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INTRODUCTION |
Homologous recombination has been demonstrated to occur frequently
during the infectious cycle of Herpes simplex virus type 1 (1-5). A
striking consequence is the inversion of the long (L) and short (S)
components of the genome relative to each other (6). Homologous
recombination also appears to be enhanced by the
HSV-11 replication machinery.
It has been suggested that the replication process generates strand
breaks that will initiate recombination (4). The physiological role of
homologous recombination during the infectious cycle has not been
studied. One might, however, speculate that it is involved in repair or
editing of replication intermediates. It might also constitute one way
to promote circularization of linear viral genomes containing extensive
directly repeated sequences at the ends (7). A recent study, on the
other hand, demonstrates that circularization of the guinea pig
cytomegalovirus genome is likely to proceed by a different mechanism
(8). So far, no gene products, viral or cellular, have been identified that directly take part in the circularization of viral genomes.
Three distinct pathways involved in the repair of double-stranded
breaks have been detected in eukaryotic cells. One of these, referred
to as nonhomologous end joining, involves Ku and the DNA-dependent protein kinase (9). Inside cells, it appears to yield products that contain an altered DNA sequence at the site of
fusion. More recently, an in vitro system that depends on
DNA ligase IV/Xrcc4 and requires Ku70, Ku86, and the catalytic subunit
of DNA-dependent protein kinase has been developed.
This system produces accurate and efficient ligation of complementary ends (10).
A second alternative is nonconservative homologous recombination using
a single strand annealing mechanism. In Saccharomyces cerevisiae, an endonuclease formed by the rad10
and rad1 gene products is strictly required (11). The
mammalian homologues are the ERCC1 and ERCC4 gene products. The loss of
the ERCC1 gene in mammalian cells results in an
increase of recombination-dependent deletions at tandemly
repeated sequences and an increased instability of genomes (12, 13).
XP-F cells that are devoid of the ERCC4 gene product show an impaired
capacity to circularize linear DNA molecules with short direct repeats,
but they can efficiently recombine long homologous sequences (14).
The third pathway is homologous recombination, which in S. cerevisiae depends on the rad51 gene product and the
other members of the rad52 epistasis group (15). The
mammalian homologue, HsRad51, appears to be essential for viability of
cells (16, 17). It is believed to act primarily in recombination repair during the late S-G2 phase of the cell cycle (18).
Overexpression of Rad51 protein increases the frequency of
recombination events and the resistance of mammalian cells to ionizing
radiation (19). It has been observed that homologous recombination in
mammalian cells is dependent on the length of the homologous regions
(14, 20, 21).
Ancient viruses that establish life-long infections in the host are
likely to interact with the cellular pathways that control the
replication and integrity of cellular chromosomes. We have performed a
series of investigations aimed at identifying interactions between the
DNA metabolism of herpes simplex virus type 1 and the infected cell.
For example, we found that inhibition of topoisomerase II by ICRF-193
prevented efficient replication of HSV-1, indicating that decatenation
of replication products was an early and essential step during virus
replication (22). We also noted that direct repeats of the HSV-1
terminal a sequence were able to promote nonconservative
homologous recombination not dependent on XP-F/ERCC4 (14). In fact,
linear DNA molecules containing the HSV-1 origin of replication, oriS,
as well as directly repeated sequences replicated as efficiently as the
corresponding circular molecule in BHK cells (14).
We have now investigated if viral DNA synthesis or viral gene products
are required for recombination to occur. We have also looked at the
efficiency of homologous recombination in different cell types. A new
assay that allowed us to monitor homologous recombination in the
absence of HSV-1 DNA replication was developed. Briefly, a plasmid that
supports the expression of luciferase was redesigned to contain the
middle portion of the coding sequence as direct repeats. The repeated
sequences were separated by a noncoding sequence derived from
bacteriophage
. Linear plasmid DNA was produced by restriction
enzyme cleavage in the
sequence and introduced by lipofection into
cells. The correct open reading frame could now be restored by
homologous recombination. This assay, referred to as
recombination-dependent expression of luciferase, was
combined with a described previously assay for
oriS-dependent amplification of recombination products to
monitor how a HSV-1 infection and the genetic background of cells
affect the efficiency of homologous recombination.
The results show that homologous recombination is mechanistically
independent of HSV-1 but dependent on the properties of the cell.
Interestingly, the small amounts of recombination products that are
formed are very efficiently amplified by the HSV-1 DNA synthesis
machinery, suggesting a structural or temporal coupling between these processes.
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MATERIALS AND METHODS |
Cells and Viruses--
BHK cells (clone 13 ATCC CCL 10) were
grown in Glasgow modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% tryptose phosphate broth, 10% newborn calf
serum, 50 units/ml penicillin, and 50 µg/ml streptomycin at 37 °C
in an atmosphere of 5% CO2. COS-7 (ATCC CRL 1651) cells
and mouse embryo fibroblasts (MEFs) were grown in Dulbecco's modified
Eagle's medium with 10% fetal bovine serum, 2 mM
glutamine, 50 units/ml penicillin and 50 µg/ml streptomycin. MEF p53
/
was from Klas Wiman (Karolinska Institute, Stockholm). Wild type
MEFs were from Maria Enge (Department of Medical Biochemistry,
Göteborg University). Balb/c 3T3 cells (ATCC CLL-163) and Balb/c
3T3.SV-T2 cells (ATCC CCL 163-1) were grown in Dulbecco's modified
Eagle's medium with 10% calf serum, 2 mM glutamine, and
4.5 g/liter glucose.
HSV-1 Glasgow strain 17 syn+ was propagated at 37 °C and the viral
stocks were prepared as described previously (14, 22). HSV-1 tsK and
tsS were obtained from Dr. Nigel Stow (MRC Virology Unit, Institute of
Virology, Glasgow, United Kingdom). Titration of all viruses was
performed on BHK cells.
Reagents--
Phosphonoacetic acid, luciferin, and camptothecin
were from Sigma.
Plasmids--
A series of plasmids containing repeated sequences
has been described (Fig. 1; Refs. 2 and 14). In some cases, the
directly repeated sequences are separated by a spacer of
DNA
derived from the plasmid pRD105 (2, 14). They were designated p2aLORI, p2bLORI, and p2hLORI, depending on the source of the repeated sequences
(Fig. 1A). The repeated sequences were the 317-bp
a sequence from the HSV-1 genome, a 341-bp sequence from BHK
cells, and a 161-bp sequence of human origin. Another set of plasmids without the
DNA spacer, p2aORI and p2bORI, was described previously (14). The plasmid pBORI lacks repeated sequences (14). The HSV-1 origin
of replication, oriS, was included to allow transient replication in
cells superinfected with HSV-1. Another series of plasmids, p2aLSV and
p2hLSV, contains repeated sequences as well as the SV40 origin of replication.
A new series of plasmids was designed to measure
recombination-dependent expression of luciferase (Fig.
1B). The functional gene was divided into three parts
starting from the 5'-end and ending with the SV40 polyadenylation
signal. They were called LucI (612 bp), LucII (776 bp), and LucIII
(1222 bp). These sequences were cloned into an expression vector,
pCMV19K, from which E1B19K and the SV40 origin were removed by
BamHI and EcoRI cleavage (23). HSV-1 oriS was
cloned downstream of the luciferase gene. pXY1 contains the intact
luciferase gene. In pXY2, LucII was duplicated, and the two copies were
separated by a
sequence. The restriction fragments Fr1, Fr2, and
Fr3 contain only parts of the luciferase gene (Fig. 1). Fr1 and Fr2
were from pXY2. Fr3 was derived from a different plasmid. It contains
LucII and LucIII but lacks the CMV promotor. The DNA molecules were
used either as circular DNA or as linear DNA produced by restriction
enzyme cleavage as shown in Fig. 1.
Transfection Protocols--
Circular or linear plasmid DNA was
introduced into cells using liposomes. BHK and COS-7 cells were
transfected as described previously (14, 22). 3T3 cells and MEFs were
transfected using LipofectAMINE PLUS as described by the manufacturer
(Life Technologies, Inc.). 0.25-1 µg of DNA was mixed with 50 µl
of Optimem and 3 µl of Plus reagent. The mixture was incubated at
room temperature for 15 min. Then 2 µl of LipofectAMINE and 50 µl
of Opti-MEM were added. The 105-µl suspension of liposomes was used
for the transfection of cells together with 400 µl of Opti-MEM in
21-mm dishes.
The frequency of transfection was determined using the plasmid pCMV
(CLONTECH) encoding
-galactosidase. The
transfected cells were stained with
5-bromo-4-chloro-3-indolyl-
-D-galactoside. For each
monolayer, the frequency of cells expressing
-galactosidase was
determined at several locations in the wells. An average of the values
obtained for each well was used to construct Table II.
Transient Replication Assay--
Subconfluent monolayers of
cells were transfected by circular or linear plasmid DNA using
liposomes for 4 h. Superinfection with HSV-1 (5 pfu/cells) or
cotransfection with a collection of expression plasmids encoding the
seven viral replication genes provided the necessary trans-acting
factors (24). Total DNA was harvested either 18 h after
superinfection or 48 h following cotransfection with expression
plasmids. DNA was digested with restriction enzymes as indicated and
subjected to electrophoresis in 1% agarose gels. The enzyme
DpnI was used to cleave input DNA. The DNA was blotted onto
Hybond-H+ membranes (Amersham Pharmacia Biotech) as described by the
manufacturer. Hybridization was performed using randomly labeled pUC 19 probes (Megaprime; Amersham Pharmacia Biotech). This probe
recognizes the oriS-containing plasmids as well as the pE series of
expression plasmids. Probes from the fragments a, b, and h were used in
the experiment shown in Fig. 2.
Expression of Luciferase-dependent on
Recombination--
The plasmid pXY2 was used as a substrate for
recombination. The middle part of the luciferase gene, LucII, was
present as direct repeats separated by a
DNA sequence (Fig.
1B). Homologous recombination between the LucII repeats will
create an intact luciferase gene, which can be monitored by a
luciferase assay. Confluent or subconfluent monolayers of BHK cells in
15-mm dishes and Balb/c 3T3 or 3T3.SV-T2 cells in 21-mm dishes were
lipofected as indicated. The cells were harvested after 24 h. The
monolayers were washed twice with cold phosphate-buffered saline. They
were suspended in 0.1 M potassium phosphate, pH 7.8, and
centrifuged at 12,000 × g at 4 °C for 2 min. The
cell pellet was resuspended in 50 µl of the same solution. This
suspension was repeatedly frozen and thawed three times. The cell
lysate was cleared by centrifugaton at 12,000 × g at
4 °C for 5 min. Luciferase activity was measured by taking 20 µl
of the supernatant mixed with 0.36 ml of a buffer containing 5 mM ATP, 15 mM MgCl2, 1 mM dithiothreitol, 25 mM potassium phosphate,
pH 7.8. Then 100 µl of a solution containing 0.4 mM
luciferase, 25 mM potassium phosphate, pH 7.8, and 0.4 mM dithiothreitol was added. Luciferase activity was
measured in a LUMAT LB 9501 luminometer. The results were expressed as relative luciferase units/µg of protein. Protein concentration of the
extract was determined using the Bio-Rad protein assay.
To verify the authenticity of luciferase, Western blotting was
performed using an anti-luciferase antibody. BHK cells in 15-mm dishes
were lipofected with plasmids containing the entire luciferase gene or
parts thereof. After 24 h post-transfection, the monolayers were
washed and suspended in cold phosphate-buffered saline. The cells were
collected by low speed centrifugation. The cell pellets were
resuspended in 40 µl of phosphate-buffered saline and 40 µl of 2×
loading buffer (50 µM Tris-HCl, pH 6.8, 0.1 M
dithiothreitol, 2% SDS, 0.1% bromphenol blue, and 10% glycerol) and
boiled for 3 min. The samples were loaded on a 10% SDS-polyacrylamide
gel. Electrophoretic transfer was done onto Immobilon-P membrane
(Millipore Corp.). The immunoreaction of the blotted membrane was
performed by using Bio-Rad Immun-Blot assay kit (goat anti-rabbit IgG,
alkaline phosphatase) and an anti-luciferase antibody (Promega).
Assays for Apoptosis--
Monolayers of cells in 21-mm dishes
were transfected with liposomes containing either circular or linear
forms of the plasmid pCMV
as indicated. The transfected cells were
incubated for 20 h and subsequently stained with
5-bromo-4-chloro-3-indolyl-
-D-galactoside as described
by the manufacturer (CLONTECH). The total number of
blue cells expressing
-galactosidase and the number of apoptotic shrunken blue cells were determined by light microscopy.
Apoptosis was also measured by staining cells with annexin V coupled to
green fluorescent protein using the ApoAlertTM kit from
CLONTECH. Balb/c 3T3 cells were grown on glass
coverslips in 35-mm dishes. The cells were lipofected at 80%
confluency with 1.6 µg of circular or linear pCMV
. The growth
medium was collected at 8 h post-transfection, and the monolayers
were rinsed three times with 1 ml of phosphate-buffered saline. The
nonadherent cells were collected by centrifugation. The adherent and
nonadherent cells were stained separately by annexin V-EFGP as
described by the manufacturer. The cells were washed after staining.
The nonadherent cells were suspended in 20 µl of phosphate-buffered
glycerol prior to fluorescence microscopy.
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RESULTS |
Coupling of Homologous Recombination and DNA Replication--
To
study mechanisms of recombination in mammalian cells, we have developed
a transient replication assay to monitor the circularization of linear
DNA (14). We constructed plasmids that contained directly repeated
sequences that in some instances were separated by a sequence of
DNA. Furthermore, origins of DNA replication from either HSV-1 or SV40
were included (Fig. 1A).
Linear DNA was produced by restriction enzyme cleavage between the
direct repeats. DNA was introduced into cells by lipofection. The use
of calcium phosphate precipitation resulted in significant degradation
of the transfected DNA, and the products of recombination were much
more heterogenous (results not shown). In the initial phase of this
investigation, we chose to work with BHK cells, since they have been
widely used to study HSV-1 replication. Properties of BHK cells that
are pertinent for the interpretation of our results are reviewed under
"Discussion."

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Fig. 1.
Plasmids used to study recombination in
infected and uninfected cells. A, plasmids used for
transient replication assays depending on HSV-1 oriS or SV40 origin of
DNA replication. The directly repeated sequences (gray
boxes) are the 317-bp HSV-1 a sequence
(a), the 341-bp BamHI fragment from BHK DNA
(b), and the 161-bp BamHI fragment from human DNA
(37) (h). The plasmids were linearized by restriction
enzyme cleavage either in the DNA sequence (striped
boxes) separating the direct repeats in p2aLORI, p2bLORI,
p2hLORI, p2aLSV, and p2hLSV or at a BamHI site between the
direct repeats in the plasmids p2aORI and p2bORI. B,
plasmids and DNA fragments used to measure luciferase expression. The
coding sequence of luciferase was divided into three parts, LucI,
LucII, and LucIII (light through dark
gray boxes). A disrupted gene was then
constructed with two copies of LucII separated by a 0.8-kilobase pair
DNA sequence (striped boxes). The plasmids
were either linearized downstream from the polyadenylation signal,
pXY1, or in the sequence, pXY2. The restriction fragments Fr1 and
Fr2 were derived from pXY2. Fr3 came from a different construct.
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We initially examined the fate of transfected linear DNA in infected
and uninfected BHK cells (Fig. 2). Total
DNA was isolated from cells undergoing a transient replication
experiment and cleaved with EcoRI prior to agarose gel
electrophoresis. The two bands in the middle of the autoradiograph
represent linear input DNA. Imprecise nonhomologous end joining or
homologous recombination produces different products (14). The
molecules formed by end joining are found in a slightly diffuse band in
the upper part of the autoradiograph. The molecules formed by
homologous recombination have lost one of the repeats as well as the
intervening
sequence. They are represented by a distinct band in
the lower part of the autoradiograph. We estimate that in the absence
of a virus infection less than 1% of the total amount of transfected
DNA was converted to circular molecules by homologous recombination in
BHK cells (Fig. 2). However, in infected cells much larger amounts of
the recombination products were seen using the plasmids p2aLORI and p2bLORI (Fig. 2). In the presence of phosphonoacetate, there was no
accumulation of recombined DNA, demonstrating that the recombination products were amplified by the HSV-1 replication machinery. Homologous recombination was sequence-independent inasmuch as the viral
a sequence and the b sequence derived from BHK
DNA gave rise to similar amounts of recombination products. We also
noted that the plasmid p2hLORI supported homologous recombination to a
lesser extent. In this instance, a significant amount of DNA recombined by nonhomologous end joining was seen (Fig. 2). This finding is in
agreement with a previous observation that homologous recombination that occurs in the absence of the XP-F/ERCC4 gene product is dependent on the length of the homologous sequences (14).

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Fig. 2.
Products of homologous recombination are
efficiently amplified by the HSV-1 replication machinery. Linear
DNA was transfected into BHK cells. The cells were then superinfected
with HSV-1 in the presence and absence of phosphonoacetate
(PAA) as indicated. DNA was isolated at 18 h after
infection and subjected to cleavage by EcoRI. The
autoradiograph represents a Southern blot of an agarose gel using
randomly labeled a, b, or h fragments
as probes. The positions of the fragments representing input DNA as
well as the products of nonhomologous end joining (EJ) and
homologous recombination (HR) are shown.
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It is possible that homologous recombination might assist in the
assembly of productive replication forks, thereby serving as a link
between replication and recombination (25). However, we have
transfected linear DNA without a viral origin of DNA replication but
still containing directly repeated a sequences into BHK
cells. We found that this DNA did not serve as a template for
origin-independent DNA synthesis (results not shown). The finding
indicates that the recombination process does not generate structures
that can assemble the HSV-1 replisome.
We have asked if the replication machineries assembled at either HSV-1
oriS or SV 40 origins of replication in BHK cells and COS-7 cells
amplify recombination products differently. DNA was isolated from the
cells and treated with DpnI to completely digest the unreplicated DNA
(Fig. 3). When circular DNA was used in
the transfection experiment only small amounts of recombination
products were produced. Linear DNA supported circularization by the end joining pathway as well as by homologous recombination. Although the
two replication systems are structurally completely distinct they
appear to amplify molecules produced by end joining and homologous recombination in similar ways. The results argue that the outcome of
the recombination reaction is not governed by the virus.

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Fig. 3.
Amplification of recombination products by
either HSV-1 or SV40 replisomes. Circular (C) or linear
(L) plasmid DNA, p2aLORI and p2hLORI, was transfected into
BHK cells. The cells were superinfected with HSV-1. DNA was harvested
at 18 h postinfection. The plasmids p2aLSV and p2hLSV were used to
transfect COS-7 cells. DNA was harvested 48 h after transfection.
The DNA samples were cleaved with DpnI to remove
unreplicated DNA and with HindIII to produce monomers. The
autoradiograph shows a Southern blot probed by randomly primed pUC19.
The position of molecules formed by nonhomologous end joining
(EJ) and homologous recombination (HR) are
shown.
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Recombination-dependent Expression of Luciferase Does
Not Require HSV-1--
To further elucidate the interdependence of DNA
replication and recombination, we devised an assay that allowed us to
measure recombination in the absence of DNA synthesis. A recombination substrate, pXY2, was made in which a luciferase gene was altered to
contain directly repeated coding sequences separated by a
DNA
spacer (Fig. 1B). Control DNA contained either a complete intact luciferase gene, pXY1, or different parts of the luciferase genes as in Fr1, Fr2, and Fr3 (Fig. 1B). First, we
introduced linear DNA molecules into BHK cells (Fig.
4A). We found that none of the
DNA fragments containing incomplete luciferase genes supported luciferase expression when transfected into BHK cells (Fig.
4A). The linear form of the plasmid pXY1 containing the
complete gene readily supported luciferase expression. We also found
that linear pXY2 containing the disrupted luciferase resulted in high
level expression of luciferase in BHK cells (Fig. 4A). This
demonstrates that homologous recombination efficiently can restore the
proper open reading frame for luciferase. We then looked at luciferase expression from circular and linear forms of the plasmids pXY1 and pXY2
(Figs. 4B and 7A). Again luciferase was
efficiently produced in cells transfected with circular and linear
pXY1. Linear pXY2 also supported luciferase expression. Circular pXY2,
on the other hand, gave rise to very little luciferase. Apparently, a
double strand break greatly stimulated homologous recombination,
leading to restoration of an intact luciferase gene. We could also
verify using SDS gel electrophoresis and Western blotting with an
anti-luciferase antibody that the molecular weights of luciferase
enzyme produced from the plasmids pXY1 and pXY2 were identical (Fig.
4C). Moreover, similar amounts of luciferase were
synthesized in cells transfected with linear pXY1 and pXY2 (Fig.
4C). Together, these results argue that homologous
recombination is able to circularize linear DNA very efficiently in BHK
cells.

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Fig. 4.
Recombination-dependent
expression of luciferase. Plasmid DNA or restriction fragments
derived from plasmids were transfected into BHK cells. The cells were
incubated for 24 h. A cell lysate was prepared, and luciferase
activity and protein concentration were measured. A, 0.5 µg of linear DNA was transfected into BHK cells. pXY1 is the intact
gene; pXY2 is the disrupted gene; and Fr1, Fr2, and Fr3 are restriction
fragments corresponding to different parts of pXY2 (see "Materials
and Methods"). B, increasing amounts of DNA were
transfected into BHK cells, and the luciferase activity was measured.
C, 0.5 µg of DNA was transfected into BHK cells. Cell
lysates were prepared at 24 h after transfection and analyzed by
SDS-polyacrylamide gel electrophoresis. Western blots were then
prepared using an anti-luciferase antibody.
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It was noted in the previous paragraph that only a very small part of
the transfected DNA was circularized by homologous recombination in the
absence of DNA synthesis (Fig. 2). We now observe that a linear intact
and a linear disrupted luciferase gene support the production of
approximately the same amount of active enzyme. When combined, these
observations argue that a subset of DNA molecules may have a high
probability of simultaneously participating in homologous
recombination, DNA replication, and transcription.
An assay based on gene expression rather than DNA amplification allowed
us to examine more directly the effects of viral DNA synthesis on
homologous recombination. We compared luciferase expression from linear
pXY1 and pXY2 in the presence and absence of HSV-1 (Fig.
5). We found that superinfection with
HSV-1 did not significantly affect recombination-dependent
expression of luciferase. We also noticed that the inhibitor of
viral DNA synthesis, phosphonoacetate, did not inhibit
recombination-dependent expression of luciferase (Fig.
5).

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Fig. 5.
Effects of HSV-1-supported DNA replication on
recombination-dependent expression of luciferase. Linear
DNA (0.5 µg) was transfected into BHK cells. The cells were then
superinfected with wild type or mutant strains of HSV-1 in the presence
and absence of phosphonoacetate (PAA). Cell lysates were
prepared, and the luciferase activity was measured. A,
effects of virus replication on luciferase expression. B, the effect of
mutant viruses at the nonpermissive temperature on luciferase
expression. The tsK virus harbors a mutation in the
ICP4 gene, and tsS virus has a mutation in the
UL9 gene.
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We also examined the effects of viral gene expression using HSV-1 tsK
and tsS strains at the nonpermissive temperature. The results
demonstrated that recombination-dependent expression of luciferase was not affected significantly by the lack of either functional ICP4 or functional UL9 origin-binding protein (Fig. 5B).
By monitoring recombination-dependent expression of
luciferase, we can examine large variations in the efficiency of
homologous recombination. The presence of a double strand break will
enhance homologous recombination more than 10-fold (Fig.
4B). In a similar way, the induction of apoptosis by linear
recombination substrates results in a dramatic decrease in luciferase
expression (see below). The variations in expression of luciferase
shown in Fig. 5 are at least 1 order of magnitude smaller than the
effect caused by a double strand break, and we believe that they most
likely are caused by nonspecific effects on transcription. Our results
therefore suggest that homologous recombination does not require HSV-1
DNA replication and gene expression.
Homologous Recombination Is Controlled by the Cell--
To
investigate how the cellular machineries for recombination act on our
model substrates we have compared oriS-dependent amplification of linear and circular DNA substrates in different cell
lines. In the first experiment, circular and linear p2aLORI, p2bLORI,
and p2hLORI were cotransfected with a set of seven expression plasmids
encoding the HSV-1 replication genes (Fig.
6). The results demonstrate that the
circular plasmids replicated readily in Balb/c 3T3 cells and BHK cells.
Linear DNA was also replicated efficiently in BHK cells. In contrast,
linear DNA replicated very poorly in Balb/c 3T3 cells (Fig. 6). The
products were as before produced either by end joining or by homologous
recombination. We also noted that neither the transfected linear nor
circular DNA was significantly degraded in 3T3 cells (Fig. 6). The
dramatic reduction of homologous recombination observed in Balb/c 3T3
cells could be explained by apoptosis induced by the presence of linear
DNA. It has been demonstrated that DNA damage activates p53 as a
transcription factor in Balb/c 3T3 cells and induces cell cycle arrest
or apoptosis (26-28). The role of p53 was therefore examined using
Balb/c 3T3.SV-T2 cells containing the SV40 large T-antigen as well as
MEFs that were either wild type or homozygous knock-outs for
p53.

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Fig. 6.
Replication of linearized plasmid DNA is
impaired in 3T3 cells. 0.25 µg of circular or linear
oriS-containing plasmid DNA, p2aLORI, p2bLORI, and p2hLORI, was
cotransfected with 0.25 µg of a collection of seven expression
plasmids encoding the essential HSV-1 replication gene products into
BHK and 3T3 cells. DNA was isolated 48 h after transfection and
cleaved with DpnI and HindIII. The DNA samples
were analyzed by Southern blotting of agarose gels using randomly
primed pUC19 as the radioactive probe. The positions of the bands
representing the products of end joning (EJ) and homologous
recombination (HR) are shown. The position of the product
formed by replication of circular DNA (Intact) coincides
with the end joining product. The bands corresponding to transfected
DNA (Input DNA) are shown in the lower
part of the gel.
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First, recombination-dependent expression of luciferase was
used to look at recombination in BHK cells and Balb/c 3T3 cells. We
found again that there was efficient expression of luciferase from
linear pXY1 as well as pXY2 in BHK cells (Fig.
7A). However, recombination-dependent expression of luciferase from pXY2
was very low in Balb/c 3T3 cells (Fig. 7B). Importantly,
luciferase expression from linear pXY1 was also reduced. These results
agree with those obtained using oriS-dependent
amplification of recombination products as the assay (Fig. 6). In
Balb/c 3T3.SV-T2 cells containing the SV40 large T-antigen, the
expression of luciferase from circular and linear pXY1 did not differ,
but recombination-dependent expression of luciferase from
pXY2 was still lower than observed in BHK cells, indicating that
inhibition of p53 activity abolishes the negative effect of linear DNA
and partially restores recombination (Fig. 7C). Mouse embryo
fibroblasts devoid of p53 (MEFp53
/
) readily supported
recombination-dependent expression of luciferase (Fig. 7D). Wild type mouse embryo fibroblasts were also examined.
In this instance, the relative levels of luciferase produced by
circular and linear plasmids containing intact genes were 100 and 70%, respectively. The amount of luciferase produced by the linear disrupted
gene was 25% when compared with the circular template (results not
shown). Together, these results demonstrate that homologous
recombination is tightly controlled by the cell. One mechanism might be
induction of p53-dependent apoptosis by linear DNA.

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Fig. 7.
Recombination-dependent
expression of luciferase is cell type dependent. Circular and
linear DNA was transfected into BHK cells (A), 3T3 cells
(B), 3T3.SV-T2 cells (C), and MEF p53 / cells
(D). Cell lysates were made 24 h after transfection,
and the luciferase activity was measured.
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Linear DNA Induces p53-dependent Apoptosis in 3T3
Cells--
As mentioned above, DNA damage activates p53 as a
transcription factor in Balb/c 3T3 cells and induces cell cycle arrest
or apoptosis (26-28). DNA-dependent protein kinase or
ATM-related protein kinases may be responsible for the
activation of p53 (26, 27, 29-31). Protein kinases or other signaling
devices may recognize DNA ends introduced into cells by transfection
and thereby initiate a process leading either to cell cycle arrest or
cell death (32). We now wanted to examine if p53 either had a direct
effect on homologous recombination or exerted an indirect effect by
promoting the induction of apoptosis by linear DNA.
The induction of apoptosis was first monitored by a morphological
assay. We used plasmid DNA encoding
-galactosidase under the control
of the HCMV promotor. Circular and linear forms of plasmid DNA were
then transfected into cells, and the morphology of the cells expressing
encoding
-galactosidase was registered after staining with
5-bromo-4-chloro-3-indolyl-
-D-galactoside. The
appearance of shrunken blue cells with pyknotic nuclei was used as an
indicator of apoptosis. The linear DNA was produced by ScaI
cleavage 1.7-kilobase pair downstream the polyadenylation signal. We
noted that linear DNA increased the frequency of apoptotic cells as
defined by morphological criteria ~4.4-fold for Balb/c 3T3 cells and
wild type mouse embryo fibroblasts (Table
I). BHK cells, Balb/c 3T3.SV-T2, and
p53-deficient mouse embryo fibroblasts were not affected by linear DNA
in the same way.
The induction of apoptosis by transfected linear DNA was also
investigated by staining of Balb/c 3T3 cells with annexin V, which is
an early indicator of apoptosis (33). First, we looked at adherent
Balb/c 3T3 cells. We found that the proportion of adherent cells
binding annexin V-EFGP did not vary significantly when nontransfected
cells and cells transfected with linear DNA were compared (results not
shown). We then collected the nonadherent cells and found that the
proportion of apoptotic cells stained by annexin V-EFGP increased
between 5- and 10-fold following transfection with linear DNA (results
not shown). We conclude that linear DNA but not circular DNA induces
apoptosis in Balb/c 3T3 cells.
Finally, we examined the effect of circular and linear DNA on the
transformation frequency. We found that circular plasmids readily
transformed all cell lines (Table II).
Experiments using linear DNA produced a different picture. The
transfection frequencies for Balb/c 3T3 cells and wild type mouse
embryo fibroblasts were dramatically reduced. In contrast,
p53-deficient mouse embryo fibroblasts and BHK cells were readily
transformed by linear DNA.
Our results suggest that linear DNA molecules with nonhomologous end
sequences induce p53-dependent apoptosis in Balb/c 3T3 cells. This finding may help to explain the low level of
recombination-dependent expression of luciferase observed
in Balb/c 3T3 cells (Fig. 7B). It should be noted that BHK
cells most probably express wild type p53 (34). Other factors therefore
must have a decisive influence on the outcome of recombination
reactions occurring in the cell.
The Structure of the Recombination Substrate Determines the
Efficiency of Homologous Recombination in 3T3 Cells--
Herpes
viruses that introduce linear genomic DNA into mammalian cells
run the risk of triggering an apoptotic response. We have shown above
that linear DNA unable to undergo homologous recombination induces
apoptosis in Balb/c 3T3 cells (Table I). As a result, linear DNA
molecules appear to replicate very poorly in nontransformed Balb/c 3T3
cells and fail to support recombination-dependent expression of luciferase (Figs. 6 and 7B). Cells appear to
face a situation where a decision has to be made between different pathways of recombination repair as well as between different fates of
the cell. The structure of the broken DNA molecule may influence this
decision. We have for this reason compared the replication and
recombination of the linearized plasmids p2aLORI, p2aORI, and p2bORI in
Balb/c 3T3 cells and BHK cells (Fig.
8A). The linearized plasmids
p2aORI and p2bORI have the repeated sequences at the ends of the DNA
molecule, and they were circularized and replicated by the HSV-1
replication machinery in both cell types. The insertion of a
DNA
insert between the directly repeated a sequences in p2aLORI
resulted specifically in a 10-fold reduction in the efficiency of
circularization and replication in Balb/c 3T3 cells (Fig.
8B). Our results suggest that Balb/c 3T3 cells transfected
with p2aORI and p2bORI may have escaped apoptosis. Two
interpretations are possible. Homologous recombination may still take
place in Balb/c 3T3 cells, but the pathway that permits recombination
repair of linear DNA containing direct repeats at an internal position
is suppressed in these cells. The alternative interpretation would
suggest that unique DNA ends induce apoptosis much more rapidly than
DNA ends containing direct repeats.

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Fig. 8.
The position of directly repeated sequences
in linear DNA determine the efficiency of homologous recombination in
3T3 cells. Circular and linear DNA containing directly repeated
sequences either with a DNA spacer, p2aLORI, or without a spacer, p2aORI and p2bORI, were transfected into BHK and Balb/c 3T3
cells. The plasmid pBORI lacks repeated sequences (14). The
transfection was carried out in the presence of the seven expression
plasmids encoding the HSV-1 replication genes (see "Materials and
Methods"). DNA was isolated 48 h after transfection and
subjected to DpnI and HindIII cleavage. Southern
blots of agarose gels were probed using randomly primed pUC19 DNA.
A, an autoradiograph of the Southern blots. B,
the autoradiograph was analyzed using a PhosphorImager. The amount of
replicated DNA obtained using circular p2aLORI in BHK cells was
assigned a value of 100% and compared with the amount of replicated
DNA from linearized plasmids in the same cells. The amount of
replicated DNA for p2aLORI was assigned a value of 100% in Balb/c 3T3
cells and compared with the amount of replicated DNA from linearized
plasmids in Balb/c 3T3 cells. The structures of the substrate molecules
are presented schematically. To facilitate comparison, a
star indicates the lanes in which the replication
of linear p2aLORI was analyzed as well as the columns that
show the relative amount of replicated p2aLORI DNA determined by
PhosphorImager analysis.
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DISCUSSION |
In this paper, we have examined the interdependence of DNA
replication and recombination during the infectious cycle of Herpes simplex virus type I. We have also looked at cellular factors and
conditions that affect homologous recombination. We find that homologous recombination occurs readily in BHK cells in the absence of
viral DNA replication and virus-encoded gene products. Furthermore, the
products of homologous recombination appear to be very efficiently utilized by replication and transcription machineries. In Balb/c 3T3
cells, linear DNA molecules with nonhomologous sequences at the ends do
not recombine, and they induce p53-dependent apoptosis. On
the other hand, linear DNA molecules with direct repeats at the ends of
the molecule support homologous recombination, and they are replicated
by the HSV-1 replisome in Balb/c 3T3 cells. Our discussion will focus
on three aspects of this work: the mechanisms for a coupling between
DNA replication and recombination in virus infected cells; the
regulation of recombination by p53; and the role of homologous
recombination during the infectious cycle of herpes viruses.
We have noted that in the absence of DNA synthesis only small amounts
of recombination products are detected. However, the products of
homologous recombination appear to be readily amplified by the HSV-1
replication apparatus and to be efficiently used by the cellular
transcription machinery. It therefore appears as if a subset of the
transfected DNA molecules display a high probability of simultaneously
undergoing homologous recombination, DNA replication, and transcription
in BHK cells. In other words, our observations suggest that these
processes are to some extent coupled. Different models can account for
the postulated coupling between homologous recombination and DNA
synthesis. In prokaryotic organisms homologous recombination may
support the formation of productive replication forks (25). However, we
have not been able to demonstrate a similar phenomenon in HSV-1
infected cells. DNA replication remained origin-dependent
under all of our experimental conditions, and homologous recombination
did not appear to stimulate DNA synthesis. A more likely explanation of
the coupling between DNA replication and homologous recombination would
be that these processes are spatially and temporally coordinated. In
its simplest form, we might assume that most DNA molecules recombine
and replicate as soon as they reach the nucleus. Another hypothesis
would state that the interactions between different pathways of DNA
metabolism depend on the nonrandom distribution of replication and
recombination proteins into nuclear foci (35-39). Still another
possibility would be that viral DNA replication and recombination are
coordinated events in the cell cycle.
It is a striking fact that homologous recombination is easily
demonstrated in BHK cells using functional assays. This is unexpected, since nonhomologous end joining is considered to be a more frequent process for double strand break repair in mammalian cells (9, 20). More
recently, however, it has been demonstrated that homology-directed repair constitutes a major repair pathway in mammalian cells (18, 40).
In this paper, we demonstrate that homologous recombination is
regulated differently in BHK cells and Balb/c 3T3 cells. For example,
DNA molecules with ends that cannot directly take part in homologous
recombination failed to recombine in Balb/c 3T3 cells, and they were
shown to induce apoptosis. In contrast, recombination and replication
of linear DNA with the direct repeats located at the very ends of the
molecules were readily detected in Balb/c 3T3 cells. In BHK
cells, on the other hand, homologous recombination did not depend on
the location of the repeated sequences.
We also demonstrated that homologous recombination in Balb/c 3T3 cells
was controlled by p53. However, p53 cannot be the only factor governing
the outcome of recombination reactions. BHK cells are immortalized
cells that appear to express wild type p53 (34). T antigen from BK
virus binds to p53 in BHK cells and greatly increases the half-life of
the protein (41). BHK cells transformed with T antigen can now form
foci in soft agar (41). Since BHK cells and Balb/c 3T3 cells react
differently to our recombination substrates, other factors must
contribute to the regulation of homologous recombination. A separate
indication that other factors must act together with p53 to regulate
recombination comes from studies on gene amplification. Drug-resistant
colonies containing amplified genes are easily selected from BHK cells,
but DNA amplification leading to drug-resistance is rare in normal
human cells (42, 43).
A functional coupling between homologous recombination and p53 has also
been observed in other instances (16, 44, 45). What are the mechanisms
connecting p53 to homologous recombination? It is tempting to speculate
that a direct communication between the apparatus responsible for
homologous recombination and p53 exists. The protein-protein
interaction between Rad51 and p53 might be involved (46).
Alternatively, the ability to distinguish between recombination
substrates and relay that information to p53 and other components of
the cell cycle machinery might rely on the use of signaling proteins
such as DNA-dependent protein kinase and ATM-related
kinases (26, 27, 29-31). A conceivable consequence might be that the
induction of apoptosis could be suppressed by successful initiation of
homologous recombination.
There are several steps in the life cycle of Herpes simplex virus type
I and other herpes viruses that may involve cellular recombination
pathways. The viral genomes are linear molecules. They have in many
instances directly repeated sequences at their termini. The viral
chromosomes have to be circularized to support a productive infection
(6). Homologous recombination may be directly responsible for the
circularization of viral genomes. Homologous recombination involving
the repeated sequences at the ends of virus chromosomes may, in fact,
be one way of evading an apoptotic response by the cell. Our
observation that only linear molecules that present repeated sequences
at the very ends of the DNA molecule are recombined and replicated in
Balb/c 3T3 cells supports this notion. It is important to remember,
however, that circular DNA molecules may also be formed by direct
ligation of complementary DNA ends. An in vitro system that
accurately performs direct ligation has recently been described (10).
DNA ligase IV/Xrcc4, Ku70, Ku86, and the catalytic subunit of
DNA-dependent protein kinase are essential components of
this system. In the event that the site of fusion would be altered, the
virus still has a possibility to select correct products in the
cleavage and packaging stages. There is also the possibility that
homologous recombination coupled to gene conversion may edit the
products of the ligation event. Cell lines that lack recombination
proteins are now becoming available. It should therefore be possible to establish genetically the pathway that leads to circularization of
herpes viruses.
A separate function of homologous recombination is to repair strand
breaks occurring during the replication of HSV-1. As a result, a high
frequency of recombination between genetic markers on different HSV-1
strains would be expected from coinfection experiments. Abundant
experimental evidence demonstrates this phenomenon (7, 47, 48).
Interestingly, however, homologous recombination between cellular
chromosomes in mitotically growing mammalian cells is very rare (49).
Possibly, chromatin structure will suppress homologous recombination in
higher cells. Studies of recombination taking place during viral
replication cycles might be a relatively accessible way of studying
mechanisms of homologous recombination in mammalian cells and
identifying the genes and gene products that control recombination.