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Plant Physiol. (1998) 118: 1029-1038
A Chloroplast DNA Helicase II from Pea That Prefers Fork-Like
Replication Structures
Narendra Tuteja* and
Tuan-Nghia Phan
International Centre for Genetic Engineering and Biotechnology,
Aruna Asaf Ali Marg, New Delhi 110 067, India
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ABSTRACT |
A DNA helicase, called chloroplast
DNA (ctDNA) helicase II, was purified to apparent homogeneity from
pea (Pisum sativum). The enzyme contained intrinsic,
single-stranded, DNA-dependent ATPase activity and an apparent
molecular mass of 78 kD on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. The DNA helicase was markedly stimulated by DNA
substrates with fork-like replication structures. A 5 -tailed fork was
more active than the 3-tailed fork, which itself was more active than
substrates without a fork. The direction of unwinding was 3 to 5
along the bound strand, and it failed to unwind blunt-ended duplex DNA.
DNA helicase activity required only ATP or dATP hydrolysis. The enzyme
also required a divalent cation
(Mg2+>Mn2+>Ca2+) for its
unwinding activity and was inhibited at 200 mM KCl or NaCl.
This enzyme could be involved in the replication of ctDNA. The DNA
major groove-intercalating ligands nogalamycin and daunorubicin were
inhibitory to unwinding (Ki approximately
0.85 µM and 2.2 µM, respectively) and
ATPase (Ki approximately 1.3 µM and 3.0 µM, respectively) activities of
pea ctDNA helicase II, whereas ellipticine, etoposide (VP-16), and
camptothecin had no effect on the enzyme activity. These ligands may be
useful in further studies of the mechanisms of chloroplast helicase
activities.
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INTRODUCTION |
A special class of DNA-interacting enzymes known as DNA helicases
catalyze the unwinding of energetically stable duplex DNA in an
ATP-dependent manner, and thus play an important role in DNA
replication, repair, recombination, and transcription (Matson et al.,
1994 ; Lohman and Bjornson, 1996; Tuteja and Tuteja, 1996; Tuteja,
1997). DNA helicases are ubiquitous enzymes now known for their analogy
to motor proteins such as myosin, kinesin, and dynein, which use ATP
hydrolysis for energy (West, 1996). All helicases contain intrinsic
DNA-dependent ATPase activity, which provides energy for the reaction
(Kornberg and Baker, 1991). Helicases generally bind in the ssDNA or in
the ss-/ds-DNA junctions and translocate unidirectionally along the
bound strand, either in the 3 to 5 or in the 5 to 3 direction. Many
DNA helicases have been isolated from bacteria, bacteriophage, virus,
and eukaryote systems (Thommes and Hubscher, 1992; Matson et al., 1994;
Tuteja and Tuteja, 1996).
Chloroplasts are highly polyploid, semiautonomous, intracellular
organelles that contain their own genetically active genomes. In higher
plants the ctDNA is a ds, circular molecule that ranges in size from
120 to 160 kb and encodes about 130 genes. The mechanism of DNA
replication is well defined in bacteria, viruses, bacteriophage, plasmids, and, to a lesser extent, yeasts (Kornberg and Baker, 1991),
but is still not understood in plant systems. DNA replication requires
the concerted assembly and activity of many proteins (Hubscher and
Spadari, 1994). Most of the studies on replication of DNA in plants
have focused on ctDNA because of the relative ease of isolating and
handling the intact chloroplast genome (Tewari, 1987; Meeker et al.,
1988). A number of enzymes that may be involved in
replication, such as DNA polymerases (Sala et al., 1980; McKown and
Tewari, 1984; Heinhorst et al., 1990), DNA topoisomerases (Siedlecki et
al., 1983; Lam and Chua, 1987; Nielson and Tewari, 1988), and DNA
helicase (Tuteja et al., 1996), have been purified from the
chloroplasts of higher plants. DNA primase activity has also been
reported from pea (Pisum sativum L.) chloroplasts (Nielson et al., 1991).
Most organisms encode multiple DNA helicases because of their
involvement in numerous biological reactions at different stages of
cell metabolism. A set of 13 different DNA helicases have been reported
in Escherichia coli (Lohman, 1992; Matson et al., 1994). Nine different DNA helicases have been purified to homogeneity from
HeLa cells (Tuteja et al., 1990, 1991, 1992, 1993, 1994, 1995, 1996).
In plants multiple DNA helicases are also expected to be present.
However, to date only one DNA helicase has been purified to homogeneity
from pea chloroplasts (Tuteja et al., 1996). Although the existence of
two other DNA helicases, one from lily (Hotta and Stern, 1978) and
another from soybean (Cannon and Heinhorst, 1990), has been reported,
these enzymes were neither purified nor well characterized.
We are studying the detailed molecular mechanism of ctDNA replication
with the underlying goals of establishing a well-defined in vitro
replication system and establishing the use of organelle DNA for the
transformation of plants. In this context we have initiated systematic
studies of the DNA helicases present in pea chloroplasts with the
objective of isolating and characterizing them and eventually cloning
their genes for functional study. Here we report the purification and
characterization of pea ctDNA helicase II, which is stimulated by
fork-like replication structures. The properties of this enzyme make it
a candidate for a DNA replicative helicase. We have also tested the
effect of different DNA-interacting ligands on the unwinding and ATPase
activities of pea ctDNA helicase II.
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MATERIALS AND METHODS |
DNA, Nucleotides, and Ligands
M13 mp19 ssDNA, dsDNA, and RNA from pea (Pisum sativum
L.) leaves were prepared as described previously (Sambrook et al., 1989). NTPs and ATP S were obtained from Boehringer-Mannheim and [-32P]ATP (185 Tbq/mmol) and
[ -32P]dCTP (approximately 110 Tbq/mmol) were
purchased from Amersham. The various oligodeoxyribonucleotides used to
construct helicase substrates were synthesized chemically using a DNA
synthesizer (model 380 A, Applied Biosystems), and purified
electrophoretically. The sequences and details of the
oligodeoxyribonucleotides were described previously (Tuteja et al.,
1994, 1996). The DNA-interacting ligands daunorubicin, ellipticine,
camptothecin, and VP-16 were purchased from Topogene (Columbus, Ohio).
Nogalamycin was from Sigma.
Preparation of DNA Helicase Substrates
The DNA substrate used in the helicase assay consisted of
32P-labeled complementary
oligodeoxyribonucleotides hybridized to M13 mp19 phage ssDNA to create
a partial duplex. A substrate with a 5-hanging tail was used for
purification and for most of the characterization unless otherwise
stated. The structures of the DNA substrates (circular or linear) were
as described previously (Tuteja et al., 1994, 1996). The 3 to 5 and
5 to 3 direction-specific substrates were constructed as described
previously (Tuteja et al., 1994, 1996).
DNA Helicase Assay
The helicase assay measures the unwinding of a labeled
oligodeoxyribonucleotide fragment from a partial duplex molecule,
catalyzed by ctDNA helicase II. The standard reaction mixture (10 µL)
consisted of 20 mM Tris-HCl (pH 8.5), 8 mM DTT,
4% (w/v) Suc, 80 µg/mL BSA, 2 mM ATP, 1.5 mM
MgCl2, 100 mM KCl, approximately 1 ng
of 32P-labeled DNA substrate (approximately 1000 cpm), and the helicase fraction. The reaction mixture was incubated
at 37°C for 30 min and the reaction was stopped by the addition of
1.5 µL of 75 mM EDTA, 2.25% SDS, 37.5% (by volume)
glycerol, and 0.3% bromphenol blue. The products were separated by
12% native PAGE, and the gel was dried and exposed to film for
autoradiography. The DNA unwinding was quantitated by excising the
radioactive bands from the gel and counting the radioactivity in
Beckman liquid-scintillation fluid. One unit of helicase activity is
defined as the amount of enzyme that unwinds 30% of the DNA helicase
substrate at 37°C in 30 min in the linear range of enzyme
concentrations. For examining the effect of DNA-interacting ligands on
helicase activity, different types were added to the helicase reaction
mixture prior to the addition of the enzyme.
DNA-Dependent ATPase Assay
The hydrolysis of ATP catalyzed by ctDNA helicase II was assayed
by measuring the formation of 32P from
[-32P]ATP. The reaction conditions were
identical to those described for the helicase reaction, except that the
32P-labeled helicase substrate was replaced by
1665 Bq [-32P] ATP. The reaction was
performed both in the presence and absence of 50 ng of M13 mp19 ssDNA,
followed by TLC and quantitation as described earlier (Tuteja et al.,
1992). For inhibition of pea ctDNA helicase II ATPase activity, the
DNA-interacting ligands were included in the reaction prior to the
addition of the enzyme, as described earlier (Tuteja et al., 1997).
Other Methods
DNA topoisomerase, polymerase, ligase, nicking, and nuclease
activities were performed as described earlier (Tuteja et al., 1990,
1991, 1992, 1993, 1994, 1995, 1996). Protein concentration was
determined using the Bio-Rad protein assay kit. SDS-PAGE was performed
by the method of Laemmli (1970), followed by silver staining of the gel
with the Bio-Rad kit.
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RESULTS |
Purification of ctDNA Helicase II
DNA helicase activity was biochemically assayed as described
previously (Tuteja, 1997). The DNA substrate used for the purification procedure and for most of the characterization consisted of a P-labeled 32-base oligonucleotide annealed
through its last 17 nucleotides (position 16-32) to M13 mp19 ssDNA.
This partial duplex DNA substrate contained a 5-end protruding tail of
15 nucleotides. The displacement of a DNA fragment was measured by
native gel electrophoresis. The results of purification are summarized
in Table I, and the purification scheme
is outlined in Figure 1. All of the
purification steps were performed at 4°C. Triton X-100-disrupted chloroplast lysate (fraction I, 660 mL) was prepared from 2.2 kg of pea
leaves (7- to 8-d-old plants) and dialyzed against buffer A (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 1 mM DTT, 1 mM EDTA, 10% glycerol, 1 mM PMSF, 1 mM sodium-metabisulfite, 1 µM pepstatin, 1 mg/mL benzamidine, and 1 µM
leupeptin) as described earlier (Tuteja et al., 1996). Fraction I was
loaded onto a 240-mL DEAE-cellulose column (DE-52, Whatman)
equilibrated with buffer A as described previously (Tuteja et al.,
1996). The flow-through and wash fractions were collected (fraction II,
1200 mL) and loaded onto an 85-mL cellulose column (CM-52, Whatman)
equilibrated with buffer A. After a thorough washing, bound proteins
were eluted with an 850-mL linear gradient of 0.05 to 1 M
KCl in buffer A. Fractions eluted at around 0.2 M KCl
contained helicase activity. The active fractions were pooled and
dialyzed against buffer A (fraction III, 95 mL). Up to this step the
activity was not quantitated due to contamination with nuclease
activity.
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| Figure 1.
Purification scheme of pea ctDNA helicase II. The
strategy used for fractionation of DNA helicase II is shown. The
numbers indicate the molarity of the KCl gradient or step elution to
elute the enzyme activity.
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Fraction III was applied to a 3-mL heparin Sepharose column
equilibrated with buffer A. Following washing with buffer A, the bound
proteins were eluted with a 36-mL linear gradient from 0.05 to 1 M KCl in buffer A. The active fractions eluted at about 0.5 M KCl, and were then pooled and dialyzed against buffer B
(fraction IV, 7.2 mL. 3600 units). Buffer B was buffer A plus 1 mM ATP and 1 mM MgCl2.
Fraction IV was loaded onto a 1.2-mL dsDNA-cellulose column
equilibrated in buffer B. The column was washed thoroughly, and bound
proteins were eluted with an 18-mL linear gradient of KCl (0.05 to 1 M) in buffer B. The activity eluted from the column at
about 0.5 M KCl (fraction V, 3 mL, 1500 units). Fraction V was first diluted with buffer B to adjust the KCl concentration to 0.05 M and was then loaded onto a 0.5-mL ssDNA-cellulose column equilibrated with buffer B. After washing the column with 10 mL of
buffer B the bound proteins were eluted in steps with 0.2, 0.4, 0.6, 0.8, and 1 M KCl in buffer B. The helicase
activity was detected only in the 0.4 M KCl fraction
(fraction VI, 1 mL, 665 units).
SDS-PAGE analysis followed by silver staining revealed the presence of
only one polypeptide of 78 kD in fraction VI (Fig. 2, lane 1), which showed that ctDNA
helicase II was purified to apparent homogeneity with a specific
activity of 3.9 × 105 units/mg (Table I).
The enzyme preparation did not contain any detectable DNA polymerase,
ligase, topoisomerase, nicking, or nuclease activities. ssDNA-dependent
ATPase activity was present at a level of 400 pmol of ATP hydrolyzed in
30 min by 5 ng of pure enzyme (fraction VI).
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| Figure 2.
SDS-PAGE of purified pea ctDNA helicase II.
Fraction VI (60 ng, lane 1) and the Mr
marker (lane 2) were separated on a 12% polyacrylamide gel and
visualized by silver staining.
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Cofactor and Reaction Requirements
The reaction requirements of pea ctDNA helicase II are shown in
Table II. The enzyme is heat labile and
loses its activity upon heating at 56°C for 1 min. Significant
unwinding activity was observed in a broad pH range (pH 7.5-9.5) with
an optimum near pH 8.5 (data not shown). The activity was completely
inhibited by trypsin (1 unit), EDTA (5 mM), potassium
phosphate (100 mM), ammonium sulfate (45 mM),
and ssDNA (30 mM as phosphate). However, dsDNA, pea leaf
total RNA, and Escherichia coli tRNA inhibited the helicase
activity to 70%, 60%, and 50%, respectively (Table II). The enzyme
showed an absolute requirement for divalent cations. Mg2+
(1.5 mM) optimally fulfilled this requirement (Fig.
3B), whereas Mn2+ and
Ca2+ at equivalent concentrations supported 62% and 20%
of the activity, respectively. However, at 12 mM
MgCl2 the activity was totally inhibited (Fig.
3B). Other divalent cations, such as Zn2+,
Cd2+, Cu2+,
Ni2+, Ag2+, and
Co2+, were unable to support the reaction (Table
II). The optimum concentration of KCl required for the helicase
reaction was 100 mM. At a greater concentration (200 mM) of KCl the activity was inhibited (Fig. 3C).
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| Figure 3.
Effect of ATP (A), MgCl2 (B), and KCl
(C) on pea ctDNA helicase II activity. In each reaction 5 ng of
fraction VI with 1 ng of the 5-tailed substrate was used with varying
concentrations of ATP, MgCl2, or KCl. Quantitative data are
displayed on the right side of each autoradiogram. The structure of the
substrate is shown on the left side of each gel. Asterisks denote the
32P-labeled end. Lanes marked "No enzyme" and
"Heated" are the reactions without the enzyme and with
heat-denatured substrates, respectively. The activity is shown as
percent unwinding.
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DNA helicase activity was totally dependent upon ATP with an optimum
concentration requirement of 2 mM (Fig. 3A). At greater than 8 mM ATP the enzyme was inhibited. dATP also supported
90% of the activity, whereas other NTPs or
deoxyribonucleoside triphosphates did not support the unwinding
activity (Fig. 4). ADP, AMP, and the
poorly hydrolyzable ATP analog ATPS were inactive (Table II).
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| Figure 4.
Preference of nucleotides for pea ctDNA helicase
II activity. The standard helicase reactions were performed with 5 ng
of fraction VI, 1 ng of 5-tailed substrate, and 2 mM NTP
or deoxyribonucleoside triphosphate. The amount of unwound DNA was
quantitated and plotted as a histogram above the autoradiogram of the
gel. Lanes 1 and 10 are the reactions without the enzyme and with the
heat-denatured substrate, respectively. Lanes 2 to 9 are reactions in
the presence of ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, and dTTP,
respectively. The structure of the substrate is shown on the left side
of the autoradiogram. The asterisk denotes the 32P-labeled
end.
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Kinetics and Titration of Helicase Activity
The kinetics of the helicase reaction under the standard assay
conditions with 5 ng of purified enzyme (fraction VI) showed a linear
rate of up to 30 min (Fig. 5A). After
further incubation it deviated from the linearity and became saturated
at 60 min. Titration of helicase activity with increasing amounts of
the pure enzyme showed an approximate linear response; up to 55%
unwinding with 5 ng of the protein and approximately 1 ng of the
substrate (Fig. 5B).
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| Figure 5.
Kinetics and concentration dependence of pea ctDNA
helicase II. The enzyme activity data from the autoradiograms (left)
were quantitated and are shown on the right. The structure of the
substrate used is shown on the extreme left. Asterisks denote the
32P-labeled end. A, The standard reaction was carried out
with 5 ng of fraction VI at the times indicated. B, An increasing
amount of fraction VI was used in the standard helicase assay. The
concentrations are indicated at the top of each lane. Lanes labeled
"No enzyme" and "Heated" are the reactions without the enzyme
and with the heat-denatured substrate, respectively.
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Helicase Activity Is Stimulated by Fork Structures
The influence of fork structures in the DNA substrate on the
unwinding activity of pea ctDNA helicase II was examined by using four
different substrates in standard assay conditions. All four substrates
had the same duplex length (17 bp) with an identical sequence but
differed in the presence of noncomplementary tails at the 3 end (Fig.
6B), 5 end (Fig. 6C), both of the ends
(Fig. 6D), or without a tail (Fig. 6A). The substrate without the tail supported helicase activity poorly (20% unwinding, Fig. 6A). In the
presence of a 3 tail, the helicase activity was stimulated and showed
more unwinding (36% unwinding) compared with the no-tail substrate
(Fig. 6B). However, substrates containing either a 5 tail alone or
both 5 and 3 tails were the most efficiently displaced. At the same
concentration of enzyme (fraction VI, 5 ng) the 5-tailed and 5- and
3-tailed fork structures showed unwinding of 52% and 50% of the
duplex, respectively (Fig. 6, C and D). The enzyme was unable to unwind
the longer duplex even if it contained both tails (Fig. 6E). The enzyme
also failed to unwind blunt-ended duplex DNA (Fig. 6H).
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| Figure 6.
Preference of forked DNA structures for unwinding
activity of pea ctDNA helicase II. The DNA helicase reactions were
performed under standard conditions using different DNA substrates that
contained either no tail (A), a 3 tail (B), a 5 tail (C), or both 3
and 5 tails (D and E). Small linear substrates with forked structures,
which also represent 3 to 5 (F) or 5 to 3 (G) direction
specificity, and a blunt-ended DNA substrate (H) were also used. The
schematic structure of each substrate is shown on the left side of the
autoradiogram of the gel. Asterisks denote the 32P-labeled
end. The percent unwinding is shown at the top of each panel. In each
panel, lane 1 is the reaction without the enzyme, lane 2 is the
reaction with the enzyme (5 ng), and lane 3 is the heat-denatured
substrate.
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Small, linear, partial-duplex substrates with 5- or 3-tailed
fork-like structures (Fig. 6, F and G) were also examined. The results
show that pea ctDNA helicase II was able to unwind only a 5-tailed
substrate (Fig. 6F) and not the 3-tailed substrate (Fig. 6G). It
should be noted that the length of the tails (15 nucleotides) was
significantly shorter, so the enzyme loaded and translocated
unidirectionally on the other free ssDNA strand. Since these are
direction-specific substrates and only the 5-tailed substrate was
unwound (Fig. 5F), the pea ctDNA helicase II must translocate in the 3
to 5 direction, as is illustrated below.
Direction of Unwinding by Pea ctDNA Helicase II
The direction of unwinding by helicase is defined by the strand to
which the enzyme binds and moves. The results in Figure 6, F and G,
show that the enzyme moves in the 3 to 5 direction (Fig. 6F) and not
in the 5 to 3 direction (Fig. 6G). This finding was further confirmed
by constructing two other direction-specific substrates. The
construction of the substrates was described earlier (Tuteja et al.,
1996), and these structures are shown in Figure 7. These substrates consisted of a
longer, linear M13 ssDNA with short stretches of duplex DNA at both
ends (Fig. 7, A and B). The results show that the pea ctDNA helicase
moved unidirectionally from 3 to 5 along the DNA strand to which it
bound (Fig. 7A). The enzyme did not show unwinding in the 5 to 3
direction (Fig. 7B).
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| Figure 7.
Direction of unwinding by pea ctDNA helicase II.
The structure of the linear substrate for the 3 to 5 direction (A)
and 5 to 3 direction (B) is shown on top of the autoradiogram. In
each gel, lane 1 is the reaction without enzyme; lane 2 is the reaction
with 8.5 ng of fraction VI; and lane 3 is the heat-denatured substrate.
Asterisks denote 32P-labeled ends.
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Influence of DNA-Interacting Ligands on DNA Unwinding and
ATPase Activities of the Enzyme
A set of five different DNA-interacting ligands, including both
nonintercalative (camptothecin, VP-16) and intercalative (ellipticine, daunorubicin, and nogalamycin), were included separately in the standard helicase and ATPase reactions to determine their effect on
these enzyme activities. The chemical structures of the ligands used
are described in Tuteja et al. (1997). Initially, each ligand was used
at a final concentration of 50 µM. The results are shown in Figure 8. Camptothecin, VP-16, and
ellipticine did not show any effect on helicase (Fig. 8A) or
DNA-dependent ATPase (Fig. 8B) activities of the enzyme. However,
anthracycline antibiotics, daunorubicin, and nogalamycin were
inhibitory to both of the enzyme activities (Fig. 8, A and B).
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| Figure 8.
Effect of DNA-interacting ligands on DNA unwinding
(A) and ATPase (B) activities of pea ctDNA helicase II. The standard
helicase reaction was performed with 5 ng of fraction VI, 1 ng of the
5-tailed substrate, and 50 µM of the compound. The name
of each compound is located on top of each autoradiogram.
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The kinetics of inhibition of helicase and ATPase activities were also
tested by using different concentrations of daunorubicin and
nogalamycin, and the results are shown in Figures
9 and 10. The apparent
Ki values for inhibition of both the
unwinding and ATPase activities of pea ctDNA helicase II by
daunorubicin were 2.2 µM (Fig. 9A) and 3.0 µM (Fig. 10A), respectively. However, the apparent
Ki values for nogalamycin as an inhibitor
of the unwinding and ATPase activities of the enzyme were 0.85 µM (Fig. 9B) and 1.3 µM (Fig. 10B),
respectively.
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| Figure 9.
Titration of inhibition of unwinding activity of
pea ctDNA helicase II by daunorubicin (A) and nogalamycin (B). The DNA
helicase reactions were performed in the presence of increasing
concentrations of the ligand using 1 ng of 32P-labeled
substrate and 5 ng of the pure enzyme. The quantitative curve is shown
on the left side of each autoradiogram. Various concentrations of each
ligand used are located at the top of each lane.
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| Figure 10.
Titration of inhibition of DNA-dependent ATPase
activity of pea ctDNA helicase II by daunorubicin (A) and nogalamycin
(B). The standard ATPase reactions were performed in the presence of
increasing concentrations of the ligand using 5 ng of the pure enzyme.
The quantitative curve is shown on left side of each autoradiogram of
the TLC plate. The positions of the Pi and ATP spots are indicated by
arrows. Various concentrations of each ligand used are indicated at the
top of each lane.
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DISCUSSION |
Green plant cells contain three separate genomes (nuclear,
mitochondrial, and chloroplast), which replicate, transcribe, and express their genetic information independently of each other (Tuteja,
1997). At least one helicase activity is required for the unwinding of
duplex DNA ahead of an advancing fork, which is an absolute requirement
for DNA replication (Kornberg and Baker, 1991). A reasonable
attribution of functions has been obtained for many of the E. coli, yeast, and viral DNA helicases. On the contrary, very little
is known about the plant DNA helicases and the in vivo role of most of
the eukaryotic DNA helicases. In this study we have described the
purification and properties of a DNA helicase II from pea chloroplast,
which requires fork structures for maximum unwinding activity and is
inhibited by major DNA groove-binding ligands.
ctDNA helicase II was fractionated on the basis of its behavior on a
DEAE-cellulose column, to which it did not bind. In this property, as
well as several others, it differs from our previously described DNA
helicase I from the same source (Tuteja et al., 1996) as shown in Table
III. However, similar activity was
observed by Cannon and Heinhorst (1990) in soybean chloroplasts, which did not adsorb to DEAE cellulose, but the enzyme was not further studied.
The pea ctDNA helicase II translocates in the 3 to 5 direction along
the bound strand in a manner similar to that of the previously
described ctDNA helicase I (Tuteja et al., 1996), human DNA helicase I,
II, III, V, VI, and (Seo and Hurwitz, 1993; Tuteja and Tuteja,
1996), simian virus-40 large tumor antigen (Stahl and Knippers, 1987),
and nDNA helicase I and II from calf thymus (Zhang and Grosse, 1991).
The most striking feature of the enzyme was its preference for
fork-like structures of the substrate, unlike ctDNA helicase I (Tuteja
et al., 1996). A 5-tailed fork structure was more stimulatory than the
3-tailed structure. Similar results were reported for human DNA
helicase (Seo and Hurwitz, 1993). The observation that the
3-tailed DNA was a relatively poor substrate compared with the 5- and
5-and 3-tailed substrate suggested that the pea ctDNA helicase II
translocated in the 3 to 5 direction, since the displacement
efficiency probably depends on the availability of ssDNA for binding
and subsequent translocation. The results reported in Figure 6, F and
G, indicate that helicase II needs more than 15 and less than 84 nucleotides of the free-loading zone of ssDNA for binding and moving
along it. The increased activity with the 3-tailed substrate (Fig. 6B)
compared with the nontailed substrate (Fig. 6A) indicates that the
enzyme needs a fork-like situation for effective binding. The failure
to unwind the 41 bp (Fig. 6E), even though it contains both the tails,
was due to the longer length of the base-paired region. It probably
needs some additional supporting protein(s) to unwind longer duplexes. The enzyme could not unwind the blunt-ended DNA duplex because of the
lack of a free ssDNA-loading zone. Chloroplast helicase II was
inhibited by dsDNA and RNA, which shows that this enzyme has the
affinity to bind them, as was also reported for human DNA helicase IV
(Tuteja et al., 1991) and ctDNA helicase I (Tuteja et al., 1996).
The DNA-unwinding activity and the intrinsic ATPase activity of the
ctDNA helicase II can be inhibited by DNA-interacting ligands that bind
DNA. In E. coli and human helicases, this inhibition has
been shown to be highly specific with respect to the ligand used
(George et al., 1992; Tuteja et al., 1997). The topoisomerase inhibitor
etoposide (VP-16) and the cytotoxic alkaloid camptothecin, which are
nonintercalating ligands and do not bind DNA directly, showed no
inhibition of the helicase or ATPase activities of ctDNA helicase II.
Camptothecin and VP-16 were also reported to not inhibit E. coli (George et al., 1992) or human DNA helicase II (Tuteja et
al., 1997). The ellipticine was reported to inhibit E. coli
DNA helicase II (George et al., 1992) and yeast topoisomerase II by
directly interacting with the protein (Froelich-Ammon et al., 1995).
The two most potent inhibitors of unwinding and ATPase activities
were daunorubicin and nogalamycin, similar to E. coli DNA helicases (George et al., 1992), human DNA helicase II (Tuteja et al.,
1997), and partially purified DNA helicase from HeLa cells (Bachur et
al., 1992). Recently, a ctDNA helicase I has also been shown to be
inhibited by daunorubicin and nogalamycin (Tuteja and Phan, 1998).
These ligands probably inhibited the helicase reaction by intercalating
into the major groove of the DNA. This presumably provides a physical
block to continued translocation by the ctDNA helicase II, causing the
unwinding reaction to be inhibited, as was also suggested by George et
al. (1992). Since these ligands were also inhibitory to the ATPase
reaction, we can conclude that ATPase requires translocation on the
DNA.
The possibility that the helicase reaction could also be inhibited by
direct binding of these ligands to the ctDNA helicase II protein was
ruled out by preincubating the helicase with inhibitory concentrations
of daunorubicin and nogalamycin prior to dilution in an unwinding
reaction. Under these conditions the unwinding activity was not
inhibited (data not shown). This further confirmed that the inhibition
was due to the formation of a ligand-DNA complex that impeded the
translocation of the protein. Overall, the data suggest the possibility
that inhibitory intercalators must place a functionality in the major
groove when bound to DNA. The exact mechanism of DNA unwinding is not
yet defined. These results may be important for understanding both the
mechanism by which the duplex DNA is unwound by a helicase and also the
mechanism by which these ligands inhibit cellular function.
The biological roles of only a few eukaryotic helicases have been
determined. Recently, a gene encoding a putative helicase was reported
in Arabidopsis by activation of a promoter trap (Wei et al., 1997). In
plants the MCM proteins/gene products from Arabidopsis (Springer et al., 1995), maize (Sabelli et al., 1996), and
Dactylis glomexata (Ivanova et al., 1994) were also shown to
contain helicase motifs. The MCM proteins, first discovered in
minichromosome maintenance mutants of yeast, were reported to be
involved in activating the origins of replication. If these MCM
proteins from plants are shown to be ATP-dependent helicases by virtue
of helicase activity (which has not been shown yet), this will prove a
role for a helicase in DNA replication. However, the simian virus-40
large tumor antigen helicase has been shown to play a role in DNA
replication (Stahl and Knippers, 1987; Goetz et al., 1988). The E1
protein, a DNA helicase from bovine papilloma virus, is also involved
in DNA replication (Seo et al., 1993). Recently, the Werner's syndrome gene product was shown to contain DNA helicase activity (Suzuki et al.,
1997). Werner's syndrome is a rare, autosomal recessive genetic
disorder causing premature aging accompanied by rare cancer. Recently,
the crystal structures of complexes of E. coli Rep
helicase bound to ssDNA and ADP (Korolev et al., 1997) and a C-terminal fragment of the bacteriophage T7 gene 4 helicase (Bird et al., 1997)
have been determined.
The replication of ctDNA was studied by analyzing the structure of
replicative intermediates in the electron microscope. In pea, ctDNA
replication was initiated by introducing two displacement loops (OriA
and OriB D-loops), which expand toward each other and initiate the
formation of Cairns replicative forked structures (Tuteja, 1997; Tuteja
and Tewari, 1998). Since ctDNA helicase II prefers thefork structures,
it seems likely that the enzyme unwinds the ctDNA through Cairns
replicative fork and is thereby involved in ctDNA replication. However,
the possibility of its role in other DNA transactions cannot be ruled
out.
| |
FOOTNOTES |
*
Corresponding author; e-mail narendra{at}icgebnd.ernet.in; fax
91-11-6162316.
Received May 4, 1998;
accepted July 30, 1998.
| |
ABBREVIATIONS |
Abbreviations:
ATPS, adenosine 5-O-(3-thiotriphosphate).
ds, double-stranded.
NTP, nuceloside triphosphate.
ss, single-stranded.
VP-16, 4-demethyl-epipodophyllotoxin- -D-ethylidene
glucoside.
| |
ACKNOWLEDGMENTS |
We thank Prof. Arthur Kornberg (Stanford University Medical
Center, CA), Dr. Robert Haselkorn (University of Chicago), Dr. Jorge
Allende (University of Chile, Santiago), Dr. Sandor Pongor (International Center for Genetic Engineering and Biotechnology, Trieste, Italy) and Prof. K.K. Tewari (International Centre for Genetic
Engineering and Biotechnology, New Delhi, India) for critical reading
of the manuscript; and R. Radha and Gita Prakash (New Delhi) and
Suzanne Kerbavcic (Trieste) for secretarial assistance.
| |
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Top
Abstract
Introduction