; also
known as replication factor-A (RF-A)) and activation inhibitor (also
known as A1, or replication factor-C (RF-C)) (
)stimulate pol
and pol
activities(4, 5) . These
replication factors are required for sequential initiation of lagging
and leading strand DNA synthesis in reconstituted SV40 replication
systems(6, 7) . It has also been suggested that
inhibition of HeLa pol
by NaCl can be overcome by the presence of
HSSB, A1, and PCNA(8) . We have investigated the effect of
these replication factors on HeLa DNA pol
processivity and
activity under a variety of salt conditions.
Previous studies of
template specificity among the three HeLa DNA pols suggested that pol
prefers activated DNA as template, and pol
prefers
alternating poly(dA-dT) as template, whereas pol
prefers long
stretches of single-stranded regions in poly(dA)
oligo(dT) as
template(9) . We have further investigated the template
specificities of these polymerases, particularly with
polyribonucleotide primers.
It has been reported that yeast pol
is involved in DNA replication (1) , and our current
interest is to determine whether HeLa pol
is involved in either
or both DNA replication and DNA repair. To address the question of DNA
replication, we compared the mRNA levels of pol
in quiescent and
proliferating HeLa cells as well as in untreated and UV-irradiated HeLa
cells and normal diploid fibroblasts.
We have also begun an
immunogenic study for which a fusion protein (staphylococcal protein A
fused to a pol fragment) was used to produce antiserum that is
able to recognize denatured pol
but can neither immunodeplete nor
neutralize enzymatic activity. Purified pol
was also used to
raise a mouse antiserum that is able to recognize different pol
subunits and neutralize pol
activity and shows no cross-reaction
with HeLa pols
,
, and
. We have now produced monoclonal
antibodies from this mouse and have obtained purified IgGs, one of
which is described here. It specifically recognizes the pol
catalytic subunit.
The protein A gene fusion vector
pRIT31 was originally constructed by Uhlen and co-workers (13) and maintained in E. coli RSB571 (derived from E. coli 697). This vector has both the lac and the temperature-inducible
C1
promoters.
The glutathione S-transferase (GST) fusion vector,
pGEX-1
T, was purchased from Pharmacia with the correct reading
frame for the pol
fragment and maintained in E. coli DH5
. The pol
fragment used for constructing the Staphylococcus aureus protein A (SpA-) fusion protein was a
435-bp BamHI/EcoRI fragment of a 1.2-kb clone
obtained in this laboratory. This pol
fragment (nucleotides
1611-2045) (14) contains the conserved region II of the
-class DNA polymerases(15, 16) .
BA-S NC nitrocellulose membranes for Northern blots were from Schleicher & Schuell. Randomly primed DNA kits for labeling probes and RNA molecular markers II (7.4, 5.3, 2.8, 1.9, and 1.6 kb) were purchased from Boehringer Mannheim. GeneClean kits were purchased from U. S. Biochemical Corp.
Processivity assays used either single primed
(dA)oligo(dT)
or single
primed M13mp18 DNA. Reactions were as above except that
[
-
P]TTP (about 5,000-8,000 cpm/pmol)
was used. The incorporation was controlled so that on average roughly
one nucleotide or less was incorporated per primer terminus. Thus the
average length of the product in each case corresponds to a single
elongation event and reflects the processivity of the enzyme.
Primase activity was assayed by coupling oligoribonucleotide
synthesis to polymerization of dNTPs by E. coli DNA polymerase
I Klenow fragment(19) . The rate of the reaction was determined
by monitoring the incorporation of labeled dNTPs into acid-insoluble
material. Reaction mixtures (50 µl) contained 50 mM Tris-HCl (pH 7.5), 0.5 mM DTT, 0.5 mg/ml BSA, 7.5 mM MgCl, 1 mM ATP, 80 µM (dT)
, 20 µM [
H]dATP (200 cpm/pmol), and 0.5 unit of
Klenow fragment. Incubations were for 30 min at 30 °C.
Thirty µg of the total RNA was
denatured and electrophoresed through 0.7% agarose, 6.3% formaldehyde,
50% formamide gels at 40 volts for about 3 h. The gels were
electroblotted onto a nitrocellulose membrane for 8-10 h in 20
SSC and then fixed at 80 °C under vacuum for 1-2 h.
The membrane was prehybridized at 42 °C for a minimum of 6 h and
then hybridized with a
P-labeled probe at 42 °C in 50%
formamide for at least 12 h in the presence of 50% formamide. After
hybridization, the membrane was washed twice in 2
SSC, 0.5% SDS
for 1 h at room temperature and then twice in 0.1
SSC, 0.5% SDS
for 1 h at 50 °C and finally twice at 65 °C with Ultra Blot
Wash (50 mM Tris-HCl (pH 8.0), 2 mM EDTA, 0.5% sodium
pyrophosphate, 1
Denhardt's solution, 1% SDS, 0.05% N-lauroylsarcosine). Membranes were exposed on Kodak XAR-5
film at -80 °C for appropriate times with an intensifying
screen, and then band intensities were quantitated with a Hoefer gel
scanner.
For pol activity measurements corresponding to the mRNA
levels, HeLa cells or F65 fibroblasts were treated as for the RNA
preparation and then harvested. Extracts were prepared as described for
the HeLa pol preparation(9) , and then protein was
fractioned with ammonium sulfate. The material that precipitated
between 30 and 50% saturation was resuspended and dialyzed against 4
liters of 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 10%
(v/v) glycerol. All autoradiograms were quantitated with a Hoefer gel
densitometer.
To obtain monoclonal antibodies,
the mouse with the best antigenic response was sacrificed. Its spleen
was removed, minced gently, and then the spleen cells were fused with
myeloma cells by electrofusion at the ratio of five spleen cells to one
myeloma cell. Immediately following the fusion, the cells were grown in
Iscove's modified Dulbecco's medium with 20% FCS and 10%
macrophage-conditioned medium. After 24 h, the unfused cells were
selected against using HAT medium, and then the fused hybridoma cells
were maintained in the same medium with half-strength HT
concentrations. Fused hybridoma cells began to form colonies in about
10 days to 2 weeks. Aliquots of supernatant from wells with a single
colony about one-third of the size of the well were screened for
antibody response by ELISA. High titer samples were tested further by
polymerase neutralization assays and Western immunoblots. Based on the
above results, interesting cell lines were subcloned by serial dilution
and dispensed into five 96-well plates such that each well would
contain only one cell. Hybridomas with positive responses, producing
monoclonal antibodies with the ability to recognize pol subunits
or to neutralize pol
activity, were then selected and scaled up
for IgG production. Cell lines were placed in Iscove's modified
Dulbecco's medium with 10% dimethyl sulfoxide and 30% FCS and
frozen in liquid nitrogen for long term storage.
Figure 1:
SDS-polyacrylamide gel analysis of HeLa
DNA pol purified by Mono S FPLC. The Mono S fraction of HeLa DNA
pol
(0.1 mg) was electrophoresed on a 7% SDS-polyacrylamide gel
and stained with silver. The positions of molecular mass markers are
shown on the left in kDa.
To identify the catalytically
active subunit(s) present in the preparation, the photolabeling
procedure of Insdorf and Bogenhagen (17) was employed. This
technique incorporates BrdUTP and [-
P]dATP
into a synthetic hairpin-primed template. The polymerase stalls at a
site where the complimentary dNTP is absent, and then UV-irradiation is
used to cross-link the pol
to the hairpin template via BrdU.
SDS-polyacrylamide gel electrophoresis is then employed to resolve the
pol
heterodimer, leaving the pol
catalytic subunit
covalently attached to the radioactive template. Autoradiography
revealed two major bands of >200 and 122 kDa (data not shown). Kesti
and Syvaöja (22) identified a 258-kDa
polypeptide in a similar DNA polymerase trap experiment which, when
cleaved by trypsin, generated an active 122-kDa fragment. Thus the Mono
S fraction had both forms of the pol
catalytic subunit.
When assayed with
(dA)oligo(dT)12-18, pol
had maximal
activity between 1 and 15 mM MgCl
, whereas pol
had the highest activity, between 10 and 15 mM MgCl
. Thus the two pols can be distinguished by their
different MgCl
optima when assayed on
poly(dA)
oligo(dT). However, when assayed on primed M13 DNA, both
pols had a maximal activity at 1 mM MgCl
.
As seen in Fig. 2A, the factors also
had little effect upon processivity of pol in these same
reactions; the enzyme was extremely processive in the presence or
absence of these factors. In fact, pol
was able to copy the
entire M13mp18 genome in the absence of factors, and PCNA had no effect
on pol
activity or the DNA product size (Fig. 3).
Figure 2:
Effect of replication factors on the
processivity of pol with single primed M13 DNA. Panel A,
reactions are those of Table 2, Experiment I. The pause site
noted near nucleotide 120 is the synthetic polylinker region of M13mp18
DNA. Panel B, reactions are those of Table 2, Experiment
II, with 1 mM MgCl
, either in the presence or
absence of 100 mM NaCl. DNA products were analyzed on 8%
polyacrylamide, 7 M urea gels.
Figure 3:
Effect of PCNA on the processivity of pol
with single primed M13 DNA. Assays (25 µl) contained 1 mM MgCl
, 2.5 pmol of primer termini, and 80 ng of HeLa
PCNA from various phenyl-Sepharose fractions and were as described
under ``Experimental Procedures.'' DNA products were isolated
and analyzed on a 0.8% alkaline agarose gel. Lane M, DNA size
markers with their apparent lengths (in nucleotides) shown on the left. Reactions for lanes 1-4 contained no PCNA
and fractions 80, 78, and 84 of PCNA, respectively. Incorporation for
the four reactions was 6.6, 5.3, 7.5, and 7.7 pmol of dNMP,
respectively.
Pol
activity was strongly inhibited by the presence of 100 mM NaCl, and the various replication factors alone had very little
effect on this salt inhibition, although this inhibition could be
slightly overcome when all three replication factors were present (Table 2; Fig. 2B).
When the assays were
repeated with single primed
(dA)oligo(dT)
, there was
also no major effect on pol
either by PCNA or by all three
factors, although RF-A and RF-C individually stimulated pol
activity slightly in either 1 or 15 mM MgCl
(see Table 3, Experiment I; Fig. 4A). When 60 mM NaCl was added, activity and processivity were both greatly
reduced, and the individual factors had little effect upon pol
.
However, when RF-A, RF-C, and PCNA were all present, pol
activity
was enhanced almost 2-fold and a somewhat longer DNA product was
observed (Table 3, Experiment II; Fig. 4B).
Similar experiments were performed with 100 mM NaCl present (Table 3, Experiment III; Fig. 4C), and again
none of the factors individually or together was able to overcome the
severe inhibition of activity or product length by the salt.
Figure 4:
Effect of replication factors on the
processivity of pol with single primed poly(dA)
oligo(dT).
Reactions were as described in Table 3, Experiments I, II, and
III. DNA products were analyzed on 8% polyacrylamide, 7 M urea
gels. The spot located on the top of the rightmost track of panel C is spurious radioactivity.
Longer exposures of the gel in panel C showed no material in
the gel, only enhanced spots at the tops of the
tracks.
When
the effects of the replication factors on pol activity with
poly(dA)
oligo(dT) in the presence of 175 mM potassium
glutamate were determined, none of the factors had any significant
effect (Table 3, Experiment IV). In effect, the pol
activity was inhibited by 90% when 175 mM potassium glutamate
was present, but this salt inhibition was not overcome by either HSSB,
A1, PCNA, or the three in combination, although PCNA and A1 showed weak
enhancement. These results are in contrast to those reported by Lee et al.(8) in which under the same conditions the
presence of the various replication factors (HSSB, A1, and PCNA)
overcame the salt inhibition.
Figure 5:
Northern blot analyses for the transcript
of pol catalytic subunit in quiescent versus serum-stimulated cells. Panel A, 30 µg of total RNA
was present in each lane. Preparations of RNA, blotting, and
quantitation were as described under ``Experimental
Procedures.'' Quiescent HeLa cells were mid-log phase cells that
were resuspended in medium containing only 0.1% FCS and held for 96 h.
Half of these serum-starved cells were then fed medium containing 10%
FCS for 30 h to obtain proliferating cells. As a control, a
800-bp
chicken actin cDNA fragment was used as a probe. The levels in all
samples were determined by gel scanning. Panel B, procedures
were as in panel A, except that cells were held in quiescence
for 48 h and then fed medium containing 10% FCS. Total RNA was prepared
at the times indicated for Northern
analyses.
The level of transcript 4 h after UV irradiation in total RNA of HeLa cells or diploid normal fibroblasts was also studied (Fig. 6, A and B, respectively), and no effects from the UV radiation were seen. Similar results were obtained when purified mRNA was probed. Hence the UV radiation does not appear to alter expression of the gene.
Figure 6:
Northern blot analyses for the transcript
of pol catalytic subunit after UV irradiation of HeLa cells and
diploid fibroblasts. Thirty µg of total RNA obtained 4 h after
irradiation for 7 s at 2 J/m
/s was used in each lane, and
all cells were at mid-log phase during treatment. The preparation for
total RNA, blotting procedures, and irradiation conditions are
described under ``Experimental Procedures.'' As a control, a
800-bp chicken actin cDNA fragment was used as a probe. The levels
in all samples were determined by gel scanning. Panel A, HeLa
cells; panel B, normal diploid human fibroblasts.
Pol
activities were measured in parallel to mRNA levels. In the presence of
the pol
antibodies, greater than 95% of the pol
activity
was depleted, allowing pol
activity to be measured specifically.
Pol
activities were also monitored for comparison and did not
change appreciably (Table 4). When DEAE fractions (9) from quiescent and proliferating cells (18 h after
quiescence) were compared, pol
activities (as normalized by the
total cell number) increased by roughly 40%, and pol
activities
increased by 62% (Table 4) as calculated from incorporation with
their preferred templates. Likewise, when the levels of catalytic
subunit in DEAE fractions from quiescent and proliferating cells were
compared by immunoblotting with pol
monoclonal antibody, they
contained a similar level of antigen per unit of activity (Fig. 7). Similar constancy was observed when assays were done
with unfractionated crude extracts (not shown). Hence in contrast to
the approximately 6-16-fold increase of mRNA, the amount of the
catalytic subunit of pol
as determined by activity or by
antigenicity changed little. This result recalls similar results with
pol
wherein mRNA levels changed without corresponding changes in
activity(23) .
Figure 7:
Immunoblotting of pol catalytic
subunit from DEAE-Sephacel fractions from quiescent and proliferating
HeLa cells. Proteins in each lane were the pooled fractions from DEAE
chromatography from extracts of either quiescent or proliferating
cells. Various amounts of the above proteins were resolved by
electrophoresis on a 7% SDS-polyacrylamide gel and probed with mouse
monoclonal IgG 3C5.1 at 1 µg/ml as described under
``Experimental Procedures.'' The secondary antibody was
horseradish peroxidase-conjugated sheep anti-mouse IgG at a 1:5,000
dilution. Lanes 1-6 were from quiescent cells. Lanes
7-12 were from proliferating cells. Polymerase activities
were: lanes 1 and 7, 0.5 unit; lanes 2 and 8, 0.33 unit; lanes 3 and 9, 0.17 unit; lanes 4 and 10, 0.07 unit; lanes 5 and 11, 0.033 unit; lanes 6 and 12, 0.017 unit. Lane 13 contained prestained protein standards with their
apparent molecular masses shown on the right in
kDa.
When pol activities from 30-50%
ammonium sulfate fractions of UV irradiation were compared, it was seen
that the UV irradiation also produced no significant change in the
activity of pol
or
with HeLa or normal fibroblast cells (Table 4).
Figure 8:
Immunoblotting of various fractions of DNA
pols ,
,
, or
with polyclonal antibody against the
SpA-pol
fusion protein. The antibody was a purified IgG (987.1)
at a concentration of 0.3 µg/ml from ascitic fluid of a mouse that
was immunized with a protein A-pol
fusion protein (SpA-pol
)
as described under ``Experimental Procedures.'' Purified HeLa
pols were resolved by electrophoresis on a 7% SDS-polyacrylamide gel
then probed with the antibody. Lanes from left to right contained 0.12 unit of the Mono S fraction of pol
,
0.15 unit of the phosphocellulose fraction of pol
, 0.11 unit of
the glycerol gradient fraction of pol
, 0.03 unit of the
hydroxylapatite fraction of pol
, and 0.15 unit of the Mono S
fraction of pol
. The positions of protein markers are indicated
on the left in kDa. The catalytic subunits of pols
,
, and
migrate with M
values of 180,000,
40,000, and 125,000, respectively. The hydroxylapatite fraction of pol
did not appear because of the low level of enzyme used. It does
show when the same amount of the same fraction is probed with a
monoclonal antibody as in Fig. 10B.
Figure 9:
Neutralization of pol with
polyclonal antibody against native HeLa DNA pol
. Assays were as
described under ``Experimental Procedures.'' Hydroxylapatite
fraction (0.05 unit) of pol
was used for each reaction. The
relative activity of 1.0 was equal to 26.7 pmol of dTMP
incorporated.
Figure 10:
Immunoblotting with a purified IgG
directed against the pol catalytic subunit. The antibody,
obtained after immunization of a mouse with HeLa pol
, was a
monoclonal purified IgG (3C5.1) (see ``Experimental
Procedures''). It was used at 1 µg/ml. Panel A,
3-µl samples of fractions from DEAE-Sephacel chromatography were
resolved on a 7% SDS-polyacrylamide gel and probed with antibody. The numbers at the top represent the fraction numbers. Panel B, purified HeLa pols
,
,
, and
were resolved by electrophoresis on a 7% SDS-polyacrylamide gel. Lanes 1 and 7, 0.12 unit of a Mono S fraction of pol
; lanes 2 and 8, 0.15 unit of a phosphocellulose
fraction of pol
; lanes 3 and 9, 0.11 unit of a
glycerol gradient fraction of pol
; lane 4, 0.03 unit of
hydroxylapatite fraction of pol
; lane 5, 0.15 unit of a
Mono S fraction of pol
; lane 10, 0.15 unit of a
hydroxylapatite fraction of pol
; lane 6 contained
rainbow marker protein standards whose apparent molecular masses and
positions are shown on the left in kDa. The catalytic subunits
of
,
, and
migrate with M
values
of 180,000, 40,000, and 125,000,
respectively.
Monoclonal lines of hybridoma cells were then
prepared by fusion of mouse spleen cells with myeloma cells, and we
have obtained several monoclonal antibodies that recognize the
catalytic subunit of pol . Using purified IgG from one of these
(3C5.1), fractions across the DEAE-Sephacel gradient were probed, and
the intensity of the 220-kDa subunit on an immunoblot correlated with
the pol
activity (Fig. 10A). Moreover, the
antibody did not recognize pol
,
, or
(Fig. 10B).
Pol from HeLa cells was initially identified in our
laboratory as a DNA repair factor for permeabilized human
fibroblasts(3) . Unlike pol
, pol
was not easily
resolved from pol
, but the utilization of Mono S FPLC
chromatography has now allowed complete separation of the two pols,
albeit still with a somewhat low yield of pol
activity.
Homogeneous HeLa pol
appears by protein staining with silver to
consist of two subunits: >200 kDa and 55 kDa, unlike yeast pol
, which has a large (>200-kDa) catalytic subunit and three
smaller subunits(25) . At present, it is not known whether
homologs of the small yeast subunits exist in human cells and interact
with human pol
. Pols
,
, and
are each
considerably less sensitive to inhibition by potassium glutamate than
by NaCl, KCl, ammonium sulfate, and K
SO
. Richy et al.(26) have demonstrated that potassium and
glutamate are major intracellular ionic osmolytes, and Griep and
McHenry found that E. coli DNA polymerase III holoenzyme could
tolerate a much higher concentration of glutamate than
chloride(27) . Moreover, specific protein-DNA interactions also
tolerate much higher glutamate than chloride
concentrations(28, 29) . Therefore, potassium
glutamate should probably be more commonly utilized as an in vitro buffer and salt.
Neither the activity nor the processivity of
our preparations of HeLa pol is greatly affected by HSSB (RF-A),
A1 (RF-C), or PCNA, individually or in combination. There is a major
pause site for pol
within the M13mp18 DNA template which results
in a band at position 120 bp and maps to the vicinity of the polylinker
(see Fig. 2). The addition of replication factors did not affect
this pausing.
Strong inhibition by salt of pol was observed,
but this inhibition could not be completely overcome by any of the
above replication factors, although when all three replication factors
were present, both pol
activity and processivity were enhanced
roughly 2-fold. These results contrast to those of Lee et al.(8) in which the three replication factors (HSSB, A1, and
PCNA) together could overcome the salt inhibition of pol
under
identical conditions used here. Different forms of pol
have been
reported in various studies: a large form of pol
has a
polypeptide that is greater than 200 kDa; but smaller forms of
catalytic subunits between 125 and 170 kDa have been
observed(1, 22, 30, 31, 32) ,
which appear to be related by proteolytic cleavage(22) . Our
preparation seemed to contain both forms, so the origin of the
differing results of this study and those of Lee et al.(8) cannot be attributable to proteolysis. Whatever the
differences are due to, they are worthy of further study, however,
since PCNA has been observed to affect DNA repair synthesis (33) , which could be attributable to pol
.
Only a
7.7-kb HeLa pol transcript has been identified, so there is no
reason to believe that shorter forms of the active subunit are
controlled at the transcriptional level. Although the levels of pol
activity and the catalytic subunit antigenicity increased only
slightly after serum stimulation of quiescent cells, the pol
transcript level increased up to 16-fold. After UV irradiation,
however, neither the mRNA nor activity levels increased. In yeast, mRNA
levels of DNA ligase and DNA pol
increased after DNA damage, yet
their enzymatic activities did not change(23) . In none of
these cases is it clear what the significance is of the mRNA increase
without a concomitant protein increase. It does not appear, however,
that pol
synthesis is increased, but the protein produced is less
active because the antibody studies showed roughly equal
antigen:activity ratios.
A comparison of pols ,
, and
for their primer-template preferences shows that all three pols
can efficiently use ribonucleotide-primed templates, so no clue to the
functions of pols
and
in replication were found in this
regard. It is curious that pols
and
can utilize RNA
primers, since one expects pol
to add DNA to its primase
products. One wonders, therefore, why pol
contains DNA polymerase
or, conversely, why pol
products appear to be extended by pols
or
.