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J. Biol. Chem., Vol. 277, Issue 17, 15182-15189, April 26, 2002
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From the Department of Biochemistry and Molecular Biology,
University of Miami School of Medicine, Miami, Florida 33101-6129
Received for publication, October 16, 2001, and in revised form, December 31, 2001
The genome of herpes simplex virus type-1
undergoes a high frequency of homologous recombination in the absence
of a virus-encoded RecA-type protein. We hypothesized that viral
homologous recombination is mediated by the combined action of the
viral single strand DNA-binding protein (ICP8) and helicase-primase.
Our results show that ICP8 catalyzes the formation of recombination
intermediates (joint molecules) between circular single-stranded
acceptor and linear duplex donor DNA. Joint molecules formed by
invasion of a 3'-terminal strand displaces the non-complementary
5'-terminal strand, thereby creating a loading site for the
helicase-primase. Helicase-primase acts on these joint molecules to
promote ATP-dependent branch migration. Finally, we have
reconstituted strand exchange by the synchronous action of ICP8 and
helicase-primase. Based on these data, we present a recombination
mechanism for a eukaryotic DNA virus in which a single strand
DNA-binding protein and helicase cooperate to promote homologous
pairing and branch migration.
Homologous recombination is a fundamental biological process. It
serves, among other purposes, to repair DNA damage such as double
strand DNA breaks or to reinitiate DNA replication at stalled replication forks. Herpes simplex virus
(HSV)1 undergoes a high
frequency of homologous recombination during its replicative cycle. One
of the earliest reports on HSV recombination dates to 1955;
co-infection of chorioallantois with HSV strains of varying virulence
led to recombinant progeny with altered virulence (1). Additional proof
for recombination of the HSV genome comes from the observation that the
subgenomic elements, UL and US, undergo
inversion with respect to each other, generating four possible isomeric
forms (2-4). This genome isomerization appears to be a consequence of
homologous recombination rather than of a site-specific event involving
cleavage at the a sequences that reside in inverted
orientations at the termini of UL and US (5). Nevertheless, the a sequences may act as recombination
hotspots. In this context, the a sequences are the sites of
double-stranded (ds) DNA breaks created either by a structure-specific
endonuclease2 or during cleavage
and packaging of the viral genome (6, 7). The terminal a
sequences have also been shown to be involved in the circularization of
the viral genome by homologous recombination (8). This process occurs
immediately after infection by the virus and appears to be mediated by
host factors (9, 10). The frequency of recombination has been estimated
to be on the order of 0.5-0.7%/kilobase of the viral genome (11).
Furthermore, a screen for replication-competent HSV-1 origins from a
library in which particular origin elements were substituted with
random sequences resulted in the isolation of wild-type origins at a frequency of 25% as a consequence of homologous recombination (12).
Insight into the mechanism by which homologous recombination proceeds
during HSV-1 replication is provided by several lines of evidence; it
was shown that plasmid-based homologous recombination was dependent on
replication initiated from the viral origin, oriS (13). Furthermore,
this recombination was also shown to depend solely on the seven
essential viral replication genes (13). In subsequent studies, it was
shown that a-sequence-mediated recombination was tightly
linked to DNA replication (14, 15). These findings establish a direct
role for DNA replication and DNA replication enzymes in the
recombination process. This notion is supported by the presence during
viral replication of highly branched DNA replication intermediates that
may arise as a consequence of strand-transfer reactions (16-18).
Escherichia coli RecA has served as a model for understanding
the enzymology of homologous recombination (19). Many organisms, ranging from bacteriophages to yeasts to metazoans encode RecA homologs
such as UvsX, RadA, Dmc1, and Rad51 (20-23). HSV-1 and other members
of the Herpesviridae, like other viruses such as bacteriophage T7, do not encode a RecA-type protein. Consequently, recombination in this class of virus may proceed by an alternate mechanism. Based on its dependence on DNA replication, we propose that
HSV-1 recombination is mediated by viral DNA replication enzymes
analogous to the mechanism in T7. In T7, two DNA replication proteins,
the viral single strand DNA-binding protein (SSB) (gene 2.5 protein)
and helicase-primase (gene 4 protein) perform pivotal roles in
catalyzing strand exchange. The SSB acts as an annealing protein,
mediating the formation of joint molecules (JM), in which complementary
strands from donor and acceptor DNA molecules are physically linked,
whereas the helicase-primase performs branch migration to complete
strand exchange (24). We hypothesize that the HSV-1 SSB (ICP8) and
either the replicative helicase-primase or the UL9 helicase perform
analogous roles in HSV-1 recombination.
ICP8 binds preferentially to single-stranded (ss) DNA with a site size
of 10 ± 1 nucleotides (25). In support of its role as a protein
that participates in homologous recombination, it has previously been
shown that ICP8 catalyzes reannealing of complementary ssDNA molecules
(26). ICP8 also acts as a helix-destabilizing protein capable of
melting DNA duplexes in a reaction that requires stoichiometric amounts
of protein (27). Furthermore, using electron microscopy to visualize
individual DNA molecules, ICP8 was shown to promote limited strand
transfer (up to 1 kilobase pair (kbp)) (28). It should be noted that
the preparation of ICP8 used in that study contained traces of a 5'-3'
dsDNA exonuclease that may have generated ssDNA regions and facilitated
subsequent ICP8-mediated annealing of complementary strands (28).
HSV-1 encodes two helicases that may fulfill the role of the branch
migration enzyme. UL9 is the viral initiator protein that possesses
3'-5' helicase activity (29). In addition, ICP8 has been shown to
associate with UL9 and to stimulate its helicase activity (29-32). The
viral replicative helicase is a hetero-trimeric complex that consists
of a core enzyme (UL5 and UL52 subunits) possessing both 5'-3' helicase
and primase activities and a loading factor (UL8 subunit) that is
required for optimal activity on ICP8-coated templates (33-36). Based
on their association with ICP8, either helicase may be suitable in
branch migration.
Given that ICP8 and the two viral helicases are essential DNA
replication proteins, it is difficult to study their role in recombination using a genetic approach. Consequently, we have examined
the role of ICP8 and its associated helicases in recombination reactions in vitro. Here we show that ICP8 can catalyze
pairing of homologous DNA molecules and that helicase-primase can
process intermediates formed by ICP8 to complete strand exchange.
Materials--
ATP (disodium salt) and
[ Proteins--
Restriction endonucleases were purchased
from New England Biolabs and Promega. Bacteriophage T4
polynucleotide kinase was obtained from New England Biolabs. Proteinase
K, creatine kinase, and calf intestinal phosphatase were purchased from
Roche Molecular Biochemicals. E. coli RecA protein and SSB
were purchased from U. S. Biochemical Corp. RecA concentrations are
expressed in moles of monomeric protein, whereas SSB concentrations are
expressed in moles of tetrameric protein. ICP8, UL9, and UL5/52 core
enzyme and UL8 subunit were purified as previously described (31, 35, 37). The preparation of ICP8 was devoid of detectable exonuclease activity. Protein concentrations, expressed in moles of monomeric protein, were determined using extinction coefficients of 82,720, 89,220, 130,390, and 171,380 M Nucleic Acids--
M13 mp18 viral circular ssDNA and M13 mp18
replicative-form DNA were purchased from New England Biolabs and Bayou
Biolabs, respectively. Linear In Vitro Recombination Assay--
Joint molecule formation and
strand exchange between homologous DNA molecules consisting of linear
M13 dsDNA (donor DNA) and circular M13 ssDNA (acceptor DNA) was
detected using an electrophoretic mobility shift assay as previously
described (39). Unless otherwise stated, reactions were performed as
follows; dsDNA (substrates A to C) (5 µM) was
preincubated with 2 µM ICP8 in 20 mM
HEPES-NaOH, pH 7.5, 5% glycerol, and 1 mM dithiothreitol
for 30 min at 37 °C. The reaction was supplemented with M13 ssDNA
(10 µM), 2.5 mM MgCl2, and 25 mM NaCl and further incubated for 30 min at 37 °C. The
final concentration of ICP8 in the reaction was 1 µM. To
examine the effect of HSV-1 helicase-primase on JM formed by ICP8
(branch migration reaction), reactions were performed as described
except that the preincubation mixture contained 0.5 mM
MgCl2, and the reactions were supplemented with 0.75 mM MgCl2, 3 mM ATP,
helicase-primase (100 nM UL5/52 and 300 nM
UL8), and no NaCl and incubated for 60 min at 37 °C. Reactions with
RecA were performed in 25 mM Tris-HCl, pH 7.2, 10 mM MgCl2, 1 mM dithiothreitol, and
5% glycerol. M13 mp18 ssDNA (10 µM) was preincubated
with 2 µM RecA for 10 min at 37 °C followed by the
addition of 0.3 µM E. coli SSB, 5 µM substrate A, 1 mM ATP, 20 mM
creatine phosphate, and 1 µg of creatine kinase and incubated at
37 °C as indicated. Reactions were quenched by the addition of
termination buffer (final concentration: 1% SDS, 25 mM
EDTA, and 0.1 mg/ml proteinase K) followed by incubation for 10 min at
37 °C. The reaction mixtures were resolved by electrophoresis
through 1% agarose-Tris acetate EDTA, pH 8.3, gels at 1.5 V/cm
for 12 h. The gels were dried onto DE81 chromatography paper
(Whatman) and analyzed and quantitated by storage phosphor analysis
with a Molecular Dynamics Storm 840. Reaction products (JM, gapped DNA
(gDNA), and linear ssDNA) are expressed as a percentage of the total radioactivity.
Helix Destabilization Assay--
dsDNA (6.6 µM)
was incubated with ICP8 as indicated in 20 mM HEPES-NaOH,
pH 7.5, 5% glycerol, and 1 mM dithiothreitol for 30 min at
37 °C. Reactions were quenched as described above and resolved by
electrophoresis through 1% agarose-Tris acetate EDTA, pH 8.3, gels at 1.5 V/cm for 12 h. The gels were dried onto DE81 chromatography paper (Whatman), analyzed, and quantitated by storage phosphorimaging analysis with a Molecular Dynamics Storm 840. Linear
ssDNA products are expressed as a percentage of the total radioactivity.
ICP8 Catalyzes Pairing between Homologous DNA Molecules--
We
have examined the ability of ICP8 to catalyze intermolecular pairing
in vitro. The substrates for this reaction were acceptor DNA, consisting of circular M13 ssDNA, and donor DNA, consisting of 5'
32P-labeled complementary linear dsDNA molecules. DNA
substrates possessing TspRI ends (9 nucleotide 3' overhang)
or cos termini (12 nucleotide 5' overhang) were used to
supply ICP8 with an appropriate loading site (25). In each case, the
strand possessing the overhang was complementary to circular M13 ssDNA.
Pairing between these substrates was measured with an electrophoretic
mobility shift assay in which products exhibited reduced mobility in a
non-denaturing agarose gel, as described under "Experimental Procedures."
The standard assay consisted of preincubation of
32P-labeled donor DNA (dsDNA) with ICP8 followed by the
addition of acceptor DNA (ssDNA). Before electrophoresis, samples were
treated with proteinase K and SDS to eliminate formation of protein-DNA
complexes. Fig. 1 shows the products of
reactions with substrate A, a 2.5-kbp fragment possessing a
9-nucleotide 3' overhang at one end. The products of the complete
reaction are shown in lane 10 and encompass a heterogeneous
population of reaction intermediates (JM) as well as complete products
(gDNA and linear ssDNA). Products formed by the action of ICP8
exhibited a mobility identical to those formed by gradual cooling of
denatured ds and ssDNA (lane 3). The electrophoretic
mobility of ICP8-catalyzed products was distinct from species formed
due to stable interactions of ICP8 with DNA substrates (lane
18). The reaction was essentially ICP8-dependent since
the amount of products formed in the absence of ICP8 (lane 6) or with bovine serum albumin (lane 17) was
negligible (~5%). The small amount of products formed in the absence
of ICP8 may be attributed to spontaneous annealing of complementary
strands. Omission of either donor or acceptor DNA eliminated product
formation (lanes 7 and 8). Preincubation of ICP8
with donor DNA was essential since simultaneous incubation of ICP8 with
both substrates or preincubation with acceptor DNA reduced the amount
of product (lanes 9 and 11). ICP8-catalyzed
pairing was dependent on MgCl2 (lane 14) and
NaCl (lane 15). However, product formation was inhibited when MgCl2 and NaCl were included in the preincubation step
(lane 12). Moreover, when the complete reaction was
performed in the absence of MgCl2 and NaCl, the predominant
product formed by ICP8 was unwound ssDNA (lane 13). The
dependence of intermolecular pairing on MgCl2 and NaCl may
be rationalized by previous data, which show that although the
helix-destabilizing properties of ICP8 are inhibited by
MgCl2 and NaCl, its annealing activity is MgCl2- and NaCl-dependent (26, 27). Thus,
preincubation of dsDNA with ICP8 in the absence of MgCl2
and NaCl permits efficient loading of ICP8 on the donor DNA, leading to
destabilization, followed by subsequent MgCl2- and
NaCl-dependent annealing of complementary strands to form
either JM, in the case of incomplete destabilization, or gDNA, in the
case of complete destabilization of the donor DNA. Similar results were
obtained with substrate B, a 1.1-kbp fragment possessing a
12-nucleotide 5' overhang at one end (data not shown).
The properties of ICP8-catalyzed intermolecular pairing with substrate
A are depicted in Fig. 2. The ICP8
titration shows that product formation was "all or none,"
indicating that the reaction is cooperative and requires stoichiometric
amounts of ICP8 (Fig. 2A). Maximal product formation
occurred at a concentration of ICP8 required to coat two thirds of the
total DNA, assuming a site size of 10 nucleotides (25), corresponding
to a ratio of ~1 ICP8 molecule to 15 nucleotides of ssDNA. The amount
of product formed was proportional to the concentration of acceptor DNA
(Fig. 2B). A 10-fold increase in acceptor DNA concentration doubled the amount of product formed, indicating that the annealing step is second order with respect to DNA concentration as previously published (26). At higher acceptor DNA concentrations (>100 µM), the amount of product ceases to increase, as
expected, presumably because the concentration of ICP8 becomes
limiting. Fig. 2C shows that the extent of product formation
was dependent on the length of preincubation as well as incubation with
acceptor DNA. The rate of product formation showed a plateau at ~30
min that corresponds to the standard incubation time. Similar results
were obtained with substrate B (data not shown).
An integral part of the mechanism by which ICP8 promotes intermolecular
pairing appears to be its helix-destabilizing activity. Therefore, the
ability of ICP8 to catalyze the destabilization of substrates A (9 nucleotide 3' overhang) and C (12 nucleotide 5' overhang) was examined.
Fig. 3 shows that ICP8 mediated limited destabilization of both substrates. Importantly, the data show that
ICP8 is capable of destabilizing DNA up to ~2.5 kbp in length regardless of the polarity of the overhang. It should be noted that the
degree of denaturation observed in our experiments is an underestimate
since we found that ~15% of the denatured DNA reannealed
spontaneously during the termination reaction.
Helicase-Primase Acts on Joint Molecules to Promote Strand
Exchange--
The previous experiments show the ability of ICP8 to
catalyze intermolecular pairing, including the formation of JM. Branch migration is required to convert recombination intermediates (JM) into
strand exchange products (gDNA and linear ssDNA). As stated in the
introduction, we hypothesize that either HSV-1 helicase-primase or UL9
participate in this step.
Fig. 4 shows the results of in
vitro recombination experiments with substrate A (9-nucleotide 3'
overhang). In these experiments, 0.5 mM MgCl2
was present during the preincubation to prevent complete destabilization of the donor duplex and thereby enrich for JM formation
(Fig. 4A, lane 4). JM were present as a variety
of species, presumably because of the heterogeneity of such molecules.
Invasion of the complementary 3'-terminal strand displaces the
non-complementary 5'-terminal strand, thereby creating a loading site
for the helicase-primase. The addition of helicase-primase to preformed
JM resulted in the disappearance of JM and the concomitant appearance
of both strand exchange products (gDNA and linear ssDNA) (compare
lanes 4 and 6). The electrophoretic mobility of
the gDNA product was indistinguishable from that formed by the action
of RecA (lane 7) and coincident with that of the major
product formed by gradual cooling of denatured dsDNA and ssDNA
(lane 3). The addition of UL9 to preformed JM did not
generate significant amounts of gDNA despite the fact that significant
DNA unwinding had occurred, as shown by the presence of linear ssDNA
(lane 5). The inability of UL9 to support branch migration
is not unexpected given the 5' overhang of the displaced strand and the
3'-5' polarity of UL9. However, given the extent of DNA unwinding
observed in the presence of UL9, the inability of ICP8 to promote
intermolecular pairing indicates that ICP8 and UL9 do not cooperate in
the pairing reaction. The formation of strand exchange products was
also examined with JM formed with substrate C. In this case, invasion
of the complementary 5'-terminal strand displaces the non-complementary
3'-terminal strand, thereby creating a loading site for UL9. No strand
exchange products were observed with either UL9 or helicase-primase
(data not shown).
The data shown in Fig. 4B provides additional support for
the role of helicase-primase and ICP8 in branch migration. Omission of
ICP8 from the reaction abolished the formation of gDNA and linear
ssDNA, indicating that the reaction is ICP8-dependent
(lane 3). The formation of strand exchange products was
reduced ~2-fold when the helicase-primase loading factor, UL8
subunit, was omitted (lane 4). Branch migration was
dependent on ATP (lane 5).
To determine the amount of helicase-primase required for optimal branch
migration, increasing concentrations of helicase-primase were added to
preformed JM. Quantitation of the reaction products indicates that
product formation was dose-dependent beyond 50 nM enzyme (Fig.
5A). It is possible that at
low enzyme concentrations (<50 nM), helicase-primase is
unable to efficiently interact with the ICP8-coated substrate (35). The
kinetics of branch migration are shown in Fig. 5B. The data
show that there was a concomitant increase in both reaction products
(gDNA and linear ssDNA). The optimal MgCl2 concentration
for the branch migration activity of helicase-primase was 1.25 mM. At 4 mM MgCl2, activity was
reduced 3-fold. Similarly, branch migration was also inhibited at
concentrations of NaCl above 50 mM (data not shown).
ICP8 and Helicase-Primase Act in Concert to Mediate Strand
Exchange--
The strand exchange reactions performed thus far can be
delineated into two parts; they are ICP8-catalyzed JM formation and ICP8/helicase-primase-mediated branch migration. Because ICP8 interacts
both physically and functionally with helicase-primase (35, 36), it is
possible that the two proteins form a multi-subunit recombinase in
which both steps are coupled. This complex would be functionally
analogous to E. coli RecA. Therefore, the ability of an
ICP8/helicase-primase complex to mediate strand exchange synchronously
was compared with that of RecA. The data in Fig. 6 show that the ICP8/helicase-primase
complex does indeed promote strand exchange, albeit not as efficiently
as RecA. Possible explanations as to the inefficiency of the reaction
may be that ICP8 becomes limiting or that in the steady state, an
equilibrium exists in which helicase-primase acts to drive strand
exchange but also disrupts JM by initiating 5'-3' DNA unwinding on the
circular region of the JM.
Here we have demonstrated that the SSB (ICP8) and replicative
helicase (helicase-primase) of HSV-1, two essential viral replication proteins, cooperate to promote strand exchange. We have used a classical in vitro recombination assay to detect pairing and
strand exchange between homologous donor (linear dsDNA) and acceptor (circular ssDNA) molecules (39). To initiate homologous pairing, the
donor DNA needs to be processed to generate ssDNA regions that are
complementary to the acceptor DNA. We have constructed DNA substrates
that possess overhangs of defined length and polarity by
endonucleolytic incisions, generating short overhangs (9-12 nucleotides) that restrict non-enzymatic intermolecular pairing. Using
such substrates, we have shown that ICP8 catalyzes pairing between
complementary DNA molecules, generating recombination intermediates
that are acted upon by helicase-primase to promote branch migration and
complete strand exchange. Importantly, we have also shown that the
concerted action of ICP8 and helicase-primase can promote strand exchange.
The intermolecular pairing activity of ICP8 appears to require
stoichiometric amounts of protein, consistent with the formation of a
nucleoprotein filament, as seen with other recombinases (40). However,
optimal pairing activity does not require saturating concentrations of
ICP8, possibly because only one strand needs to be coated.
In contrast to previous work in which the helix-destabilizing activity
of ICP8 was restricted to short DNA fragments (27), we found that ICP8
can promote extensive destabilization of fragments up to 2.5 kbp. This
may be rationalized by the fact that the substrates used in our current
work provided ICP8 with an appropriate loading site (25).
To complete strand exchange, JM formed by the action of ICP8 need to be
processed by branch migration. We hypothesized that this role may be
performed by either of the two HSV-1 helicases, UL9 or
helicase-primase. We find that only helicase-primase is active in this
capacity. Thus, invasion of the complementary 3'-terminal strand
displaces the non-complementary 5'-terminal strand, thereby creating a
loading site for the helicase-primase to enable branch migration. In
addition, helicase-primase is suitable for this task since it has been
shown to interact functionally and physically with ICP8 (35, 36). This
association is dependent on the UL8 subunit, which when omitted from
our in vitro reactions, significantly decreased branch
migration activity. Our results indicate that UL9 does not support
branch migration despite its close functional and physical association
with ICP8 (29-32). Conceptually, UL9 should be active on JM that
possess a 3'-terminal loading site (formed by the transfer of a
complementary 5'-terminal strand). However, it is possible that such
ICP8-coated JM are not efficiently recognized by UL9, especially at
coating concentrations of ICP8 (30).
Our findings are summarized schematically in Fig.
7. Depending on reaction conditions,
products consisted of either recombination intermediates (JM), when the
helix-destabilizing activity of ICP8 was inhibited (Steps I
and III), or strand exchange products (gDNA and linear
ssDNA), when ICP8 was allowed to completely destabilize the donor DNA
duplex (Steps II and III). In both cases, pairing was presumably mediated by the renaturation activity of ICP8 (26). To
complete strand exchange, JM formed by the action of ICP8 need to be
processed by branch migration. Transfer of a 3'-terminal strand
displaces the non-complementary 5'-terminal strand, thereby creating a
loading site for the helicase-primase to enable branch migration
(Step IV). Strand exchange products may also be generated by
an additional pathway in which the combined action of helicase-primase and ICP8 on the donor DNA increases the pool of linear ssDNA available for pairing with the acceptor DNA (Step V). It should be
noted that during viral replication in vivo, some
recombination may also be mediated by host enzymes. Moreover, invasion
by a 3'-terminal strand would act to prime DNA synthesis, resulting in
the coupling of recombination and replication, aiding in the rescue of
collapsed replication forks (41). DNA synthesis that is primed by
invading strands could account for the branched intermediates that are prevalent during viral replication (16-18).
Our results directly implicate DNA replication proteins in HSV-1
homologous recombination. We propose that an SSB (ICP8) performs an
active role in HSV-1 recombination by mediating intermolecular pairing.
This function is analogous to that of bacteriophage T7 SSB (gene 2.5 protein), which also acts as a pairing protein (24). We propose that
the HSV-1 helicase-primase participates in recombination by promoting
branch migration. This function would be analogous to that of the
E. coli branch migration enzymes RuvAB and RecG, which also
exhibit helicase activity (42, 43). Similarly, the replicative
helicases of T4 (gene 41 protein) and T7 (gene 4 protein) also catalyze
branch migration (24, 44). However, in contrast to T7 helicase, which
catalyzes branch migration in the absence of its cognate SSB (45),
branch migration in HSV-1 appears to be ICP8-dependent.
Initiation of homologous recombination by strand invasion requires the
presence of ssDNA ends. During viral replication, ssDNA ends may be
created due to the collapse of replication forks at gaps in the viral
genome (16). The combined action of ICP8 and helicase-primase may
subsequently extend regions of ssDNA to initiate intermolecular
pairing. Alternatively, dsDNA exonucleases may process the DNA from
dsDNA ends (e.g. the ends of the rolling-circle intermediate
or breaks at a sequence hotspots). Processing at these sites
may be carried out by the HSV-1 5'-3' exonuclease (UL12). After
exonucleolytic processing of a DNA end, the known association between
UL12 and ICP8 may serve to recruit ICP8 to promote intermolecular
pairing (46). This situation would be analogous to the mechanism of
red-mediated homologous recombination in *
This work was supported by National Institutes of Health
Grant GM62643 and Florida Biomedical Research Program Grant BM 022.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, February 6, 2002, DOI 10.1074/jbc.M109988200
2
K.-J. Huang and I. R. Lehman, personal communication.
The abbreviations used are:
HSV, herpes simplex
virus;
kbp, kilobase pair(s);
ds, double-stranded;
ss, single-stranded;
gDNA, gapped DNA;
JM, joint molecule(s);
SSB, single strand DNA-binding
protein.
In Vitro Strand Exchange Promoted by the Herpes
Simplex Virus Type-1 Single Strand DNA-binding Protein (ICP8)
and DNA Helicase-Primase*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (4,500 Ci/mmol) were purchased from Sigma
and ICN Biomedicals, respectively. Creatine phosphate and Sephadex G-25
(fine) Quick spin columns were purchased from Roche Molecular
Biochemicals. The Wizard DNA clean-up system and Ultrafree-DA-agarose
gel DNA extraction devices were purchased from Promega and Millipore, respectively.
1
cm1 at 280 nm for ICP8, UL9, UL8, and UL5/52, respectively.
DNA was purchased from New England
Biolabs. Recombinant M13 containing cos
(M13-cos) was constructed by inserting a 1163-bp
DraI fragment (positions 47401-120) derived from
circularized DNA into the SmaI site of M13 mp18 DNA.
M13-cos viral circular ssDNA was prepared according to the
method of Sanger et al. (38). M13 mp18 replicative-form DNA
was digested with DraI (position 4621) to create one blunt
end and TspRI (position 2105) to generate a 2516-bp linear
dsDNA with a 9-nucleotide 3' overhang (substrate A). Similarly,
substrates B and C were generated by digesting DNA with
DraI (position 47,429) and BsrBI (position 45,991) to create 1,073- and 2511-bp cos-terminal
(12-nucleotide 5' overhang) fragments, respectively. In all cases, the
strand possessing the overhang was complementary to viral circular M13 ssDNA. All fragments were dephosphorylated using calf intestinal phosphatase and resolved by agarose gel electrophoresis followed by
extraction and purification with the Ultrafree-DA and Wizard clean up
systems. Purified DNA substrates were labeled at their 5' ends with
[-32P]ATP and polynucleotide kinase. Unincorporated
nucleotides were removed by spin column chromatography, and DNA
concentrations were determined spectrophotometrically using an
extinction coefficient of 6,600 M1
cm1 at 260 nm. DNA concentrations are expressed in moles
of nucleotides.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (40K):
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Fig. 1.
ICP8 catalyzes pairing between homologous DNA
molecules. Recombination products were formed using substrate A as
described under "Experimental Procedures," except that
electrophoresis was performed at 4.5 V/cm for 3 h. Storage
phosphorimage shows: lane 1, heat-denatured dsDNA;
lane 2, renatured dsDNA; lane 3, products formed
by gradual cooling of denatured dsDNA and ssDNA; lane 4,
dsDNA; lane 5, ssDNA; lane 6, reaction without
ICP8; lane 7, reaction without ssDNA; lane 8,
reaction without dsDNA; lane 9, complete reaction without
preincubation; lane 10, complete reaction; lane
11, ICP8 preincubated with ssDNA instead of dsDNA; lane
12, preincubation with MgCl2 and NaCl, lane
13, reaction without MgCl2 and NaCl; lane
14, reaction without MgCl2; lane 15,
reaction without NaCl; lane 16, complete reaction with ICP8
and bovine serum albumin (1 µM); lane 17,
reaction with bovine serum albumin (1 µM) only;
lane 18, complete reaction without termination. The
positions of dsDNA, ssDNA, recombination products, and ICP8-DNA
complexes are as indicated. Substrates and reaction products are
schematically represented on the left side of the figure.
The asterisk indicates the position of a 5' 32P
label. The substrate contained a minor contaminant (1.8% of total)
that originated from 32P-labeled linear M13 ssDNA, with a
slightly faster electrophoretic mobility than the recombination
products (lane 4).
View larger version (8K):
[in a new window]
Fig. 2.
Properties of ICP8-catalyzed pairing.
Recombination products were formed with substrate A as described under
"Experimental Procedures." Quantitation of products formed with the
following variables ICP8 (A) and acceptor M13 ssDNA
(B) are shown. C, times of preincubation ( 
)
and incubation (
).
View larger version (35K):
[in a new window]
Fig. 3.
Helix-destabilizing activity of ICP8.
Helix destabilization was measured as described under "Experimental
Procedures." A, storage phosphorimage showing activity of
ICP8 with substrates A (lanes 1-5) and C (lanes
6-10). Lanes 1-5 and 6-10, 0, 0.5, 1, 2, and 4 µM ICP8, respectively. B, quantitation
of the data in A. , substrate A; , substrate C.
View larger version (58K):
[in a new window]
Fig. 4.
Branch migration mediated by helicase-primase
and ICP8. Reactions with ICP8/helicase-primase, ICP8/UL9, or RecA
were performed with substrate A as described under "Experimental
Procedures." A, storage phosphorimages show the role of
helicase-primase and UL9 in branch migration. Lane 1, dsDNA;
lane 2, heat-denatured dsDNA; lane 3, products
formed by gradual cooling of denatured dsDNA and ssDNA; lane
4, branch migration reaction without helicase-primase
or UL9; lane 5, branch migration reaction with 200 nM UL9; lane 6, branch migration reaction with
helicase-primase; lane 7, RecA-catalyzed strand exchange
(180 min incubation). B, requirements for
helicase-primase-catalyzed branch migration. Lane 1,
RecA-catalyzed strand exchange (120-min incubation); lane 2,
complete branch migration reaction; lane 3, branch migration
reaction without ICP8; lane 4, branch migration reaction
without UL8 subunit; lane 5, branch migration reaction
without ATP. The positions of dsDNA, ssDNA, JM, and gDNA are as
indicated and schematically represented. The asterisk
indicates the position of a 5' 32P label. The substrate
contained a minor contaminant (1.8% of total) that originated from
32P-labeled linear M13 ssDNA, with a slightly faster
electrophoretic mobility than gDNA (panel A, lane
1).
View larger version (13K):
[in a new window]
Fig. 5.
Properties of strand exchange mediated by
ICP8 and helicase-primase. Branch migration reactions were
performed with substrate A as described under "Experimental
Procedures." A, concentration of helicase-primase. The
indicated concentrations refer to those of UL5/52. UL5/52 and UL8 were
present in a molar ratio of 1:3. B, time of incubation. ,
gDNA; , linear ssDNA.
View larger version (58K):
[in a new window]
Fig. 6.
Kinetics of strand exchange mediated by the
concerted action of ICP8 and helicase-primase. Branch migration
reactions with RecA or ICP8/helicase-primase were performed with
substrate A as described under "Experimental Procedures," with the
exception that there was no preincubation for the ICP8/helicase-primase
reaction. A, storage phosphorimage showing reaction
products. Lanes 1-6 and 7-12: 0, 5-, 10-, 30-, 60-, and 120-min incubation with RecA and ICP8/helicase-primase,
respectively. Samples were loaded immediately after termination, giving
rise to the staggered pattern of the image. The positions of dsDNA,
ssDNA, JM, and gDNA are as indicated and schematically represented. The
asterisk indicates the position of a 5' 32P
label. The substrate contained a minor contaminant (1.8% of total)
that originated from 32P-labeled linear M13 ssDNA, with a
slightly faster electrophoretic mobility than gDNA (lanes 1 and 7). B, quantitation of gDNA in A.
, ICP8/helicase-primase; , RecA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 7.
A model for homologous recombination mediated
by ICP8 and helicase-primase. ICP8 interacts with ssDNA regions of
the donor duplex DNA. Step I, in the presence of salts
(MgCl2 and NaCl), ICP8 promotes partial unwinding of the
donor duplex DNA. Step II, in the absence of salts, ICP8
promotes complete unwinding of dsDNA. Step III, on
supplementing complementary circular ssDNA and salts, ICP8 catalyzes
the formation of a joint molecule (JM) and/or gapped circular DNA
(gDNA). Step IV, helicase-primase, with the aid
of ICP8, acts on JM and promotes branch migration to form gDNA and
linear ssDNA. Step V, ICP8-coated dsDNA is a suitable
substrate for helicase-primase, resulting in further unwinding of the
donor DNA. The asterisk indicates the position of a 5'
32P label.
and related
bacteriophages (47). However, earlier studies with UL12
mutants showed that they were capable of supporting DNA
replication-mediated homologous recombination and exhibited branched
DNA replication intermediates that presumably arose from invading DNA
replication forks (48), indicating that homologous recombination in
HSV-1 is unlikely to proceed by a red-type mechanism. Nevertheless, UL12 may participate in substrate processing.
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, University of Miami School of Medicine, P. O.
Box 016129, Miami, FL 33101-6129. Tel.: 305-243-2934; Fax: 305-243-3955; E-mail: pboehmer@molbio.med.miami.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Wildy, P.
(1955)
J. Gen. Microbiol.
13,
346-360 2.
Mocarski, E. S.,
Post, L. E.,
and Roizman, B.
(1980)
Cell
22,
243-255[CrossRef][Medline]
[Order article via Infotrieve] 3.
Smiley, J. R.,
Fong, B. S.,
and Leung, W. C.
(1981)
Virology
113,
345-362[CrossRef][Medline]
[Order article via Infotrieve] 4.
Mocarski, E. S.,
and Roizman, B.
(1982)
Proc. Natl. Acad. Sci. U. S. A.
79,
5626-5630 5.
Martin, D. W.,
and Weber, P. C.
(1996)
J. Virol.
70,
8801-8812[Abstract] 6.
Sarisky, R. T.,
and Weber, P. C.
(1994)
J. Virol.
68,
34-47 7.
Dutch, R. E.,
Zemelman, B. V.,
and Lehman, I. R.
(1994)
J. Virol.
68,
3733-3741 8.
Yao, X. D.,
Matecic, M.,
and Elias, P.
(1997)
J. Virol.
71,
6842-6849[Abstract] 9.
Garber, D. A.,
Beverley, S. M.,
and Coen, D. M.
(1993)
Virology
197,
459-462[CrossRef][Medline]
[Order article via Infotrieve] 10.
Yao, X. D.,
and Elias, P.
(2001)
J. Biol. Chem.
276,
2905-2913 11.
Umene, K.
(1999)
Rev. Med. Virol.
9,
171-182[CrossRef][Medline]
[Order article via Infotrieve] 12.
Hammarsten, O.,
and Elias, P.
(1997)
Nucleic Acids Res.
25,
1753-1760 13.
Weber, P. C.,
Challberg, M. D.,
Nelson, N. J.,
Levine, M.,
and Glorioso, J. C.
(1988)
Cell
54,
369-381[CrossRef][Medline]
[Order article via Infotrieve] 14.
Dutch, R. E.,
Bruckner, R. C.,
Mocarski, E. S.,
and Lehman, I. R.
(1992)
J. Virol.
66,
277-285 15.
Dutch, R. E.,
Bianchi, V.,
and Lehman, I. R.
(1995)
J. Virol.
69,
3084-3089[Abstract] 16.
Friedmann, A.,
Shlomai, J.,
and Becker, Y.
(1977)
J. Gen. Virol.
34,
223-234 17.
Severini, A.,
Morgan, A. R.,
Tovell, D. R.,
and Tyrrell, D. L.
(1994)
Virology
200,
428-435[CrossRef][Medline]
[Order article via Infotrieve] 18.
Severini, A.,
Scraba, D. G.,
and Tyrrell, D. L.
(1996)
J. Virol.
70,
3169-3175[Abstract] 19.
Shinohara, A.,
and Ogawa, T.
(1999)
Mutat. Res.
435,
13-21[Medline]
[Order article via Infotrieve] 20.
Yonesaki, T.,
Ryo, Y.,
Minagawa, T.,
and Takahashi, H.
(1985)
Eur. J. Biochem.
148,
127-134[Medline]
[Order article via Infotrieve] 21.
Seitz, E. M.,
Brockman, J. P.,
Sandler, S. J.,
Clark, A. J.,
and Kowalczykowski, S. C.
(1998)
Genes Dev.
12,
1248-1253 22.
Bishop, D. K.,
Park, D., Xu, L.,
and Kleckner, N.
(1992)
Cell
69,
439-456[CrossRef][Medline]
[Order article via Infotrieve] 23.
Shinohara, A.,
Ogawa, H.,
and Ogawa, T.
(1992)
Cell
69,
457-470[CrossRef][Medline]
[Order article via Infotrieve] 24.
Kong, D.,
and Richardson, C. C.
(1996)
EMBO J.
15,
2010-2019[Medline]
[Order article via Infotrieve] 25.
Gourves, A. S.,
Tanguy Le Gac, N.,
Villani, G.,
Boehmer, P. E.,
and Johnson, N. P.
(2000)
J. Biol. Chem.
275,
10864-10869 26.
Dutch, R. E.,
and Lehman, I. R.
(1993)
J. Virol.
67,
6945-6949 27.
Boehmer, P. E.,
and Lehman, I. R.
(1993)
J. Virol.
67,
711-715 28.
Bortner, C.,
Hernandez, T. R.,
Lehman, I. R.,
and Griffith, J.
(1993)
J. Mol. Biol.
231,
241-250[CrossRef][Medline]
[Order article via Infotrieve] 29.
Boehmer, P. E.,
Dodson, M. S.,
and Lehman, I. R.
(1993)
J. Biol. Chem.
268,
1220-1225 30.
Boehmer, P. E.
(1998)
J. Biol. Chem.
273,
2676-2683 31.
Boehmer, P. E.,
and Lehman, I. R.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8444-8448 32.
Arana, M. E.,
Haq, B.,
Tanguy Le Gac, N.,
and Boehmer, P. E.
(2001)
J. Biol. Chem.
276,
6840-6845 33.
Crute, J. J.,
Mocarski, E. S.,
and Lehman, I. R.
(1988)
Nucleic Acids Res.
16,
6585-6596 34.
Crute, J. J.,
Tsurumi, T.,
Zhu, L. A.,
Weller, S. K.,
Olivo, P. D.,
Challberg, M. D.,
Mocarski, E. S.,
and Lehman, I. R.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
2186-2189 35.
Tanguy Le Gac, N.,
Villani, G.,
Hoffmann, J. S.,
and Boehmer, P. E.
(1996)
J. Biol. Chem.
271,
21645-21651 36.
Falkenberg, M.,
Bushnell, D. A.,
Elias, P.,
and Lehman, I. R.
(1997)
J. Biol. Chem.
272,
22766-22770 37.
Sampson, D. A.,
Arana, M. E.,
and Boehmer, P. E.
(2000)
J. Biol. Chem.
275,
2931-2937 38.
Sanger, F.,
Nicklen, S.,
and Coulson, A. R.
(1977)
Proc. Natl. Acad. Sci. U. S. A.
74,
5463-5467 39.
Cox, M. M.,
and Lehman, I. R.
(1981)
Proc. Natl. Acad. Sci. U. S. A.
78,
6018-6022 40.
Egelman, E. H.,
and Stasiak, A.
(1986)
J. Mol. Biol.
191,
677-697[CrossRef][Medline]
[Order article via Infotrieve] 41.
Marians, K. J.
(2000)
Curr. Opin. Genet. Dev.
10,
151-156[CrossRef][Medline]
[Order article via Infotrieve] 42.
Kowalczykowski, S. C.
(2000)
Trends Biochem. Sci.
25,
156-164[CrossRef][Medline]
[Order article via Infotrieve] 43.
Lloyd, R. G.,
and Sharples, G. J.
(1993)
Nucleic Acids Res.
21,
1719-1725 44.
Salinas, F.,
and Kodadek, T.
(1995)
Cell
82,
111-119[CrossRef][Medline]
[Order article via Infotrieve] 45.
Kong, D.,
Griffith, J. D.,
and Richardson, C. C.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2987-2992 46.
Thomas, M. S.,
Gao, M.,
Knipe, D. M.,
and Powell, K. L.
(1992)
J. Virol.
66,
1152-1161 47.
Muyrers, J. P.,
Zhang, Y.,
Buchholz, F.,
and Stewart, A. F.
(2000)
Genes Dev.
14,
1971-1982 48.
Martinez, R.,
Sarisky, R. T.,
Weber, P. C.,
and Weller, S. K.
(1996)
J. Virol.
70,
2075-2085[Abstract]
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