A 43 kDa DNA binding protein from the pea chloroplast interacts with and
stimulates the cognate DNA polymerase
A 43 kDa DNA binding protein from the pea chloroplast interacts with and stimulates the cognate DNA polymerase
Weiliang
Chen
,
Amos
Gaikwad
,
Sunil Kumar
Mukherjee*
,
Nirupam Roy
Choudhary
1
,
Dhirendra
Kumar
and
Krishna K.
Tewari
2
International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali
Marg,
New Delhi
110067,
India
,
1
Genetic Engineering Unit, CBT, J.N.U.,
New Delhi
110067,
India
and
2
Department of Molecular Biology and Biochemistry, University of California,
Irvine
, CA 92717,
USA
Received July 8, 1996
;
Revised and Accepted September 2, 1996
ABSTRACT
A DNA binding protein with DNA polymerase `accessory activity' has been identified and purified to apparent homogeneity from pea
chloroplasts. This protein consists of a single subunit of 43 kDa and binds to
DNA regardless of its base sequence and topology. It increases cognate DNA
polymerase-primase activity in a dose dependent manner. Using solid phase protein-protein interaction trapping and co-immunoprecipitation techniques, the purified protein was found to associate with the chloroplast DNA polymerase. The chloroplast DNA polymerase also binds directly to the
radioiodinated 43 kDa protein. The specific interaction between 43 kDa protein
and chloroplast DNA polymerase results in the synthesis of longer DNA chains.
The 43 kDa protein, present abundantly in the pea chloroplast, appears to
increase processivity of the chloroplast DNA polymerase and may play an
important role in the replication of pea chloroplast DNA.
INTRODUCTION
DNA metabolism, namely replication, repair, and recombination, requires the
concerted assembly and activity of multiprotein complexes (
1
). DNA synthesis, in particular, is the result of an interplay between the core
DNA polymerases and associated accessory proteins. Accessory proteins are
either subunits of holoenzymes or those that interact with the DNA polymerases
for processive movement of the replication fork. For example, the
E
.
coli
DNA
Pol
III core enzyme requires the so-called [gamma]-[beta] complex (holoenzyme subunits) along with the homologous single
stranded DNA binding protein (SSB) to achieve fork movement at a rate of 700
nucleotides (nt) per second at 37oC, a value close to
in vivo
fork movement of 1000 nt per second (
2
,
3
). Eukaryotic DNA polymerases [alpha] and [delta] also have their respective holoenzyme-like structures (
4
,
5
). Here again, the eukaryotic SSBs, namely Replication Protein A (RPA), interact
with the polymerases to increase processivity (
6
). In addition to SSBs, some general DNA binding proteins such as UL42 protein
of Herpes Simplex Virus are also reported to associate with and accentuate the
homologous DNA polymerizing activity (
7
).
The mechanisms of DNA replication are fairly well understood in many prokaryotic
and lower eukaryotic systems. On the other hand, DNA replication in plants is
poorly studied (
8
-
10
). Many studies on plant DNA replication center around those on chloroplast DNA (ctDNA) because of the relative ease of isolating and handling the intact
chloroplast genome (
11
-
13
). A partially purified
in vitro
system from pea chloroplast that can faithfully replicate plasmids containing
replication origin sequences of pea ctDNA has been developed (
14
). The enzymes, namely DNA polymerase, DNA topoisomerase and a nuclease have
been identified from this system and purified to homogeneity (
15
-
17
). In our continuing effort to study the
in vitro
replication system, we now report the identification, purification and
characterization of an accessory protein of pea ctDNA polymerase.
The purified accessory protein is a monomer of 43 kDa and exhibits non specific binding to DNA. Cognate DNA polymerase/ primase activity is stimulated several-fold depending upon the nature of template and amounts of the 43 kDa
protein. The protein binds to the pea ctDNA polymerase in a specific manner and
causes synthesis of longer DNA chains. This protein is found only in green
tissues of the plant and is localized within the chloroplasts.
MATERIALS AND METHODS
Purification of the 43 kDa protein
Triton disrupted chloroplasts (Fraction I) were prepared from 750 g of pea leaves (
18
) and loaded on to a 50 ml DEAE cellulose column (DE 52, Whatman) equilibrated
with buffer A (50 mM Tris, pH 8, 10 mM [beta]-mercaptoethanol, 10% glycerol and five protease inhibitors namely 1
mg/ml benzamidine, 10 mM sodium-metabisulfite, 10 [mu]M pepstatin A, 2 [mu]g/ml leupeptin and 100 [mu]M phenylmethylsulfonylfluoride) and 100 mM NaCl. The column
was washed with 100 ml of buffer A. The flow-through and wash fractions were concentrated by ultrafiltration and
equilibrated to buffer B (25 mM sodium acetate, pH 5.2, 10% glycerol plus the
protease inhibitors) using the Amicon RC 8400 system (10 kDa cut off, Amicon).
This was called Fraction II and was applied on a 50 ml CM cellulose column (CM
52, Whatman) equilibrated with buffer B containing 25 mM NaCl. After thorough
washing, bound proteins were eluted with a 500 ml linear gradient of 25-600 mM NaCl in buffer B. Active fractions were identified by southwestern hybridization (
19
) with radiolabelled denatured DNA (specific activity = 5 * 10
7
c.p.m./[mu]g) and were found to elute at 400-475 mM NaCl. The active fractions were pooled and dialysed against
buffer A containing 50 mM NaCl and 5 mM MgC1
2
. Dialysed proteins (Fraction III) were then loaded onto a 10 ml calf thymus
ssDNA cellulose column (U.S.B.). Following washing with buffer C (buffer A with
5 mM MgC1
2
) containing 75 mM NaCl, the bound proteins were eluted with a 50 ml linear
gradient of 75-750 mM NaCl in buffer C. The active fractions eluting between 250-350 mM NaCl were pooled, concentrated and dialysed against buffer
C containing 25 mM NaCl. This concentrate (Fraction IV) was subsequently chromatographed on a Sephadex G-100 (Superfine, Sigma) column (50 * 1 cm). The eluted active fractions were pooled, concentrated by
ultrafiltration (Fraction V) using the centricon membrane (30 kDa cut off,
Amicon) and either stored at -70oC or used immediately. All the chromatographic steps were performed at 4oC. The 43 kDa protein obtained as fraction V was used for all biochemical
studies.
DNA binding
Appropriate amounts of different forms of DNA and 43 kDa protein were incubated in 20 [mu]l of reaction buffer containing 50 mM Tris, pH 7.5 and varying concentrations of NaCl (as mentioned in the text)
at 37oC for 30 min. Some of the binding reactions were also performed in presence
of 2 mM ATP or other substrates as described in the text. Bound complexes were
processed for agarose-gel electrophoresis, filter binding assay or electron microscopy.
For south-western blots, autoradiograms containing 43 kDa protein were used to
calculate the DNA binding units, described in Table
1
. Blots were exposed for sufficient time intervals to ensure linearity of
autoradiographic bands in the densitometric scale (
20
). Unit activity was taken to correspond to an arbitrarily chosen amount of band
intensity. Based on this measurement, the intensity of bands in lanes 1 and 5
of Figure
1
B would represent 2 and 10 units respectively.
DNA synthesis
DNA synthesis was carried out as described by Reddy
et al.
(
14
). One unit of DNA polymerase activity was defined as the amount responsible for
incorporation of 1 * 10
5
c.p.m. of [
3
H]TMP (specific activity of [
3
H]TTP = 5 * 10
13
c.p.m./mmol) into acid insoluble radioactivity. The reactions were usually
carried out at 37oC for 30 min using 1 [mu]g of activated calf thymus DNA as template. Under similar conditions, 1 [mu]g of unprimed M13 ssDNA template yielded an insoluble radioactivity of about 20 000 c.p.m.
The unprimed ssDNA template was used for monitoring activity of DNA primase
coupled DNA polymerase activity. Wherever necessary, synthesis was also carried
out using [
35
S]dATP (specific activity = 10
7
c.p.m./nmol) and appropriate templates.
Solid phase assay for protein-protein interactions
The 43 kDa protein was allowed to bind to the polystyrene surface of the wells of an ELISA plate. The binding was carried out in 50 [mu]l B1 buffer (50 mM Tris pH 8, 10 mM [beta]-mercaptoethanol, 10% glycerol and protease inhibitors) with 150 mM NaCl for 2 h at 37oC. The unbound proteins were removed, followed by two
washes with 100 [mu]l B1 buffer containing 150 mM NaCl. The wells were then blocked with 1% BSA
for 1 h at 37oC in 200 [mu]l B1 buffer containing 150 mM NaCl. After three washes as above, salt-free Fraction 2A containing 1-2 U of DNA-polymerase activity in 50 [mu]l B1 buffer with 50 mM NaCl was added to the
coated wells. Incubation was continued at 25oC for 20 min in the presence or absence of various reagents such as 2 mM ATP or 1 [mu]g of M13ssD or 40 [mu]g polyclonal anti-43 kDa antibodies. Unbound enzymes were removed by washing
three times with 200 [mu]l B1 buffer containing 200 mM NaCl. The bound enzymes were eluted with 400
mM NaCl in 50 [mu]l B1 buffer. The eluted enzyme (25 [mu]l) was added to 75 [mu]l of DNA synthesis reaction. Care was taken to keep the salt
concentration below 125 mM NaCl, the inhibitory limit for the DNA polymerase
activity (
15
).
Co-immunoprecipitation
Fraction 2A containing 1.5 U DNA-polymerase activity was mixed with 5 [mu]g of 43 kDa protein (or 5 [mu]g of BSA as control) at 37oC in 30 [mu]l of Bl buffer containing 150 mM NaCl. After 30 min, 50 [mu]g of rabbit anti-43 kDa antibodies were added, incubated at 25oC for 15 min, followed by the addition of
15 [mu]l of Protein G-Sepharose (capacity = 19 [mu]g of rabbit IgG/[mu]l, Pharmacia). The mixture was shaken mildly at 37oC for another 30 min. The immunoprecipitate was removed
by centrifugation at 3000
g
for 10 min and the supernatant was assayed for DNA polymerase activity. The presence of DNA polymerase within the immunoprecipitate was also
established by a DNA polymerase activity gel (
22
).
Radioiodination of 43 kDa protein
Approximately 30 [mu]g of 43 kDa protein was conjugated with 0.5 mCi of Na[
l25
I] (specific activity = 1500 Ci/mmol, Amersham) using preloaded iodobeads and published protocol (Stratagene). Radioiodinated protein (6 [mu]g in 150 [mu]l) at a specific activity of 1.8 * 10
7
c.p.m./[mu]g was obtained after removing unincorporated radioactivity by gel filtration
chromatography. This was used as a probe for far western hybridization as
described below.
Various fractions of ctDNA polymerase were electrophoresed in a SDS-10% PAGE and transferred onto nitrocellulose membrane. The membrane was incubated with a blocking buffer containing phosphate buffered solution (PBS), 0.3% Tween 20, 1% BSA (Sigma) for 1-2 h at room temperature followed by incubation with [
125
I]-43 kDa protein in the same buffer (2 [mu]l of radioiodinated protein/ml of blocking buffer) for 16 h. The membrane was then washed 3-4 times with buffer containing 1.5 * PBS and 0.3% Tween 20, dried and exposed for
autoradiography.
Size determination of the nascent DNA
Five units of Fraction 2A ctDNA polymerase and 10 [mu]Ci of [
35
S]dATP (specific activity = 10
7
c.p.m./nmol) were used
in each DNA synthesis reaction with DNA templates and substrates as described in
the text. While 80% of the synthesised DNA was processed to check trichloroacetic acid (TCA) insoluble incorporation, the rest was analysed for size distribution on a urea-8% PAGE at 45oC. The gel was fixed, dried and exposed for autoradiography.
The autoradiogram was scanned by a laser densitometer. The intensity (I) and the
Molecular weight (M) of each DNA species was plotted to generate an I versus M curve for each lane of the gel. Molecular weight (M) of nascent DNA species was calculated using the
relationship between the electrophoretic mobility ([mu]) and the molecular weight (M) i.e. log M = K
1
[mu] + K
2
(K
l
and K
2
are the empirical constants, the values of which were determined from standard markers). The average molecular weight (M bar) was found as M bar =[integral]IdM/[integral]dM from the plots of I versus M.
Others
Other procedures such as purification of antibodies from rabbit antisera,
agarose gel electrophoresis, labelling of probes by nick translation and 3'-end labelling, western blotting using anti 43 kDa rabbit polyclonal IgG or antisera (dilution 1:20 000) etc. were
carried out using published procedures (
23
).
RESULTS
Purification of 43 kDa protein
A 43 kDa DNA binding protein was purified 800-fold to apparent homogeneity from the pea chloroplasts using a combination
of ion exchange, affinity and gel filtration chromatography (Table
1
). The enrichment of the protein was followed up by south western hybridization
with radiolabelled denatured DNA. Due to its basic nature (pI > 9, not shown),
the 43 kDa protein did not bind to DEAE cellulose but was retained on a CM
cellulose column at pH 5.2. Purity of the protein was enhanced significantly by
affinity chromatography over ssDNA cellulose where the only co-eluant was a 16 kDa protein as shown in lane 4 of Figure
1
A. The 16 kDa protein however did not bind DNA on a southwestern blot (Fig.
1
B) and could be separated from the 43 kDa protein by a subsequent step of gel
filtration chromatography. It is important to note that the proteins of ~33 kDa in size were abundantly present in Triton X 100 lysate (Fig.
1
B, lane 1) and have been reported earlier to be the RNA binding proteins (
24
). These were removed from the 43 kDa protein following DEAE cellulose
chromatography.
The molecular mass of the purified protein was similar when analysed either by
gel filtration chromatography or SDS-PAGE. Therefore the 43 kDa protein is a monomer in its native state. The
DNA binding activity of 43 kDa protein was stable at
4oC for 2-3 weeks and for 6 months at -70oC when stored at a concentration of 1 mg/ml. The
activity was lost when treated at 60oC for 15 min. The purified 43 kDa protein did not have any detectable DNA
polymerase, DNA-primase, DNA topoisomerase, DNA helicase, ATPase or nuclease activity.
Purified 43 kDa protein binds to any sequence and all structural forms of DNA
DNA binding properties were evaluated by gel retardation, filter-retention and electron microscopic visualization of the DNA-protein complexes. These experiments revealed that the 43 kDa protein could
complex with any topological form and sequence of DNA. At low salt
concentration (<50 mM NaCl), strong binding was observed with either the supercoiled,
topoisomers, linear, nicked, or single stranded form of any plasmid DNA and the
complex could easily be detected by agarose gel electrophoresis. Lane 2 of
Figure
2
A shows the retarded mobility of the complex between the protein and supercoiled
DNA. The retardation depended on the amount of 43 kDa protein and salt
concentration. Addition of anti-43 kDa polyclonal antibody during the complex formation (or after complex formation) induced a `supershift' of the complex (lanes 3 and 4). The DNA-protein interaction was, however, sensitive to high salt and was
reduced to the extent of 80% in presence of 400 mM NaCl (lane 8). The smear in lanes 5-10 may reflect either the gradual loss of complex-forming ability during electrophoresis or formation of heterogenous species in the
presence of high salt.
The 43 kDa protein stimulates ctDNA polymerase/ primase activity
Many DNA binding proteins are known to stimulate their homologous DNA polymerase activities. Hence the role of the 43 kDa protein on DNA synthesis was examined. Since attempts were not made to purify
the DNA polymerase to homogeneity, due to its extreme low abundance in pea
chloroplasts, the chromatographic fractions specifically enriched for DNA polymerase activity
(Materials and Methods) were used. Of the various fractions tested, stimulation of DNA synthesis by the 43 kDa protein was
maximal with the DNA polymerase Fraction 2A.
Fraction 2A contained proteins other than the pea ctDNA polymerase and the
primase but was devoid of 43 kDa protein as detected by western blotting. This
fraction was capable of synthesising DNA chains on unprimed M13mpl9ssDNA template. A kinetic analysis of DNA synthesis (Fig.
3
A) revealed that synthesis was rapid, reaching a plateau within 10 min.
Inclusion of 43 kDa protein in the synthesis reaction resulted in a gradual
enhancement of DNA synthesis though the plateau was not reached before 25 min.
The stimulation of DNA synthesis in the presence of 43 kDa protein depended
upon the amount of DNA polymerase. For example, the DNA synthesis increased 6-fold with 0.2 U DNA polymerase in presence of 3 [mu]g of 43 kDa protein. Under similar experimental conditions using 1 U
DNA polymerase, the increase was only 2.5-fold (not shown). The increase of DNA synthesis was reversed by anti-43 kDa antibodies as shown in Figure
3
A. When the antibodies were added after 5 min of initiation of DNA synthesis, inhibition occurred within 1-2 min. In a control reaction, pre-immune rabbit antibodies did not show any significant effect on DNA-synthesis (not shown).
DNA synthesis by Fraction 2A was reduced by 50% in the absence of rNTPs (Fig.
3
B). The presence of endogenous rNTPs within Fraction 2A and/or the ability of
the primase to partially utilize dNTPs for formation of primers (
25
) may account for the residual DNA synthesis. Figure
3
B shows that the stimulation of DNA synthesis by 43 kDa protein still occurred
in such a situation. Figure
3
C demonstrates that the stimulatory effect on ctDNA polymerase/primase activity
was optimal at about 10 [mu]g of 43 kDa protein. Higher concentrations were inhibitory for stimulation
of DNA synthesis.
To investigate whether the enhancement of DNA polymerase/primase activity was
specific for 43 kDa protein, the effect of nonhomologous DNA binding proteins on pea ctDNA polymerase mediated DNA
synthesis was also studied. As shown in Table
2
,
Escherichia coli
SSB and calf thymus histone Hl proteins were inhibitory rather than stimulatory
for ctDNA polymerase/primase activity. Stimulatory activity of other
stabilizing proteins like BSA was also marginal. Table
2
also shows that the 43 kDa protein did not have any inherent DNA polymerase
activity. Since dideoxynucleotides (ddNTPs) were poor chain terminators for the
ctDNA polymerase, a 10-fold molar excess of ddNTPs over dNTPs was required to reduce the
stimulatory activity by 60%. This reduction was also consistent with the
inference that 43 kDa protein acts as an accessory to the ctDNA
polymerase/primase activity.
The 43 kDa protein binds to the ctDNA polymerase
The data of Figure
3
and Table
2
suggested that the purified protein might interact with the ctDNA polymerase. Since homogeneously purified ctDNA polymerase was not available in sufficient amounts, we used
three independent approaches to verify this.
The first approach employed a solid phase assay in which 43 kDa protein was coated onto the wells of an ELISA plate. The binding of 43 kDa
protein to the wells was strong with no disruption even at a concentration of
500 mM NaCl. After blocking nonspecific sites on the plate, Fraction 2A DNA
polymerase was applied to the wells and incubated. Subsequently, proteins bound
by 43 kDa protein were eluted and the eluant was assayed for DNA polymerase
activity.
Chloroplast DNA polymerase/primase activity is enhanced specifically by the 43
kDa protein
No.
Reaction
Addition
Synthesis (c.p.m. * 10
-3
)
at
t
= 30 min
(a)
Standard reaction
None
0.35
without Fraction 2A
DNA polymerase
(b)
Same as (a)
4.0 [mu]g p43
0.34
(c)
Standard reaction
None
4.20
with Fraction 2A
DNA polymerase
(d)
Same as (c)
ddNTP:dNTP(10:1)
2.00
(e)
Same as (c)
4.0 [mu]g p43
25.0
(f)
Same as (c)
4.0 [mu]g p43
9.20
+ddNTP:dNTP(10:1)
(g)
Same as (c)
0.25 [mu]g
E.coli
ssB
3.50
(h)
Same as (c)
1.0 [mu]g
E.coli
ssB
2.90
(i)
Same as (c)
4.0 [mu]g
E.coli
ssB
0.40
(j)
Same as (c)
0.25 [mu]g histone H1
3.40
(k)
Same as (c)
1.0 [mu]g histone H1
2.90
(l)
Same as (c)
4.0 [mu]g histone H1
0.40
(m)
Same as (c)
4.0 [mu]g BSA
4.50
(n)
Same as (c)
10.0 [mu]g BSA
5.40
Components of a standard DNA synthesis reaction included 1 [mu]g M13ssD, 100 [mu]M each of rNTPs, 50 [mu]M each of dATP, dCTP, dGTP and 1 [mu]Ci of [
3
H]TTP in a 100 [mu]l reaction volume as described in (14). A dose of 0.2 U Fraction 2A DNA polymerase was employed for primase mediated DNA synthesis. p43, 43 kDa protein.
Figure
4
shows that the polystyrene-bound 43 kDa protein was able to trap ctDNA polymerase. When 1.5 U
Fraction 2A was used, 0.25 U DNA polymerase activity could be retained by 43 kDa protein under the experimental conditions. Interestingly, addition of 1 [mu]g of M13mpl9ssDNA along with the Fraction 2A enzymes increased the retention
of ctDNA polymerase 3-fold. On the other hand, addition of 40 [mu]g of polyclonal anti-43 kDa antibodies inhibited the interaction to the extent of
50%. Similiar amount of pre-immune antibodies did not have any effect. In a separate assay, the above
mentioned eluant was found incapable of DNA synthesis with unprimed ssDNA
template. This suggests that no significant interaction between the coated 43
kDa protein and the DNA primase could be detected by this technique.
Furthermore, no polymerase binding could be detected in control wells coated
with 1% BSA alone.
The possibility of interaction of DNA polymerases from other sources with
purified 43 kDa protein was also examined. The results are summarized in Table
3
. Commercially available DNA polymerases of bacteriophages T4 and T7 did not
interact with the 43 kDa protein. Similarly a crude preparation of aphidicolin
sensitive (
26
) pea nuclear DNA polymerase (ctDNA polymerase is aphidicolin resistant) also
did not show any binding. In these cases, even a 1.0 M NaCl eluant did not show
any DNA polymerase activity, thereby ruling out the possibility of any stronger
interaction. On the other hand, binding could be observed with
E.coli
DNA
Pol
I. Here also, addition of 1 [mu]g of ssDNA along with the
E.coli Pol
I enzyme resulted in an increased recovery of the enzyme. It is worthwhile to note that 5 [mu]g of 43 kDa protein also stimulated the
E.coli
DNA
Pol
I (1 U) activity by at least 1.5-fold (not shown). Weak binding to 43 kDa protein was also observed with
Klenow fragment of
E.coli
Pol
I. (Table
3
).
In co-immunoprecipitation studies, a complex of 43 kDa and the ctDNA polymerase
along with other Fraction 2A proteins could be specifically precipitated by
anti-43 kDa antibodies but not with pre-immune antibodies. The presence and molecular weight of ctDNA polymerase in the immunoprecipitate was identified by a DNA polymerase activity gel (
22
) and the polymerase activity remaining in the supernatant was measured as mentioned in Materials and Methods. Densitometric quantitation of the autoradiogram shown in Figure
5
A demonstrated that about 0.4 U 70 kDa ctDNA polymerase was present in the immunoprecipitate (lane 2) when 1.5 U Fraction 2A enzyme was mixed with 5 [mu]g 43 kDa protein. Figure
5
B shows that a comparable amount was depleted in the supernatant fraction (lane
2). When BSA instead of the 43 kDa protein was present during co-immunoprecipitation, almost no DNA polymerase could be detected in the
precipitate as expected (Fig.
5
A, lane 1). If anti-43 kDa antibody was omitted in the reaction with the purified protein,
very little DNA polymerase activity was detected in the precipitate (Fig.
5
A, lane 3) whereas the DNA polymerase activity of the supernatant increased (Fig.
5
B, lane 3). The latter increase in activity could be attributed to the accessory
activity of 43 kDa protein present in the supernatant.
Interaction of various DNA polymerases with the 43 kDa protein
DNA polymerases
>
Amount of
3
H TMP incorporated (c.p.m.)
R* (%)
Interaction
Input (*10
-5
)
Eluant/50 [mu]l
Unbound (*10
-5
)
Fraction 2A
1.81
24012
1.02
13.3
++
T4 phage
1.75
710
1.71
0.4
-
T7 phage
1.84
620
1.73
0.3
-
Klenow
1.56
8000
1.42
5.1
+
E.coli
1.73
22000
1.34
12.7
++
Crude preparation
1.42
832
1.21
0.6
-
of pea nuclear DNA polymerases
R*, retention of DNA polymerase activity in the wells of an ELISA plate coated
with the 43 kDa protein. Approximately 7.5 [mu]g 43 kDa protein was added to coat each of the wells.
The size of the nascent DNA chains increases in the presence of 43 kDa protein
Three independent approaches were taken to address the possibility of
interaction between the DNA polymerase and the purified protein. While the
approach of a solid phase assay could reveal the interaction between the partners, no binding of the 43 kDa protein was observed with DNA polymerases from either T4 or T7 bacteriophages.
Furthermore, the binding of pea ctDNA polymerase was inhibited by polyclonal anti-43 kDa antibody. Thus the solid phase assay detected a somewhat specific interaction
between pea ctDNA polymerase and the 43 kDa protein. Binding was also observed
between
E.coli
DNA
Pol
I and the 43 kDa protein. The latter observation is indicative of the
evolutionary linkage (
27
) between the two polymerases.
In co-immunoprecipitation studies, a 70 kDa DNA polymerase was observed to
interact with the 43 kDa protein. It is important to mention that a prolonged
autoradiographic exposure of the polymerase activity gel containing only the
Fraction 2A enzymes revealed a weak band of DNA polymerase at 90 kDa (
15
) in addition to the one at 70 kDa (A. Gaikwad, unpublished). At present, it is
unclear how these two DNA polymerases are related but it could be safely
assumed that the domain for interaction with 43 kDa protein resides in the 70
kDa form.
The involvement of other protein(s) within the Fraction 2A mediating the
interaction cannot be ruled out by any of the two approaches above. Hence the
direct interaction was established on a far western blot. In spite of this
direct interaction, one could argue about the non-obligatory participation of intermediary protein(s) for the stimulatory
activity.
The mechanism of activation by 43 kDa protein remains to be elucidated. The
average size of DNA chains synthesized in presence of the 43 kDa protein
increased by about 2.5-fold while the total DNA synthesis increased by a factor of 13. These data
indicate that either higher amounts of DNA polymerases were made available at
all stages of DNA synthesis or the affinity of the DNA polymerase for the
template increased in presence of the 43 kDa protein resulting in increased processivity. These possibilities can be explored with the purified DNA polymerase and primase from the pea
chloroplast.
An obvious explanation for the accessory activity would be that the 43 kDa
protein is itself a component of the DNA polymerase holoenzyme. It does not
appear to be true for various reasons. Firstly, the protein was not detectable
in the Fraction 2A DNA polymerase which was fully competent at carrying out DNA
synthesis including pea ct-ori specific DNA replication (
14
). The DNA synthesis was also unaffected by anti-43 kDa antibodies. Secondly, the 43 kDa protein, unlike most subunits of
polymerase holoenzyme from other sources (
28
), was never observed to copurify with the polymerase activity. Rather, its high
pI of ~9 permitted its complete separation from DNA polymerase activity by a DEAE-cellulose chromatographic step. Thirdly, the subunit components of
DNA polymerase are usually present in low abundance (
29
) whereas the supplementary accessory activities like SSBs (
30
) are produced in high quantities within the relevant cells. Prior estimates
have determined that about 5 * 10
7
cells (
31
) with about 70 chloroplasts per cell (
32
) are present in 1 g tissue from 7-8 day old pea leaves. Taking into account about 88% loss of 43 kDa
protein during purification (this manuscript, Table
1
), the calculated copy number of 43 kDa protein in each chloroplast will be at
least 10 000 which is a large excess over that of pea ctDNA polymerase. Thus
for the reasons outlined above, the 43 kDa protein probably represents a
nonholoenzyme accessory factor for the pea ctDNA polymerase.
ACKNOWLEDGEMENTS
We are thankful to Drs M. Kunni and Nasreen Ethesham for their generous gifts of
Fraction 2A ctDNA polymerase and crude preparation of Pea nuclear DNA
polymerase. We acknowledge the help given by Drs K.V.S. Rao, S. Jameel, R.
Bhatnagar, E. Hasnain for critical reading of the manuscript.
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