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INTRODUCTION |
The most abundant adduct generated by the interaction of the
antitumor drug cis-diamminedichloroplatinum (II)
(cis-DDP or cisplatin)1 with DNA is the
1-2-intrastrand d(GpG) cross-link (cis-DDP-d(GpG)) (1). The
capacity of this adduct to affect cellular DNA replication significantly contributes to the cytotoxicity and mutagenicity of
cisplatin (2, 3). The effects of intrastrand cisplatin lesions on
several DNA helicases implicated in DNA recombination and repair have
been examined (4-6). In addition, we have recently studied the effect
of a site-specific cis-DDP-d(GpG) adduct on a DNA helicase
that is required for DNA replication, the herpes simplex virus type-1
(HSV-1) origin-binding protein (UL9 protein) (7). In this study, we
have examined the effect of a site-specific cis-DDP-d(GpG)
adduct on the helicase activity of the HSV-1 DNA helicase-primase.
Unlike the UL9 protein, which is required for origin-specific DNA
unwinding during replication initiation (8-10), the helicase-primase
translocates along the lagging strand, unwinding the DNA at the
replication fork (8, 11). The helicase-primase core enzyme consists of
the 99-kDa UL5 and 114-kDa UL52 gene products and
possesses 5'-3' DNA helicase, DNA-dependent nucleoside
triphosphatase, and primase activities (8). A third subunit, the 80-kDa
UL8 gene product, is an essential component of the
helicase-primase complex. Its precise role is still unclear, but it has
been reported to stimulate both the primase and helicase activities of
the core enzyme in the presence of the HSV-1 single-strand DNA-binding protein, ICP8 (12, 13). These studies predicted an
interaction between ICP8 and the helicase-primase heterotrimer
mediated by the UL8 protein, which has subsequently been demonstrated
(14).
To study the effect of a cisplatin lesion on the progression of a DNA
helicase at the replication fork, we examined the activity of the HSV-1
helicase-primase holoenzyme with a fork-like DNA substrate containing a
site-specific cis-DDP-d(GpG) adduct. We also examined the
ability of ICP8 to modulate the effect of the cisplatin lesion. The
results show that the activity of the helicase-primase was inhibited by
the adduct. ICP8 significantly increased the activity of the
helicase-primase with cisplatin-damaged DNA. We propose that ICP8
stimulates translesion DNA unwinding by recruiting the helicase-primase
to the damaged DNA.
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MATERIALS AND METHODS |
Proteins--
ICP8, UL5/52 core enzyme, and UL8 protein were
purified to near homogeneity as described (12). Escherichia
coli SSB was purchased from Amersham Pharmacia Biotech. T4
polynucleotide kinase was obtained from New England Biolabs, Inc.
Restriction endonucleases HaeIII and AciI and
proteinase K were purchased from Boehringer Mannheim.
Chemicals--
cis-diamminedichloroplatinum (II),
99.99% pure, was purchased from Aldrich. ATP was from Boehringer
Mannheim. [
-32P]ATP (6000 Ci/mmol) was from NEN Life
Science Products.
DNA Substrates--
Unmodified or cisplatin-modified substrates
B and C (Fig. 1) were constructed as
described (15). The oligodeoxyribonucleotides employed to construct
substrate A were synthesized on an Applied Biosystem DNA synthesizer
and purified by electrophoresis through denaturing polyacrylamide gels.
To obtain unmodified substrate A, equimolar concentrations of 90- and
59-mers were annealed to produce the Y-shaped partially duplex
molecule. The 59-mer necessary for the construction of platinated
substrate A was obtained by ligation of a 22-mer (residues 1-22,
starting from the 3'-end of the 59-mer), a platinated 12-mer (residues
23-34), and a 25-mer (residues 35-59). Cisplatin modification of the
12-mer (3'-TCCGGTCCCTTT-5'), which contains only one d(GpG)
modification site for cisplatin, was performed as follow: cisplatin was
dissolved in 5 mM sodium perchlorate at a concentration of
0.5 mg/ml and then incubated in 5 mM Tris-HCl, pH 7.8, 1 mM sodium perchlorate at a 2-fold molar excess with the
12-mer for 48 h at 37 °C. The platinated 12-mer was then
precipitated twice in ethanol in order to remove the unreacted drug,
and its concentration determined by absorbance at 260 nm. The
platinated 12-mer was ligated to the 22- and 25-mers using a 41-mer
(complementary to residues 9-49 of the 59-mer) as a scaffold in a
reaction containing 10 units of T4 DNA ligase per 5'-end. The resulting
platinated 59-mer was purified by denaturing polyacrylamide gel
electrophoresis, 5' 32P-labeled, and hybridized to an
equimolar concentration of the complementary 90-mer to yield platinated
substrate A.

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Fig. 1.
DNA substrates used in this study.
Underlined GG residues indicate the target site for
cis-DDP modification. Boxes show the
HaeIII site in A (substrate A) and the
AciI site in B and C (substrates B and
C, respectively).
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A 28-mer complementary to residues 1-28 of the 90-mer of substrate A
and a 27-mer complementary to residues 1-27 of the 90-mer of
substrates B and C were also synthesized and purified (see under
"Helicase Assay").
To determine the extent of cisplatin modification of substrate A, we
took advantage of the fact that the modification site is part of a
HaeIII restriction endonuclease site, which upon modification is rendered resistant to cleavage by the enzyme (4). 5 ng
of 32P-labeled unmodified or platinated substrate A was
digested with 10 units of HaeIII under standard conditions.
Fig. 2 shows that the vast majority of
the platinated 90/59-mer was resistant to cleavage, whereas the
unmodified substrate was digested nearly to completion. PhosphorImager
quantitation indicated that 95% of the platinated substrate was
resistant to HaeIII cleavage and therefore contained the
cis-DDP-d(GpG) adduct. Similar experiments performed with
substrates B and C, and AciI restriction endonuclease indicated that 90% of the substrates were platinated (data not shown).

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Fig. 2.
Extent of cis-DDP-modification of
substrate A. Unmodified or cisplatin-modified substrate A was
digested with HaeIII as described under "Materials and
Methods." The arrow indicates the position of the
HaeIII fragment. Pt, cisplatin-modified
DNA.
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Helicase Assay--
DNA helicase reactions were performed at
37 °C in 20 mM HEPES-NaOH, pH 7.5, 1 mM
dithiothreitol, 10% glycerol, 4.5 mM MgCl2, 3 mM ATP, 0.1 mg/ml bovine serum albumin. Incubation times,
DNA helicase-primase (equimolar concentrations of UL5/52 and UL8
proteins), ICP8, E. coli SSB, and DNA substrate
concentrations were as indicated in the figure legends. Assays included
a 10-40-fold molar excess of unlabeled 28-mer or 27-mer to prevent
reannealing of the unwound DNA strand. In control helicase assays,
performed in the absence of 28-mer, DNA unwinding was only 30% of the
reaction with the 28-mer (data not shown). The reactions were
terminated by addition of 0.2 volumes of 150 mM EDTA, 1%
SDS and then digested for 20 min at 37 °C with 2 mg/ml proteinase K. 3 µl of 0.5% bromphenol blue and 40% glycerol were then added, and
the reaction mixtures were resolved by electrophoresis through 15%
nondenaturing polyacrylamide gels. The gels were scanned using a
PhosphorImager, and the release of single-stranded DNA was quantitated
using the ImageQuant software. DNA unwinding activity is expressed as
the percentage of displaced fragments and is corrected for background
activity by subtracting the percentage of unwinding obtained in control
incubations without protein. Typically such background represented no
more than 10% of the total radioactivity for incubations of 60 min.
DNA unwinding data represent the average of two or more independent
experiments.
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RESULTS |
Effect of the cis-DDP-d(GpG) Adduct on DNA Unwinding by the
Helicase-primase--
The HSV-1 DNA helicase-primase is believed to be
responsible for unwinding DNA at the replication fork (8, 11). The
enzyme requires a single-stranded DNA loading site in order to initiate DNA unwinding in the 5' to 3' direction (11). We define the strand to
which the helicase binds and along which it translocates as the
template strand. As shown in Figs. 3-6, and in agreement with our
previously published work (12), the helicase-primase can unwind the
fork-like substrates depicted in Fig. 1. In order to obtain efficient
unwinding and prevent reannealing of the unwound, labeled primer
strand, an excess of unlabeled oligodeoxyribonucleotide, complementary
to the double-stranded regions of substrates A, B, or C, was present.
Consistent with previous reports (7, 12, 16), we found that a
10-40-fold molar excess of unlabeled oligodeoxyribonucleotide was
necessary to achieve optimal unwinding of the substrates.
The presence of a single cis-DDP-d(GpG) adduct on the 59-mer
template strand inhibited DNA unwinding by the helicase-primase (Fig.
3). As shown in Fig. 3, increasing DNA
unwinding activity was observed with both unmodified and platinated
DNA, reaching a plateau at ~250 nM enzyme. However, the
presence of the cis-DDP-d(GpG) adduct reduced DNA unwinding
by a factor of 3-4, and the extent of inhibition remained unchanged
over a range of enzyme concentrations from 50 to 500 nM. It
is possible that a significant fraction of the total DNA unwinding
observed with the platinated substrate may be due to the presence of a
small percentage (~5%) of unmodified substrate (see under
"Materials and Methods"). The appearance of a faint band at an
intermediate position between substrate and product was occasionally
observed with the platinated DNA (data not shown). We believe that this
band represents a hairpin structure that is induced by the
cis-DDP-d(GpG) adduct in a fraction of the 59-mer
population, thus preventing its hybridization to the 90-mer
oligodeoxyribonucleotide. The capacity of cisplatin to induce such
hairpin structures in oligodeoxyribonucleotides has been documented
(17). Fig. 4 shows that the lesion
inhibited DNA unwinding during the entire course of the reaction,
suggesting that the adduct blocks helicase action. However, the lesion
did not represent a permanent obstacle to the helicase-primase because addition of fresh enzyme resulted in increased DNA unwinding activity (Fig. 5).

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Fig. 3.
Effect of the cis-DDP-d(GpG)
adduct on the helicase activity of the helicase-primase: DNA unwinding
of substrate A as a function of protein concentration. Unmodified
or cis-DDP-modified substrate A (11 nM
molecules) was incubated for 60 min as described under "Materials and
Methods" with the indicated concentrations of helicase-primase and a
10-fold molar excess of unlabeled 28-mer. A, autoradiogram
of the reaction products. Lanes 1-8, unmodified substrate
A. Lane 1, no protein; lanes 2-7, 12.5, 25, 50, 100, 250, and 500 nM helicase-primase, respectively;
lane 8, heat-denatured substrate. Lanes 9-16,
cis-DDP-modified substrate A. Lane 9, no protein;
lanes 10-15, 12.5, 25, 50, 100, 250, and 500 nM
helicase-primase, respectively; lane 16, heat-denatured
substrate. The positions of the 90/59-mer substrates and of the unwound
59-mers are as indicated. B, quantitation of the data shown
in A. , unmodified DNA; , cis-DDP-modified
DNA.
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Fig. 4.
Effect of the cis-DDP-d(GpG)
adduct on the helicase activity of the helicase-primase: kinetics of
DNA unwinding. Unmodified or cis-DDP-modified DNA
substrate A (11 nM molecules) was incubated with 250 nM helicase-primase and a 10-fold molar excess of unlabeled
28-mer as described under "Materials and Methods." At the indicated
times, reactions were stopped and helicase activity measured. ,
unmodified DNA; , cis-DDP-modified DNA.
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Fig. 5.
Effect of the addition of fresh
helicase-primase on the unwinding of unmodified and
cis-DDP-modified substrate A. Unmodified or
cis-DDP-modified substrate A (8.5 nM molecules)
was incubated with 250 nM helicase-primase and a 20-fold
molar excess of unlabeled 28-mer as described under "Materials and
Methods." At the time indicated by the arrows (30 min),
additional 250 nM helicase-primase was added to both
unmodified and cis-DDP-modified substrates. At the indicated
times, reactions were stopped, and helicase activity was measured. ,
unmodified DNA; , unmodified DNA after addition of fresh enzyme;
, cis-DDP-modified DNA; , cis-DDP-modified
DNA after addition of fresh enzyme.
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The data in Fig. 5 also show that the helicase-primase is unstable
during the course of the reaction because addition of fresh enzyme
resulted in increased DNA unwinding activity. Preincubation of the
helicase-primase in the absence of DNA substrate showed that its
half-life was approximately 30 min at 37 °C. In addition, we
observed that preincubation of the helicase-primase with DNA substrate
significantly increased its stability. In contrast, ICP8, in either the
absence or presence of DNA substrate, was not able to increase the
stability of the helicase-primase (data not shown).
In contrast to the data obtained with substrate A, the helicase-primase
unwound unmodified and cisplatin-modified substrates B and C with
comparable efficiency (Fig. 6). As can be
seen, regardless of whether the lesion was placed 13 or 7 nucleotides
from the fork junction (substrates B and C, respectively), its position on the primer strand had no effect on the activity of the
helicase-primase. We found that the helicase-primase exhibited greater
activity with substrate A than substrates B and C. This may be due to
the higher G-C base pair content of substrates B and C, thus making them more difficult to be unwound by the helicase-primase.
Nevertheless, it is clear by comparing Figs. 3, 4, and 6 that the
cis-DDP-d(GpG) adduct inhibits the helicase-primase only
when it is present on the template strand.

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Fig. 6.
Effect of the cis-DDP-d(GpG)
adduct on the helicase activity of the helicase-primase: strand
specificity. A, unmodified or cis-DDP-modified
DNA substrate B (11 nM molecules) was incubated for 60 min
as described under "Materials and Methods" with the indicated
concentrations of helicase-primase and a 20-fold molar excess of
unlabeled 27-mer. B, unmodified or
cis-DDP-modified DNA substrate B (11 nM
molecules) was incubated with 250 nM helicase-primase and a
20-fold molar excess of unlabeled 27-mer as described under
"Materials and Methods." At the indicated times, reactions were
stopped, and helicase activity was measured. C, unmodified
or cis-DDP-modified DNA substrate C (11 nM
molecules) was incubated for 30 min as described under "Materials and
Methods" with the indicated concentrations of helicase-primase and a
20-fold molar excess of unlabeled 27-mer. D, unmodified or
cis-DDP-modified DNA substrate C (11 nM
molecules) was incubated with 400 nM helicase-primase
and a 20-fold molar excess of unlabeled 27-mer as described under
"Materials and Methods." At the indicated times, reactions were
stopped, and helicase activity was measured. , unmodified DNA; ,
cis-DDP modified DNA.
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Effect of ICP8 on the Unwinding of cis-DDP-modified Substrate A by
the Helicase-primase--
The data in the preceding section indicate
that the major cisplatin DNA lesion, when located on the template
strand of a fork-like substrate, is an obstacle to the progress of the
helicase-primase.
We have previously shown that the HSV-1 single-strand DNA-binding
protein, ICP8, stimulates the DNA unwinding activity of the
helicase-primase with unmodified substrate B. Although ICP8 could
stimulate the activity of the core enzyme (UL5/52 proteins), optimal
stimulation required the presence of the UL8 subunit (12). Consequently, we investigated the effect of ICP8 on the unwinding of
cis-DDP modified substrate A by the helicase-primase.
Because ICP8 has been shown to possess helix-destabilizing activity
(18), we determined its capacity to unwind substrate A under our
experimental conditions. As can be seen in Fig.
7, a low level of displacement was
detected only at 1000 and 1200 nM ICP8 for the unmodified and cisplatin-modified substrates, respectively. Quantitation by
PhosphorImager analysis indicated that the amount of displacement was
10 and 15% for 1000 and 1200 nM ICP8, respectively.
Estimates of the DNA-binding site size of ICP8 fall in the range of 1 ICP8 to 12-22 nucleotides of single-stranded DNA (8, 18-20). By
assuming an ICP8 binding site size of 14-15 nucleotides and by
considering the total concentration of DNA (substrate plus unlabeled
28-mer) in the reaction, we estimated that the concentration of ICP8
necessary to completely cover the DNA substrate (coating concentration) was ~800 nM. As shown in Fig. 7 (lanes 4 and
11), no strand-displacement activity was detected at this
concentration of ICP8.

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Fig. 7.
Unwinding of unmodified or
cis-DDP-modified substrate A by ICP8. Unmodified or
cis-DDP-modified substrate A (8.5 nM molecules)
was incubated for 30 min as described under "Materials and Methods"
with a 40-fold molar excess of unlabeled 28-mer and the indicated
concentrations of ICP8. Lanes 1-7, unmodified substrate A. Lane 1, no protein; lane 2, 400 nM
ICP8; lane 3, 600 nM ICP8; lane 4,
800 nM ICP8; lane 5, 1000 nM ICP8;
lane 6, 1200 nM ICP8; lane 7,
heat-denatured substrate. Lanes 8-14,
cis-DDP-modified substrate A. Lane 8, no protein;
lane 9, 400 nM ICP8; lane 10, 600 nM ICP8; lane 11, 800 nM ICP8;
lane 12, 1000 nM ICP8; lane 13, 1200 nM ICP8; lane 14, heat-denatured
substrate.
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Fig. 8 shows that addition of ICP8
stimulated the DNA unwinding activity of the helicase-primase with
platinated DNA up to 6-fold. Maximal stimulation was observed at
coating concentrations of ICP8 (800 nM). The stimulatory
effect of ICP8 on the helicase-primase is likely to be a consequence of
specific protein-protein interactions because it was not observed in
the presence of the heterologous E. coli SSB (see Fig. 8,
columns 11-20).

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Fig. 8.
Effect of ICP8 and E. coli
SSB on DNA unwinding by the helicase-primase. Unmodified
( ) or cis-DDP-modified ( ) DNA substrate A (8.5 nM molecules) was incubated for 30 min as described under
"Materials and Methods" with 250 nM helicase-primase, a
40-fold molar excess of unlabeled 28-mer, and the following
concentrations of ICP8 or E. coli SSB. Columns 1, 6, 11, and 16, helicase-primase alone; columns
2 and 7, helicase-primase plus 200 nM ICP8;
columns 3 and 8, helicase-primase plus 400 nM ICP8; columns 4 and 9,
helicase-primase plus 600 nM ICP8; columns 5 and
10, helicase-primase plus 800 nM ICP8;
columns 12 and 17, helicase-primase plus 200 nM E. coli SSB; columns 13 and
18, helicase-primase plus 400 nM E. coli SSB; columns 14 and 19,
helicase-primase plus 600 nM E. coli SSB;
columns 15 and 20, helicase-primase plus 800 nM E. coli SSB.
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Effect of Competitor DNA on the Unwinding of Unmodified or
Platinated Substrate A by the Helicase-primase in the Presence or
Absence of ICP8--
Addition of a 4-fold molar excess of unlabeled
substrate A to an ongoing helicase reaction with unmodified substrate A
in which the concentration of single-stranded 5' DNA ends was in excess
over helicase-primase resulted in a significant decrease in DNA
unwinding activity (Fig. 9A).
This result suggests that the helicase-primase readily dissociates from
the DNA substrate and that it is distributive. A higher concentration
of unlabeled 28-mer (40-fold molar excess) was used in the competition
experiments in order to prevent reannealing of the unwound labeled DNA
strand in the presence of the competitor DNA.

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Fig. 9.
Effect of competitor DNA on the unwinding of
unmodified or cis-DDP-modified substrate A in the presence
or absence of ICP8. A, unmodified 32P-labeled
substrate A (8.5 nM molecules) was incubated with 250 nM helicase-primase and a 40-fold molar excess of unlabeled
28-mer as described under "Materials and Methods." At the time
indicated by the arrow (2 min), a 4-fold molar excess (34 nM molecules) of unlabeled unmodified substrate A was
added. At the indicated times, reactions were stopped, and helicase
activity was measured. B, unmodified 32P-labeled
substrate A (8.5 nM molecules) was incubated with 250 nM helicase-primase, 800 nM ICP8, and a 40-fold
molar excess of unlabeled 28-mer as described under "Materials and
Methods." At the time indicated by the arrow (2 min), a
4-fold molar excess (34 nM molecules) of unlabeled
unmodified substrate A was added. At the indicated times, reactions
were stopped, and helicase activity was measured. C,
platinated 32P-labeled substrate A (8.5 nM
molecules) was incubated with 250 nM helicase-primase, 800 nM ICP8, and a 40-fold molar excess of unlabeled 28-mer as
described under "Materials and Methods." At the time indicated by
the arrow (2 min), a 4-fold molar excess (34 nM
molecules) of unlabeled unmodified substrate A was added. At the
indicated times, reactions were stopped, and helicase activity was
measured. , , and , no competitor DNA added; , , and
, competitor DNA added.
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To investigate the mechanism by which ICP8 stimulates DNA unwinding, we
performed competition experiments in the presence of coating
concentrations of ICP8. Fig. 9B shows that addition of ICP8
to a helicase reaction with unmodified substrate A did not
significantly alter the level of competition, suggesting that it does
not prevent dissociation of the helicase-primase from the substrate and
that it does not stimulate the helicase-primase by increasing its
processivity.
We then examined the effect of coating concentrations of ICP8 in
competition experiments performed with cisplatin-modified substrate A. We observed an increased level of competition with the platinated DNA
compared with the unmodified substrate (compare Fig. 9, B
and C), suggesting that the lesion induces the
helicase-primase to dissociate rapidly, even in the presence of
ICP8.2 Consistent with this
conclusion, Fig. 10 shows that even in
the presence of ICP8, the helicase-primase was more efficiently
competed from the platinated DNA than from the unmodified DNA
substrate, thus confirming that the cis-DDP-d(GpG) adduct
results in dissociation of the helicase-primase. In addition, the data
in Fig. 10 reiterate that increasing concentrations of competitor DNA
led to a similar reduction in DNA unwinding activity with unmodified
substrate A both in the absence or presence of ICP8.

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Fig. 10.
Effect of increasing concentrations of
competitor DNA on the unwinding of unmodified or
cis-DDP-modified substrate A in the presence or absence of
ICP8. Unmodified or cis-DDP-modified substrate A (8.5 nM molecules) was incubated with 250 nM
helicase-primase or 250 nM helicase-primase plus 800 nM ICP8 and a 40-fold molar excess of unlabeled 28-mer as
described under "Materials and Methods." After 2 min of incubation
at 37 °C, unmodified unlabeled substrate A was added at the
indicated concentrations. After 18 more min at 37 °C, reactions were
stopped, and DNA helicase activity was measured. 100% DNA unwinding
represents the activity observed in the absence of competitor DNA. ,
unmodified substrate A and helicase-primase; , unmodified substrate
A, helicase-primase, and ICP8; , cis-DDP-modified
substrate A, helicase-primase, and ICP8.
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DISCUSSION |
In this work, we have studied the capacity of a replicative DNA
helicase, the HSV-1 DNA helicase-primase, to unwind fork-like DNA
substrates that contain the major cisplatin-DNA adduct,
cis-DDP-d(GpG). The role of the HSV-1 single-strand
DNA-binding protein, ICP8, in modulating the activity of the
helicase-primase was also examined.
Our results show that the DNA unwinding activity of the
helicase-primase was significantly reduced when the
cis-DDP-d(GpG) adduct was located on the template strand to
which the enzyme binds. In contrast, a lesion located on the opposite
strand did not affect DNA unwinding (Figs. 3-6). Inhibition was
observed throughout the time course of DNA unwinding and at high (500 nM) enzyme concentrations. A comparison of these results
with previously published data (7) shows that the activity of the
helicase-primase was inhibited more strongly by the
cis-DDP-d(GpG) adduct than the helicase activity of the
HSV-1 origin-binding protein (UL9 protein), the action of which only
appeared to be retarded by the lesion. This difference in behavior may
reflect the capacity of UL9 protein to oligomerize into a large complex
that is capable of unwinding past the lesion. However, even in the case
of the helicase-primase, the cis-DDP-d(GpG) adduct did not
represent an impassable obstacle, because addition of fresh enzyme
during the course of the reaction resulted in increased DNA unwinding
(Fig. 5). It is interesting to note that, consistent with our results,
it has recently been shown that the DNA unwinding activity of the
helicase-primase is also inhibited by two UV-induced DNA lesions, the
cis-syn cyclobutane thymine dimer and the (6-4)
thymine-thymine lesion, when located on the template strand of the DNA
substrate but not on the primer
strand.3
Previous studies have shown the existence of functional and physical
interactions between ICP8 and the helicase-primase (12-14). Therefore,
we examined the effects of ICP8 on the unwinding of unmodified and
platinated substrates by the helicase-primase. Addition of
equimolar ICP8 stimulated the helicase-primase on unmodified DNA
and significantly reduced the inhibitory effect of the
cis-DDP-d(GpG) adduct on platinated DNA (Fig. 8). However, maximal stimulation of the helicase-primase on damaged DNA was observed only with coating concentrations of ICP8 (Fig. 8). The requirement for coating concentrations of ICP8 may be explained by the
fact that the lesion perturbs the structure of the DNA (21-23) and
presumably obstructs the recognition of the substrate by the
helicase-primase. Accordingly, high concentrations of ICP8 would be
necessary to recruit the helicase-primase to the site of the
cis-DDP-d(GpG) adduct and to allow efficient unwinding. The
stimulation was specific for ICP8 because no such effect was observed
with a heterologous SSB, E. coli SSB, suggesting that it
involves specific protein-protein interactions.
To obtain insight into the mechanism by which ICP8 stimulates the
helicase-primase, we performed the competition experiments depicted in
Figs. 9 and 10. Addition of excess challenger DNA to ongoing helicase
reactions with unmodified DNA in the absence of ICP8 decreased DNA
unwinding, indicating that the helicase-primase was efficiently
competed from the DNA substrate and therefore nonprocessive (Fig.
9A). It has recently been suggested that ICP8 stimulates the
DNA helicase activity of the UL9 protein by preventing its dissociation
from the DNA substrate, thereby increasing its processivity (24). A
similar mechanism may explain the stimulatory effect of ICP8 on the
helicase-primase. However, we found that even high concentrations of
ICP8 failed to prevent the dissociation of the helicase-primase from
its substrate (Fig. 9B). Moreover, the level of competition
was similar in the absence or presence of ICP8 (Fig. 10), indicating
that ICP8 does not increase the processivity of the
helicase-primase.
Interestingly, the level of competition observed with the platinated
substrate in the presence of ICP8 was greater than for the unmodified
substrate (Figs. 9C and 10). These results suggest that the
cis-DDP-d(GpG) lesion increases the dissociation rate of the
helicase-primase from the DNA. Taken together with the relatively short
half-life of the helicase-primase in the absence of DNA, these findings
may in part explain the inhibitory nature of the
cis-DDP-d(GpG) lesion (Figs. 3-5).
It is possible that ICP8 stimulates DNA unwinding by increasing the
stability of the helicase-primase. However, we found no evidence in
support of this mechanism. Rather, we favor a model in which ICP8
recruits the helicase-primase by direct protein-protein interactions
(14), thereby increasing the association rate of the helicase-primase
with DNA and allowing it to unwind the platinated or unmodified DNA.
The salient features of our model are depicted in Fig.
11. In the absence of ICP8, the
distributive nature of the helicase-primase causes the enzyme to cycle
on and off unmodified DNA substrate. The presence of a
cis-DDP-d(GpG) lesion induces the enzyme to dissociate and
also prevents the helicase-primase from binding to the substrate. In
the presence of ICP8, specific protein-protein interactions between
ICP8 and the helicase-primase (14) lead to recruitment of the
helicase-primase to the ICP8-covered DNA substrate. Recruitment of the
helicase-primase to the DNA substrate also occurs with the
cisplatin-damaged DNA, thereby permitting the enzyme to unwind past the
lesion.

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Fig. 11.
Model for ICP8-mediated stimulation of the
helicase-primase. See under "Discussion" for details.
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Because DNA helicases are among the first components of the DNA
replication fork machinery that would encounter a site of DNA damage,
it is important to understand how these enzymes interact with specific
DNA lesions. Our previous (7) and current studies show that specific
interactions between the helicase-primase or UL9 protein DNA helicase
and ICP8 allow unwinding of cisplatin damaged DNA. In HSV-1 DNA
replication, it has been shown that specific protein-protein
interactions between ICP8, UL9 protein, and the subunits of the
helicase-primase are required for the assembly of these proteins into
prereplicative sites and that recruitment of the HSV-1 DNA polymerase
into these sites is mediated by the UL42 subunit of the DNA polymerase
(25, 26). In addition, the recent finding of an interaction between the
UL8 subunit of the helicase-primase and the UL30 subunit of the DNA
polymerase (27) and the known interaction of ICP8 with both enzymes
(14, 28-31) suggest that a complex of DNA polymerase,
helicase-primase, and ICP8 may function at the replication fork and may
eventually lead to some replicative bypass of a
cis-DDP-d(GpG) adduct.