The Herpes Simplex Virus Type-1 Single-strand DNA-binding Protein
(ICP8) Promotes Strand Invasion*
Amitabh V.
Nimonkar and
Paul E.
Boehmer
From the Department of Biochemistry and Molecular Biology,
University of Miami School of Medicine, Miami,
Florida 33101-6129
Received for publication, December 9, 2002
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ABSTRACT |
ICP8, the herpes simplex virus type-1
single-strand DNA-binding protein, was recently shown to promote strand
exchange in conjunction with the viral replicative helicase (Nimonkar,
A. V., and Boehmer, P. E. (2002) J. Biol.
Chem. 277, 15182-15189). Here we show that ICP8 also catalyzes
strand invasion in an ATP-independent manner. Thus, ICP8 promotes the
assimilation of a single-stranded donor molecule into a homologous
plasmid, resulting in the formation of a displacement loop. Invasion of
a homologous duplex by single-stranded DNA requires homology at either
3' or 5' end of the invading strand. The reaction is dependent on the
free energy of supercoiling and alters the topology of the acceptor
plasmid. Hence, strand invasion products formed by ICP8 are resistant
to the action of restriction endonucleases that cleave outside of the
area of pairing. The ability to catalyze strand invasion is a novel
activity of ICP8 and the first demonstration of a eukaryotic viral
single-strand DNA-binding protein to promote this reaction. In this
regard ICP8 is functionally similar to the prototypical prokaryotic
recombinase RecA and its eukaryotic homologs. This strand invasion
activity of ICP8 coupled with DNA synthesis may explain the high
prevalence of branched DNA structures during viral replication.
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INTRODUCTION |
Herpes simplex virus type-1
(HSV-1)1 is a double-stranded
DNA virus with a genome of ~152 kbp (1). The HSV-1 genome undergoes a
high frequency of homologous recombination in a process that is
temporally linked to viral DNA replication (2). Recently, we proposed a
model for HSV-1 recombination in which strand exchange is mediated by
two essential DNA replication proteins and follows a single-strand
annealing and helicase-mediated heteroduplex extension mechanism (3).
In particular, ICP8, the viral single-strand DNA-binding protein (SSB)
utilizes its helix destabilizing and reannealing activities to promote
intermolecular pairing of homologous DNA. Heteroduplex DNA
intermediates formed in this fashion are further processed by
helicase-mediated branch migration catalyzed by the replicative DNA
helicase-primase.
Viral DNA replication intermediates include a high prevalence of
branched structures that presumably arise due to strand invasion coupled to DNA synthesis (4). To account for this phenomenon, we
examined the ability of ICP8 to promote strand invasion. Here we
describe the novel finding that ICP8 promotes assimilation of
single-stranded (ss) DNA into homologous supercoiled acceptor DNA,
resulting in the formation of a displacement loop (D-loop).
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EXPERIMENTAL PROCEDURES |
Enzymes and Reagents--
Escherichia coli SSB
(E-SSB), RecA, and exonuclease I were purchased from U. S. Biochemical
Corp. RecA concentrations are expressed in moles of monomeric protein,
while those of E-SSB are expressed in moles of tetrameric protein. DNA
topoisomerase I (calf thymus) and proteinase K were purchased from
Amersham Biosciences and Roche Molecular Biochemicals,
respectively. Bacteriophage T4 polynucleotide kinase, E. coli RecJ, and all restriction endonucleases were obtained from
New England Biolabs. ICP8 was purified as described previously (5). Its
concentration, expressed in moles of monomeric protein, was determined
using an extinction coefficient of 82,720 M
1
cm
1 at 280 nm calculated from its predicted amino acid
sequence (6). ATP (disodium salt) and chloroquine (diphosphate salt)
were purchased from Sigma. [
-32P]ATP (4,500 Ci/mmol)
and H332PO4 were purchased from ICN Biomedicals.
Nucleic Acids--
The following oligodeoxyribonucleotides, each
with the indicated region of complementarity to the minus strand of
pUC18, were synthesized by Sigma: PB9, 22-mer (422-444) (7); PB11,
100-mer (379-478) (7); PB136 (5'-GTA AAA CGA CGG CCA GTG CCA AGC TTG CAT GCC TGC AGG TCG ACT CTA ATG TGA CTG GTA ACT GTC CGT CAG TCG CAG CTC
CAT ACA CTC CAA AGT GCT C), 100-mer with 50 nucleotides 5'
complementarity (379-428); PB137 (5'- ATG TGA CTG GTA ACT GTC CGT CAG
TCG CAG CTC CAT ACA CTC CAA AGT GCT C GAG GAT CCC CGG GTA CCG AGC TCG
AAT TCG TAA TCA TGG TCA TAG CTG TTT C), 104-mer with 50 nucleotides 3'
complementarity (429-478); and PB142 (5'-GTT ATT GCA TGA AAG CCC GGC
TGA CTC TAG AGG ATC CCC GGG TAC GTT ATT GCA TGA AAG CCC GGC TG), 68-mer
with 22 nucleotides internal complementarity (422-444). Their
concentrations were determined using extinction coefficients at 260 nm
of 208918.6, 939208.1, 951107.4, 980646.5, and 653064.2 M
1 cm
1 for PB9, PB11, PB136,
PB137, and PB142, respectively. Oligonucleotides were
5'-32P-labeled with T4 polynucleotide kinase and purified
using Sephadex G-25 (fine) Quick spin columns (Roche Molecular
Biochemicals). Form I pUC18 and pUC19 DNA were prepared from E. coli JM109 using the Promega Wizard Plus DNA purification system
followed by ethanol precipitation. Uniformly 32P-labeled
form I pUC18 was prepared following overnight growth in LB broth
containing H332PO4 (5 µCi/ml).
All DNA concentrations are expressed in moles of molecules.
Strand Invasion Assay--
Unless otherwise stated, ICP8 (0.25 µM) was preincubated with oligonucleotides (10.5 nM) at 37 °C for 8 min in a buffer containing 25 mM Tris acetate, pH 7.5, 10 mM magnesium
acetate, 1 mM dithiothreitol, 1 mM ATP, and 100 µg/ml bovine serum albumin. Strand invasion was initiated by adding
pUC18 form I DNA (3.5 nM), and incubation was continued for
30 min at 30 °C. RecA-mediated strand invasion was performed under
the same conditions using 3.5 µM protein. The ability of
E-SSB to promote strand invasion was also examined under the same
conditions using 0.25 µM protein. Reactions were quenched
by the addition of termination buffer (final concentration: 2% SDS, 50 mM EDTA, and 3 µg/µl proteinase K) followed by
incubation for 10 min at 30 °C. The reaction mixtures were resolved
by electrophoresis through 1% agarose-Tris acetate EDTA, pH 7.6, gels
at 8 V/cm for 1.5 h. The gels were dried onto DE81 chromatography
paper (Whatman), analyzed, and quantitated by storage phosphor analysis
with a Amersham Biosciences Storm 820. When necessary, prior to
drying, gels were stained with 0.1 µg/ml ethidium bromide (EtBr) for
45 min and analyzed by ultraviolet transillumination using a Bio-Rad VersaDoc 1000 imaging system.
Chloroquine-Agarose Gel Electrophoresis--
Strand invasion
reaction products were extracted using the Promega Wizard DNA cleanup
system and electrophoresed through 1% agarose-Tris acetate EDTA, pH
7.6, gels containing 0, 2, 4, 10, 25, 100, and 200 µg/ml chloroquine
at 4.5 V/cm for 3.5 h (8). Chloroquine was also included in the
electrophoresis and loading buffers at the respective concentrations.
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RESULTS |
ICP8 Catalyzes Strand Invasion--
We have examined the ability
of ICP8 to catalyze strand invasion in vitro. The substrates
for this reaction were acceptor DNA, consisting of form I pUC18 DNA,
and donor DNA, consisting of a homologous 5'-32P-labeled
100-mer (PB11). Pairing between these substrates was measured with a
electrophoretic mobility shift assay of deproteinized reaction products
as described under "Experimental Procedures."
As shown in Fig. 1A,
ICP8 results in the assimilation of the 100-mer into homologous form I
acceptor DNA, presumably leading to the formation of a D-loop
(lane 1). The concentration of ICP8 used was 2.5-fold in
excess to that required to coat the 100-mer (9). Under these
conditions, up to 15% of the form I acceptor DNA participated in
D-loop formation. Strand invasion does not require a high energy
co-factor, since D-loops were formed in the absence of ATP (lane
2). ICP8 and homologous acceptor DNA are indispensable for D-loop
formation, since products failed to form in the absence of ICP8
(lane 3) or pUC18 (lane 4). Substitution with a
heterologous acceptor DNA (pUC19) also prevented D-loop formation
(lane 5). The reaction is greatly stimulated by
Mg2+, since its omission resulted in drastic reduction in
D-loop formation (lane 6). At a concentration of E-SSB
equivalent to that of ICP8 and 4-fold in excess to that required to
coat the 100-mer (assuming a site size of 40 nucleotides/tetramer),
only negligible amounts of D-loops were formed (lane 7).
Likewise, no significant D-loops were formed with coating
concentrations of human replication protein A (RP-A) or T4 gp32 protein
(data not shown). The products of ICP8-mediated strand invasion
exhibited a electrophoretic mobility identical to those formed by the
action of RecA (lane 8).

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Fig. 1.
ICP8 catalyzes strand invasion.
Reactions with ICP8, RecA, or E-SSB were performed as described under
"Experimental Procedures." A is a storage phosphor image
showing: ICP8-mediated D-loops (complete reaction) (lane 1),
complete reaction in the absence of ATP (lane 2), complete
reaction without ICP8 (lane 3), complete reaction in the
absence of acceptor DNA (lane 4), complete reaction with
heterologous acceptor DNA (pUC19) (lane 5), complete
reaction in the absence of Mg2+ (lane 6), strand
invasion reaction with E-SSB (lane 7), and strand invasion
reaction with RecA (lane 8). The reaction is schematically
represented above A. B and C are
EtBr-stained and storage phosphor images of the same gel, respectively,
that compare the ability of ICP8 to form D-loops with form I
(lane 1), form IV (lane 2), and form III
(lane 3) DNA. Form IV and III DNA were generated by treating
pUC18 form I DNA with topoisomerase I and AlwNI,
respectively. A schematic of each form of DNA is drawn above the
corresponding lanes. The positions of 100-mer, D-loops, and the three
forms of DNA are as indicated.
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Fig. 1, B and C, are EtBr-stained and storage
phosphor images of the same gel that compare the ability of three
topological forms of acceptor DNA to participate in D-loop formation.
D-loops were formed with form I (lane 1) but not with forms
IV or III (lanes 2 and 3) DNA, indicating that
strand invasion is dependent on the free energy of supercoiling.
ICP8 Promotes Complete Assimilation of a 100-mer into a Homologous
Plasmid--
To determine whether ICP8 can facilitate complete
invasion of the donor 100-mer into the acceptor DNA, we examined the
susceptibility of the resulting D-loops to digestion with ssDNA
exonucleases. D-loops were resistant to digestion with exonuclease I
(3'-5' exonuclease) as well as RecJ (5'-3' exonuclease) when analyzed by electrophoresis through a 1% agarose gel (Fig.
2A). Quantitative analysis
indicates that the amount of 100-mer protected from both exonuclease I
and RecJ digestion is equal to the amount of 100-mer participating in
D-loop formation (~5%). It should be noted that exonuclease
digestion products (5'-32P-labeled dGMP and d(pGpT) for
RecJ and exonuclease I, respectively) migrate more slowly through 1%
agarose gels than the 100-mer. Fig. 2B confirms that the
concentrations of exonucleases used were sufficient to completely
degrade the donor 100-mer. Since the 32P label of the
invading 100-mer is at the 5' end, resistance to RecJ demonstrates that
the 5' end of the 100-mer was stably assimilated into the acceptor DNA
(Fig. 2A, lane 3). To ascertain that the 3' end
of the 100-mer was fully incorporated into the donor DNA, exonuclease I
digestion products were also resolved by denaturing polyacrylamide gel
electrophoresis. Fig. 2C shows that the 100-mer involved in
D-loops was not shortened by treatment with exonuclease I, thereby
indicating that the 3' end of the 100-mer was also stably integrated
into the acceptor DNA (compare lanes 3 and 4). The amount of 100-mer that was resistant to degradation in Fig. 2C, lane 4 (~5%), corresponds to the amount of
100-mer participating in D-loop formation (~5%) (Fig. 2A,
lane 1).

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Fig. 2.
ICP8 promotes complete assimilation of a
100-mer into a homologous plasmid. A, D-loops were
formed as described under "Experimental Procedures" and extracted
using the Promega Wizard DNA purification system. Deproteinized D-loops
were split into three equal parts and treated for 60 min and analyzed
by 1% agarose gel electrophoresis as follows: lane 1, mock
treatment; lane 2, exonuclease I (25 units); lane
3, RecJ (25 units); lane 4, 100-mer digested with
exonuclease I (25 units); lane 5, 100-mer digested with RecJ
(25 units); lane 6, mock-treated 100-mer. B, 10.5 nM 100-mer was split into three equal parts and treated for
60 min and analyzed by 6% polyacrylamide gel electrophoresis as
follows: lane 1, mock treatment; lane 2,
exonuclease I (25 units); lane 3, RecJ (25 units).
C, analysis of exonuclease I digested D-loops by 8 M urea, 10% polyacrylamide gel electrophoresis. 10.5 nM 100-mer was extracted using the Promega Wizard DNA
purification system and split into two equal parts and treated for 60 min as follows: lane 1, mock treatment; lane 2,
exonuclease I (25 units). D-loops were formed as described under
"Experimental Procedures" and extracted using the Promega Wizard
DNA purification system and split into two equal parts and treated for
60 min as follows: lane 3, mock treatment; lane
4, exonuclease I (25 units). Only the areas of the image
corresponding to the 100-mer and its degradation product are shown as
indicated by the broken line to the left of the
figure. The positions of 100-mer, degraded 100-mer, and D-loops are as
indicated on the storage phosphor images.
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ICP8-mediated D-loop Formation Requires a Homologous
End--
D-loop formation was examined with a variety of
oligonucleotide donors that were either completely homologous to the
acceptor plasmid (PB11, 100-mer and PB9, 22-mer) or possessed
heterology at their 3' (PB136, 100-mer), 5' (PB137, 104-mer), or both
(PB142, 68-mer) ends. Fig. 3 shows that
long oligonucleotides possessing heterology at either 3' (PB136) or 5'
(PB137) ends were nevertheless capable of participating in D-loop
formation compared with the standard 100-mer (PB11) (compare
lanes 2, 4, and 6). Similar results were obtained with shorter oligonucleotides possessing heterology at
either 3' or 5' ends (data not shown). However, oligonucleotide PB142
(68-mer) possessing a 23-nucleotide heterology at both ends failed to
participate in D-loop formation (lane 8). A control oligonucleotide (PB9) with an equivalent length of homology as PB142
(22 nucleotides) was efficiently assimilated into the acceptor plasmid,
indicating that the failure of PB142 to form D-loops was not due to
insufficient homology but rather due to the lack of homology at the
ends (lane 10).

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Fig. 3.
ICP8-mediated D-loop formation requires a
homologous end. D-loops were formed as described under
"Experimental Procedures" at room temperature in the absence
(odd numbered lanes) or presence (even numbered
lanes) of ICP8 using the following donor oligonucleotides:
lanes 1 and 2, homologous 100-mer (PB11);
lanes 3 and 4, 100-mer with heterology at 3' end
(PB136); lanes 5 and 6, 104-mer with heterology
at 5' end (PB137); lanes 7 and 8, 68-mer with
heterology at 3' as well as 5' ends (PB142); lanes 9 and
10, homologous 22-mer (PB9). The positions of D-loops and
oligonucleotides are as indicated on the storage phosphor image.
Schematics of the oligonucleotides used are drawn above the
corresponding lanes. The zigzag lines represent regions of
heterology.
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ICP8-promoted Strand Invasion Perturbs the Topology of the
Supercoiled Acceptor Plasmid--
Upon electrophoresis through a 1%
agarose gel, D-loops and acceptor DNA have a similar mobility (Fig.
4A, lanes 2 and
3). To resolve differences in topology between these two
species, we examined their migration through 1% agarose gels
containing varying concentrations of chloroquine. Intercalation of
chloroquine into negatively supercoiled DNA leads to DNA unwinding
(10). This at first reduces the superhelicity (relaxation) of the
plasmid, concomitantly reducing its electrophoretic mobility (compare
lanes 2 of Fig. 4, A-D). However, as more
chloroquine intercalates into the DNA, the relaxed plasmid is converted
into positively supercoiled DNA, increasing its electrophoretic
mobility (compare lanes 2 of Fig. 4, D-G).
Chloroquine does not reduce the electrophoretic mobility of D-loops,
indicating that they are not unwound by the intercalating agent. On the
contrary, D-loops migrate faster with increasing chloroquine
concentrations (compare lanes 3 of Fig. 4, A-G).
This suggests that assimilation of the 100-mer alters the superhelical
density by overwinding the acceptor plasmid. Electrophoresis in the
presence of chloroquine also resolves an additional species. We believe
that this species is form X DNA, which is presumably generated due to
ICP8-mediated unwinding of the acceptor DNA involved in D-loops (Fig.
4G, lane 3). This observation is similar to that
hypothesized for RecA where extensive RecA filament formation extending
from the D-loop into the duplex portion of the plasmid causes unwinding
and accumulation of positive supercoils in the plasmid (11, 12).

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Fig. 4.
ICP8-promoted strand invasion alters the
mobility of the supercoiled acceptor plasmid.
A-G, storage phosphor images showing the effect
of chloroquine on D-loop migration. D-loops were formed as described
under "Experimental Procedures" and extracted using the Promega
Wizard DNA purification system. Samples were resolved on a 1% agarose
gel (A) and 1% agarose gels containing 2, 4, 10, 25, 100, and 200 µg/ml chloroquine (B-G). Lane
1 of each panel, 32P-labeled -HindIII
marker; lane 2 of each panel, 32P-labeled pUC18
form I; lane 3 of each panel, D-loop reaction. The
concentration of chloroquine is indicated below each panel. The
solid lines indicate the migration of the ~23 kbp
(upper) and 2 kbp (lower) -HindIII
fragments. The dashed line indicates the positions of form I
and D-loops in A, in the absence of chloroquine. The
positions of D-loops and of form X DNA are indicated in
G.
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Since D-loops exhibit a modified topology (Fig. 4), we predicted that
they would show altered sensitivity to restriction endonucleases. D-loops were digested with the following enzymes: HindIII
and EcoRI that cleave in the region of pairing at
co-ordinates 399 and 450, respectively, AlwNI and
AflIII that cleave outside the region of pairing at
co-ordinates 1217 and 806, respectively. The EtBr-stained image serves
as an internal control to show that the acceptor DNA (pUC18) was
linearized by all four enzymes (Fig. 5A, lanes 1,
2, 4, and 5). Storage phosphor
analysis of the same gel shows that acceptor DNA involved in D-loops
was cleaved by HindIII and EcoRI but not by
AlwNI or AflIII (Fig. 5B, lanes
1, 2, 4, and 5). In addition,
D-loops were resistant to cleavage by BsrBI and
DraI both of which have three recognition sites outside of
the region of pairing at coordinates 498, 739, and 2540 and 1565, 1584, and 2276, respectively (Fig. 5, C and D). This
indicates that the donor 100-mer undergoes Watson-Crick base pairing
with the complementary strand of the acceptor DNA, thereby retaining the recognition sites for the enzymes encompassing the region of
pairing. On the other hand, invasion of the 100-mer, resulting in a
change in topology, disrupts the recognition sequences of those enzymes
that cleave distally to the site of pairing, leading to resistance to
cleavage. Two additional enzymes whose recognition sites are within the
region of pairing, SmaI (433) and BamHI (429), cleaved D-loops, while two other enzymes whose recognition sites are
outside of the region of pairing, BsrFI (1779) and
AatII (2617), failed to cleave the D-loops (data not shown).
It should be noted that cleavage by restriction endonucleases in the
region of pairing was observed with deproteinized D-loops as well as
with ongoing D-loop reactions (i.e. in the presence of
ICP8), indicating that triple helical structures, which would
presumably interfere with cleavage, are not the stable end
products.

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Fig. 5.
ICP8-promoted D-loops are resistant to the
action of restriction endonucleases. Susceptibility of D-loops to
restriction endonuclease digestion. D-loops were formed as described
under "Experimental Procedures" and digested with 20 units of
enzyme for 1 h. A and B are EtBr-stained and
storage phosphor images of the same gel, respectively. Lanes
1, 2, 4, and 5,
HindIII, EcoRI, AlwNI, and
AflIII, respectively; lane 3, untreated D-loops.
C and D are EtBr-stained and storage phosphor
images of the same gel, respectively. Lanes 1-3, untreated
D-loops, DraI, and BsrBI, respectively. The
positions of 100-mer, D-loops, and of DNA forms I and III are as
indicated. indicate the relative cleavage sites of
HindIII ( ), EcoRI ( ), AlwNI
( ), and AflIII ( ). The block in cleavage for
AlwNI and AflIII is as indicated. The
schematics shown above A and B depict the
topologies of form I DNA and D-loops. The arrowheads at the
right of C indicate the positions of the
DraI and BsrBI digestion products. It should be
noted that the 1.9-kbp BsrBI digestion product co-migrates
with pUC18 form I.
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DISCUSSION |
Using a classical in vitro assay, we have demonstrated
that ICP8 promotes strand invasion. This reaction is distinct to its helix destabilizing and reannealing activities and to its ability to
promote strand exchange in conjunction with the viral replicative helicase (3). To our knowledge, ICP8 is the first eukaryotic viral SSB
shown to mediate this reaction. It has previously been pointed out that
several thermodynamic parameters of ICP8 are closer to those of known
recombinases (e.g. E. coli RecA and T4 UvsX
protein) than they are to other SSBs (e.g. E-SSB, RP-A, and T4 gp32) (9). Thus, like bona fide recombinases, ICP8
stretches ssDNA, possibly to facilitate the search for homology, forms
complexes with ssDNA that are stable at high salt concentrations and
exhibits both weaker and lower cooperativity ssDNA binding than other
SSBs. Although ICP8 promotes D-loop formation, its ability to do so is
relatively weak (up to 15% product formation) when compared with the
prototypical E. coli RecA recombinase (~40% product
formation under our reaction conditions that lack an ATP regenerating
system). However, the efficiency of the reaction promoted by ICP8 is
comparable with that achieved by eukaryotic RecA counterparts such as
yeast Rad51, which only promotes ~2% product formation (13).
An important feature of ICP8-mediated strand invasion is that it does
not require ATP. This distinguishes ICP8 from the RecA-type recombinases (e.g. E. coli RecA and eukaryotic
Rad51) that require ATP binding to catalyze strand invasion (14, 15).
ICP8 presumably utilizes the energy stored in the negatively
supercoiled form I DNA to drive the reaction. D-loop formation was
minimal in the absence of Mg2+, whereas concentrations
ranging from 5 to 50 mM Mg2+ greatly stimulated
the reaction. The low efficiency of D-loop formation in the absence of
Mg2+ can be rationalized by the fact that ICP8 is more
proficient at helix destabilization in the absence of Mg2+
and may therefore lead to dissociation of D-loops under these conditions (3). In addition, stimulation by Mg2+ may be
related to the fact that ICP8-mediated pairing is
Mg2+-dependent (3).
Our data indicate that ICP8 mediates complete assimilation of a donor
100-mer into the acceptor plasmid, which, to our knowledge, has not
been demonstrated with other strand invasion proteins such as RecA or
Rad51. The stability of D-loops formed by ICP8 following
deproteinization indicates that these structures entail plectonemic DNA
interactions. The results obtained with oligonucleotides possessing
heterology at 5' or 3' ends provides evidence that ICP8-mediated D-loop
formation can initiate at either end. Moreover, donor DNA with
heterology at both ends did not form D-loops, thereby indicating the
need for a homologous end. It may be possible for a substrate with
double heterology to form paranemic D-loops. However, such
intermediates are unstable and would dissociate upon deproteinization.
The lack of bias toward an end is in contrast to either RecA or Rad51,
which preferentially lead to assimilation of 3' and 5' ends,
respectively (13).
Strand invasion by ICP8 alters the topology of the acceptor plasmid as
evidenced by anomalous migration in chloroquine-containing gels and
resistance to restriction endonucleases that cleave outside the area of
pairing. ICP8 also generates form X DNA, which has previously been
described for RecA-mediated D-loop formation (11, 12). Generation of
form X DNA requires protein-induced unwinding of the acceptor DNA,
starting at the point of assimilation of the donor DNA. This generates
compensatory positive supercoils allowing such structures to be
resolved from normal D-loops by agents (e.g. chloroquine)
that alter the writhing number.
The exact mechanism for strand invasion remains unclear. Two competing
mechanisms have been proposed (reviewed in Ref. 16). According to the
first mechanism (R-form hypothesis), the donor nucleoprotein filament
forms a canonical DNA triple helix involving non-Watson-Crick base
pairing. The second mechanism (base-flipping model) states that the
donor nucleoprotein filament induces base-flipping in one strand of the
acceptor DNA, thereby permitting a homology search between the protein
bound oligonucleotide and the flipped bases based on Watson-Crick
interactions. Our data suggest that ICP8-mediated D-loop formation
occurs by the later model. This conclusion is based on the
susceptibility of D-loops to restriction endonucleases that cleave in
the region of pairing, whereas they would otherwise be resistant if the
D-loops were to possess triple helical character (17).
Another protein that has been shown to promote strand invasion is
E. coli RecT (18). Although RecT- and ICP8-mediated strand invasion occur under similar conditions and at similar protein to ssDNA
ratios, the ICP8-mediated reaction is greatly stimulated by
Mg2+, while the RecT-mediated reaction is
Mg2+-independent and inhibited with increasing
Mg2+. The major difference between these two proteins,
however, is that ICP8 functions as an SSB, while the RecT-mediated
reaction is dependent on its interaction with both ss and duplex
DNA (1, 18).
During its replicative cycle, multiple concatemeric HSV-1 genomes are
generated by rolling circle replication. Highly branched networks of
DNA are prevalent at later times during replication (4). These may
arise from intra- and interconcatemeric recombination. The strand
invasion activity of ICP8 may be pivotal in the formation of such
structures. Thus, we envisage that ICP8 mediates the invasion of ssDNA
into homologous regions. Invading 3'-terminal strands would presumably
prime DNA synthesis and lead to the formation of replication forks that
would ultimately result in the formation of branched DNA structures.
This process may be initiated by double-strand DNA breaks that arise
due to a variety of reasons (e.g. collapsed replication
forks, endonuclease G cleavage at viral a sequences (19),
and DNA damaging agents). ssDNA required for strand invasion may be
generated by the helix-destabilizing activity of ICP8, possibly in
conjunction with the viral replicative helicase or by exonucleolytic
processing of broken DNA ends (3).
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FOOTNOTES |
*
This work was supported by Grant GM62643 from the National
Institutes of Health and Grant BM 022 from the Florida Biomedical Research Program.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.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, University of Miami School of Medicine, R-629,
P. O. Box 016129, Miami, FL 33101-6129. Tel.: 305-243-2934; Fax:
305-243-3955; E-mail: pboehmer@molbio.med.miami.edu.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M212555200
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ABBREVIATIONS |
The abbreviations used are:
HSV-1, herpes
simplex virus type-1;
D-loop, displacement loop;
SSB, single-strand
DNA-binding protein;
E-SSB, E. coli single-strand
DNA-binding protein;
ss, single-stranded;
RP-A, replication
protein A.
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and Lehman, I. R.
(1992)
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3.
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