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Repair of Psoralen-treated DMA by Genetic Recombination in Human
Cells Infected with Herpes Simplex Virus1
Jennifer D. Hall2 and Karen Scherer
Department of Cellular and Developmental
Biology, University of Arizona, Tucson, Arizona 85 721
ABSTRACT
Herpes simplex virus type 1 was treated with 4,5',8-trimethylpsoralen (psoralen) plus near-ultraviolet light in order to
produce lesions (monoadducts and DMA cross-links) in the
viral DNA. Human fibroblasts were infected by damaged virus
under conditions in which either a single virus particle or
several particles entered a given cell, and the fraction of virusproducing cells was determined. This fraction was significantly
greater for multiply infected cells than for singly infected cells,
indicating that the psoralen lesions are repaired more efficiently
in the presence of homologous, damaged DNA (multiplicity
reactivation).
Evidence is presented that herpes simplex virus may code
for functions which participate in its own repair, both during
multiplicity reactivation and during repair which occurs in singly
infected cells: (a) host cells deficient in repair of lesions induced
by psoralen (xeroderma pigmentosum) or the DNA cross-link
ing agent mitomycin C (Fanconi's anemia) exhibited normal
levels of multiplicity reactivation of psoralen-treated herpes
virus; (b) while xeroderma pigmentosum cells have been pre
viously shown to be deficient in repair of psoralen-treated
adenovirus under conditions of single infection, herpes virus is
repaired at near normal levels in these same cells.
Recombination levels between genetically marked pairs of
herpes viruses were found to increase after treatment of the
parental viruses with psoralen, suggesting that psoralen dam
age stimulates genetic recombination. This stimulation provides
convincing evidence for a repair pathway in which genetic
recombination between damaged viral genomes can lead to
the production of viable virus.
INTRODUCTION
Psoralen3 reacts with DNA in the presence of near-UV3 light
to produce both monoadducts and diadducts (cross-links) (4).
Both types of lesions are repaired in prokaryotic and mamma
lian systems (1, 5). While repair of monoadducts appears to
involve excision repair pathways, there is considerable evi
dence that cross-links are repaired by pathways involving both
excision of adducts and genetic recombination (6). Genetic
recombination is presumably required since both strands of a
DNA duplex are damaged by the cross-link.
In Escherichia coli, the mechanism of cross-link repair has
been elucidated with genetic mutants deficient in excision
repair of UV-damaged DNA and/or in genetic recombination.
' Support by Grant AG01689 from the NIH.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: psoralen, 4,5',8-trimethylpsoralen;
XP, xero
derma pigmentosum; FA, Fanconi's anemia; HSV-1, herpes simplex virus type 1.
Received May 21, 1981 ; accepted July 30, 1981.
These studies (6) suggest that excision enzymes cut one strand
of the damaged DNA on both sides of a cross-link to remove
the lesion from this strand (half-excision). The gap produced
by excision is filled with homologous DNA from a sister chro
mosome. Finally, the psoralen adduct is removed from the
second strand by excision followed by DNA synthesis to fill this
second gap. A role for genetic recombination in cross-linked
repair has been further suggested by the ability of cross-links
to stimulate genetic recombination (16).
Damage produced by cross-linking treatments (such as by
psoralen or mitomycin C) in bacterial systems is also repaired
by a recombinational mechanism called multiplicity reactiva
tion. In this mechanism, damaged bacteriophages are repaired
more extensively in cells infected with several viral genomes
than in singly infected cells (5, 15), providing that a functional
recombination mechanism exists (15,17). Furthermore, cross
links, as opposed to monoadducts, in bacteriophage \ DNA
are not repaired unless 2 or more homologous viral genomes
are present in the same infected cell (5), indicating the absolute
requirement for multiplicity reactivation in repair of cross-links.
Repair of psoralen-induced DNA cross-links in mammalian
cells also appears to involve excision and recombination func
tions. A role for genetic recombination has been suggested
since cross-linking treatments induce recombination in lower
eukaryotes (14, 21 ) and stimulate sister chromatid and nonsister homolog exchanges in mammalian cells (24, 27). The fact
that psoralen-induced cross-links in adenovirus are not re
paired during infection of human cells (8), under conditions of
single infection, further implies that cross-links cannot be re
paired in the absence of homologous DNA.
Excision functions also appear to be required for repair of
cross-links in human cells, since at least one cell line from a
patient with XP3 is deficient in half-excision of psoralen-induced
cross-links (18). These XP cells are also deficient in excision
of UV-induced damage from DNA (12). FA3 cells are deficient
in half-excision of cross-links induced by mitomycin C (9) but
appear proficient in psoralen cross-link repair (18). These
observations suggest that different types of cross-links may be
repaired by different pathways in human cells.
Monoadducts formed by psoralen treatment are presumably
repaired by excision repair functions in bacteria (6). Excisiondeficient human XP cells show abnormally low survival of
psoralen-treated adenovirus when infected by single viral par
ticles (8). Since, under these conditions, viral DNA cross-links
were not repaired, the excision function deficient in these XP
cells appears to be required for repair of psoralen-induced
monoadducts.
Genetic recombination has been shown previously to be
involved in the repair of UV-irradiated herpes simplex virus (10,
26). These experiments demonstrated multiplicity reactivation
of irradiated virus and showed that UV irradiation of the virus
DECEMBER 1981
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5033
J. D. Hall and K. Scherer
stimulated genetic recombination between viral genomes. The
role of genetic recombination in repair of psoralen-induced
lesions in herpes-infected cells has not been studied previ
suspended in a small volume of culture medium containing 1% calf
serum, and sonicated. The virus suspensions were then clarified by
centrifugation and stored at -80°.
ously.
The goal of the present experiments was to investigate
whether psoralen damage in human cells can be repaired by
genetic recombination. HSV-13 was used as a probe in these
The procedure for plaque assays with intact virus on human or
cells has been described (10).
Psoralen Treatment. Psoralen was dissolved in ethanol at 1.5
ml and frozen at —20°.Before use, the psoralen suspension
heated to 47° for 30 min to redissolve the psoralen precipitate
studies because genetically marked viral mutants are available
to study damage-induced recombination levels. In addition, the
multiplicity of infections can be controlled so that repair of
damaged virus can be compared in singly infected and multiply
infected cells (i.e., under conditions in which genetic recom
bination between viral genomes either cannot or can occur).
We present evidence that repair of psoralen damage is sub
stantially increased in multiply infected cells, suggesting that
genetic recombination is involved in this repair. We have further
shown that psoralen damage to viral DNA increases the yield
of viral recombinants, providing further support for the idea
that genetic recombination is involved in repair of psoraleninduced lesions.
MATERIALS
saline (NaH2PCvH2O,
mg/
was
and
0.34 g/
liter; Na2HPO4, 1.93 g/liter; NaCI, 8.5 g/liter) to give a final concen
tration of 50 iig/ml. Virus was diluted 1:10 into the psoralen-buffer
mixture and irradiated at ice temperature in a liquid layer approximately
1 mm thick. Samples were kept in the dark for 10 min prior to irradiation
to allow the psoralen time to intercalate into viral DNA. This time was
more than adequate to allow maximum photosensitization.
Irradiation
was carried out in plastic Retri dishes with the tops on or in plastic
tubes to minimize exposure to shorter wavelengths. Controls (not
shown) indicated that exposure of virus suspensions to irradiation in
the absence of psoralen does not significantly alter the viability of the
virus at the doses used. Irradiation was carried out with a Sylvania
Blacklite blue lamp (F15T8). The dose rate was 8 to 10 J/sq m/sec as
measured by a Blak-Ray UV meter (Ultraviolet Products, Inc., San
Gabriel, Calif.). The suspension was swirled periodically during irradia
tion to ensure a uniform exposure dose.
UV Irradiation. UV irradiation of HSV-1 was performed as described
AND METHODS
Cell Lines and Virus Stocks. The characteristics of the cells and
virus strains used are described in Tables 1 and 2. Cells were grown
in Dulbecco's modified Eagle's medium supplemented with 20% fetal
calf serum (human cells) or 10% calf serum (monkey cells) and incu
bated in an atmosphere of 10% CO2 at 37°.
Virus stocks were prepared on Vero cells at 37°(wild type) or 33°
(temperature sensitive) by inoculating progeny from a single plaque
onto cells at a multiplicity of infection of 0.01 plaque-forming unit/cell.
When cytopathic
then diluted into phosphate-buffered
Vero
effects were observed, infected cells were harvested,
previously (10) using a General Electric germicida! lamp with an output
maximum at 254 nm.
Infective Center Assay. The infective center assay has been de
scribed previously (10). Briefly, human cells were infected with a known
number of virus particles. Cells infected with irradiated virus received
the same total number of viral particles (damaged plus residual undam
aged) as cells infected with unirradiated virus. Infected cells were then
washed, trypsinized, and seeded with an excess of uninfected Vero
cells in the presence of human y globulin. The ability of the initially
infected cells to produce virus was then determined from the numbers
Table 1
Cell lines
Source
Line
CRL 1220
XP12BEorCRL1223
American Type Culture Collection
(Rockville, Md.)
American Type Culture Collection
Genetic characteristics
Human skin fibroblasts from a normal
15-yr-old male
Human skin fibroblasts from a 7-yr-old female
with XP (complementation Group A)
skin fibroblasts from a 6-yr-old male
with FA
American Type Culture Collection
Established cell line derived from African
green monkey kidney cells
Gift of P. Henson (Harvard University)Human
Established
proficientTable
cell line derived from African
green monkey kidney cellsPresumably
Type Culture Collection
CCL 122
Vero or CCL 81
TC-7American
Ability to repair UV- or psoralen-induced
age
dam
Presumably proficient
Deficient in excision repair of UV damage (19),
psoralen cross-links (18), psoralen monoadducts (8)
proficient
Presumably proficient
Presumably
2
strainsComplementa
HSV-1
tion group
characteristicsTemperature
(25)A16
Strain
positionMaps
map
1-1SourceGift
F17
1-6
BU"-A
PAA"-5
CI 101
5034
I-4
of P. Schaffer (Harvard Univer-Genetic
sensitive; DMA-negativePhysical
near mutants Cand D on genetic map
(22) C and D map at 0.386-0.418
(3)
sity)
phenotype at nonpermissive tempera
ture (2)
Gift of P. Schaffer
Temperature sensitive; DNA-positive
Same complementation group as F18 which
maps at 0.086-0.103
(23)
phenotype at nonpermissive tempera
ture (2)
Spontaneous mutant of the KOS
Resistant to bromodeoxyuridine; presum
Presumably maps in the thymidine kinase
strain of HSV-1 (J. Hall, unpub
gene at 0.300-0.309
(11)
ably deficient in viral thymidine kinase
lished results)
Gift of D. Coen (Harvard University)
Maps in the DNA polymerase gene at 0.400Resistant to phosphonoacetic acid
0.418(3)
Originally obtained from S. Kit (Bay
Wild type
lor College of Medicine)
CANCER
RESEARCH
VOL.
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41
Repair of Psoralen Damage in Herpes-infected
Cells
of plaques in this assay. The average multiplicities of infection were
calculated as described previously (1 0) by comparing the virus titers in
virus suspensions that were removed from cells infected with unirradiated virus and in suspensions prior to infection. The amount of
adsorbed virus was then compared to the number of cells per dish to
determine the average multiplicity of infection.
Recombination Assay. The recombination assay has been de
scribed previously (1 0). Briefly, human cells were multiply infected with
pairs of genetically marked viruses. The viruses were treated with
psoralen and irradiated prior to infection as indicated. Infected cells
were incubated for 1 hr at 37° to allow viral adsorption. The viral
suspensions were then removed, and 1 ml of fresh medium was added
per plate. Incubation was continued at 33° (temperature-sensitive
viruses) for 24 hr or at 37° (temperature-resistant
viruses) for 18 hr,
and the viral progeny were harvested. The progeny viruses were then
titered to determine the recombination frequency. When temperaturesensitive parental viruses were used, this frequency was determined
by measuring the relative yields of recombinant (temperature-resistant)
viruses at 39°and total viruses (temperature resistant plus temperature
sensitive) at 33° on Vero cells. When drug-resistant parental viruses
were used, this frequency was determined by measuring the relative
yields of recombinant viruses [those able to form plaques in the
presence of phosphonoacetic
acid (100 /ig/ml) and bromodeoxyuridine (100 fig/ml)] and the total viral yield in the absence of these
drugs. The assays with drug-resistant viruses were performed on TC7 cells. Reversion of temperature-sensitive
viruses or production of
spontaneous drug-resistant mutants occurred in these assays at very
low levels and did not affect the calculation of recombination frequen
cies (data not shown). Cellular thymidine kinase (present at low levels
in monolayer cells) did not affect the results of these measurements
since similar results were obtained with iododeoxycytidine,
a com
pound which is phosphorylated
by the viral, but not the cellular,
thymidine kinase. The percentage frequency of recombination (RF)
was calculated as:
Recombinant
—
virus titer
Total virus titer
X ¿.DU
RESULTS
In order to determine whether genetic recombination might
be involved in repair of psoralen damage in human cells, HSV1 was treated with psoralen plus light and allowed to infect
cells at various multiplicities of infection. This approach facili
tated comparison of repair in multiply infected cells, in which
genetic recombination between viral genomes could occur,
and in singly infected cells, in which genetic recombination is
absent.
In this assay, human fibroblasts were first infected at the
appropriate multiplicities and then seeded with an excess of
uninfected monkey kidney cells. The survival of plaque forma
tion of the infected cells was then determined (Chart 1). Multi
plicities of infection were determined from control infections
with undamaged virus. Cells infected with psoralen-treated
virus received the same number of viral particles (damaged
plus residual plaque forming) as cells in these control infec
tions. Two multiplicities of infection were used. Multiplicities
less than 2 x 10~2 plaque-forming unit/cell were chosen to
ensure that the cells would be singly infected. Since herpes
virus preparations typically contain 10 to 100 defective parti
cles for every infectious virus, this multiplicity should also
minimize multiple infections with defective particles. A multi
plicity of infection of approximately 3 plaque-forming units/cell
was also used.
DECEMBER
1981
300
600
300
600
IRRADIATION DOSE ( J/tq m )
600
900
Chart 1. Survival of plaque formation by human fibroblasts infected at various
multiplicities with herpes simplex virus treated with psoralen plus light. The
infective center assay was performed as described in "Materials and Methods."
Human fibroblasts used in these experiments were: A, CRL 1220 (normal); B,
CRL 1223 (XP, Group A); and C, CCL 122 (FA). The average multiplicities of
infection (plaque-forming units/cell) for cells infected with unirradiated virus are
indicated in parentheses in the figures. Cells infected with irradiated virus were
infected with the same number of viral particles as cells infected with unirradiated
virus. Plating efficiencies of infective centers were greater than 34%.
,
theoretical survival curves for plaque formation by cells infected at the higher
multiplicity of each graph, assuming that plaque formation occurred only by
infection of cells with undamaged virus. The equation from which these curves
were generated has been derived previously (10), assuming a Poisson distribution
for the multiplicity of infection by both damaged and undamage virus particles.
Theoretical values were calculated to an accuracy of 0.3%.
A comparison of the survivals of plaque formation by singly
or multiply infected normal human fibroblasts is shown in Chart
ÃŒA.
Clearly, the survival for multiply infected cells is substan
tially greater than for singly infected cells. Theoretical curves
based on the expected survival at the higher multiplicity due to
infection by residual undamaged viral particles are presented
in Chart 1 as dashed lines. These theoretical values are lower
than the observed survivals for multiply infected cells at irradia
tion doses of 300 J/sq m or greater. This result suggests that
survival in multiply infected normal human cells occurs by
pathways not available in singly infected cells. This increased
repair will be referred to as multiplicity reactivation.
Two other human fibroblast cells lines were tested for repair
of psoralen-damaged herpes virus as described above. The XP
cell line, CRL 1223, has been previously reported to be defi
cient in repair of psoralen-induced cross-links (18). Certain FA
cells have been found to be deficient in repair of mitomycin Cinduced cross-links (9), while another cell line was found to
repair psoralen cross-links normally (18). Both the FA (CCL
122) and XP (CRL 1223) cell lines showed increased viral
repair in multiply infected cells as compared to singly infected
cells (Chart 1, ßand C), suggesting that they are proficient in
multiplicity reactivation. Apparently, the host functions deficient
in these mutant cells are not required for multiplicity reactiva
tion of psoralen-damaged herpes virus.
Repair of psoralen-induced monoadducts appears to involve
excision repair functions in bacteria (6) and is substantially
reduced in human XP cells singly infected with psoralen-dam
aged adenovirus (8). To determine whether cellular excision
functions might also be utilized during repair of monoadducts
in herpes simplex virus DMA,we compared the relative abilities
of various human host cells to repair psoralen-damaged herpes
virus under conditions of single infection. The results in Table
3 show that for FA cells (CCL 122) the survival of plaque
5035
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J. D. Hall and K. Scherer
Table 3
Survival of HSV-/ plaques on human fibroblasts
I.O
(A)
Human fibroblasts were infected with HSV-1, as described in the legend to
Chart 2. D37values for the curves shown in Chart 2 and for the CCL 122 cell line
(curve not shown) were determined as the doses giving 1/e survival.
lineCRL Cell
, •
for psoralen-treated
virus192(1.00)"
1220 (normal)
CRL1223(XP)
CCL 122 (FA)O
Numbers in parentheses,
1220) cells.
152(0.79)
282 (1.47)D37
for virus39.5(1.00)
UV-irradiated
O.I
13.0(0.33)
Not done
relative D,,- values compared
to normal (CRL
Õ
formation, as indicated by the D37 value, was similar to that
seen for normal cells. Survival on XP (CRL1223) fibroblasts
showed only a slight reduction compared to normal cells. This
result suggests either that repair of psoralen damage in singly
infected cells does not require the repair functions deficient in
FA or XP cells or that viral functions can complement these
deficiencies.
Previous studies have shown that repair of HSV-1 damaged
by UV is abnormally low in singly infected XP cells (10, 20,
26), suggesting that host cell excision functions are required
for repair of UV-induced lesions in herpes DMA. In contrast, as
shown above, host excision functions are not required for
repair of psoralen damage in herpes DNA. We wished, there
fore, to confirm this difference by comparing the role of host
excision functions in repair of psoralen-treated and UV-irradi
ated HSV-1 under the same experimental conditions and with
the same cell lines. For these studies, both the normal human
fibroblast line (CRL 1220) and the XP line (CRL 1223) were
used. As shown in Chart 2 and Table 3, psoralen-treated virus
was repaired at near normal levels in XP (CRL 1223) cells. In
contrast, UV-irradiated virus was repaired at significantly lower
levels in XP (CRL 1223) cells than in normal cells (CRL 1220).
The D37 value for survival of UV-irradiated HSV-1 on XP (CRL
1223) cells was 3.0-fold less than that seen on normal cells
(CRL 1220). This result suggests that, under conditions of
single infection, host cell excision repair functions are utilized
during repair of UV-irradiated HSV-1 in normal human cells but
that psoralen-treated virus does not require these same func
tions for repair.
In bacterial systems, genetic recombination is induced by
DNA cross-links (16). In addition, genetic recombination of
herpes virus is stimulated by UV irradiation (10). If genetic
recombination is also involved in multiplicity reactivation of
psoralen-treated herpes virus, then psoralen damage may also
stimulate genetic exchanges between herpes genomes.
To test this possibility, cells were infected with pairs of
psoralen-treated herpes mutants and the formation of recom
binant progeny was measured (Chart 3). Cells were multiply
infected with each mutant to allow recombination between
damaged viral genomes to take place. Infection was conducted
with complementing mutants to avoid any effect of defective
viral functions on the recombination levels measured. The
results in Chart 3A show that, with 2 different temperaturesensitive viral mutants (A16 and F17), psoralen treatment stim
ulated the yields of temperature-resistant
recombinants. In
Chart 36, 2 mutants resistant either to bromodeoxyuridine
[BUR-A, deficient in the viral thymidine kinase (28)] or to phosphonoacetic acid [PAAR-5, carrying a mutation in the viral DNA
polymerase (13)] were tested for the production of recombi-
5036
O.I
O
4OO
800
IRRADIATION DOSE( J/«qm)
Chart 2. Survival of plaque formation by human fibroblasts infected with HSV1 treated with psoralen plus light or with UV light. Virus was treated with UV (A)
as described (10) or with psoralen plus light (B) as described in "Materials and
Methods." Human fibroblasts were then infected with virus in a plaque assay,
and the survival of plaque formation was determined. Curves in B and upper
curve in A represent composites of 2 experiments. Lmes indicate the least
squares fit of an exponential curve to the points. •,CRL 1220 (normal); O, CRL
1223(XP).
1.0
(B)
(A)
0.5
OX)
600
0
300
600
IRRADIATION DOSE ( J/sq m )
Chart 3. Yield of temperature-resistant
herpes virus recombinants in human
fibroblasts infected with virus treated with psoralen plus light. Crosses between
pairs of viral mutants and calculation of the recombination frequencies were
performed as described in "Materials and Methods." In each experiment, only
300
one parental virus was exposed to psoralen and/or light. Spontaneous recom
bination frequencies in each experiment are shown by error bars. A, A16 x F17;
A16 was treated with psoralen and/or light; B, BUB-A x PAAn-5, PAAR-5 was
treated with psoralen and/or light. •,virus treated with psoralen plus light; O,
untreated virus; G, virus treated with light in the absence of psoralen. Plaqueforming units (titers determined prior to irradiation) added per cells were: 8.2
(A16); 1.2(F17); 3.2 (BUR-A); 3.2 (PAAB-5).
nants resistant to both drugs. Again, the recombination fre
quency increased following psoralen damage. These results
clearly demonstrate that psoralen damage stimulates viral re
combination and may be involved in repair of these lesions.
CANCER
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SÉF-
Repair of Psoralen Damage in Herpes-infected
DISCUSSION
The present results demonstrate that repair of psoralendamaged herpes simplex virus occurs more readily in multiply
infected human cells than in singly infected cells, a phenome
non called multiplicity reactivation. In addition, under condi
tions of multiple infection, psoralen damage stimulates the
production of viral genetic recombinants. Since these pro
cesses were both observed under the same experimental con
ditions, it seems probable that they are functionally related.
Taken together, these results strongly suggest that genetic
recombination is involved in the repair of psoralen-induced
lesions, if multiple copies of genomes are available. A role for
genetic recombination in multiplicity reactivation is made more
plausible by the requirement previously established for recom
bination in multiplicity reactivation in bacterial systems (15,17)
and in human cells infected with UV-irradiated herpes virus
(10).
While psoralen-induced cross-links are removed from cellu
lar DNA in diploid human cells (18), they are not removed from
adenovirus DNA under conditions in which human cells are
infected with single-viral particles (8). These results suggest
that repair of DNA cross-links in mammalian cells may require
genetic recombination between homologous DNA duplexes, as
found previously for bacterial systems (5, 6). Repair of monoadducts, on the other hand, occurs extensively in the absence
of genetic recombination, as indicated by the substantial re
duction in repair of psoralen-treated adenovirus in singly in
fected XP cells compared to normal cells (8). Consequently,
our results with psoralen-treated HSV-1 suggest that the in
creased survival of virus in multiply infected cells is due pri
marily to an ability of these cells to repair DNA cross-links in
viral DNA. However, we cannot rule out the possibility that
psoralen-induced monoadducts also are repaired by multiplic
ity reactivation and stimulate recombination in herpes-infected
cells. We are presently measuring the yield of psoralen-induced
cross-links produced under our conditions of treatment to
distinguish between repair of monoadducts and cross-links.
XP cells have been shown to be deficient in repair of psora
len-induced cross-links (18). However, in the present study,
these cells exhibit extensive multiplicity reactivation of psora
len-treated herpes virus. Since multiplicity reactivation of
herpes virus probably repairs DNA cross-links, the proficiency
with which XP cells carry out multiplicity reactivation of herpes
virus suggests that cross-links in viral DNA are repaired nor
mally in these cells. This result further suggests that cross
links in viral DNA are repaired by a different mechanism than
are cross-links present in cellular chromosomes, possibly due
to the presence of viral-induced repair functions.
Repair of psoralen-damaged HSV-1 is only slightly reduced
in singly infected XP cells compared to wild-type cells. In
contrast, repair of psoralen-treated adenovirus is substantially
reduced in XP cells (8). These results suggest that herpes virus
but not adenovirus might provide one or more functions which
can complement the deficiency in XP cells during repair of
psoralen damage. Since it is probable that monoadducts, not
cross-links, are repaired under conditions of single infection,
these postulated herpes repair functions appear to act on
monoadducts. Alternatively, structural differences in herpes
genomes (such as a paucity of histones) might allow the repair
of psoralen damage in XP cells, while similar lesions in either
DECEMBER
1981
Cells
adenovirus or cellular chromosomes might be inhibited.
Unlike repair of psoralen damage, repair of UV-irradiated
herpes virus is significantly reduced in singly infected XP cells
relative to wild type. Repair of UV-irradiated adenovirus, like
wise, is inhibited in XP cells (7). Apparently, herpes functions
cannot complement the excision repair deficiency in XP cells
for repair of UV-induced lesions. XP cells are thought to be
deficient in the UV endonuclease which initiates excision repair
oadducts (8). To explain the results with herpes virus, we
hypothesize that herpes repair functions are more specific than
the corresponding host function and act on psoralen adducts
but fail to recognize UV damage.
In summary, multiplicity reactivation of herpes simplex virus
can best be explained as involving a genetic recombination
mechanism. Multiplicity reactivation of HSV-1 has previously
been observed for repair of UV damage (10, 26) and alkylation
damage." Therefore, this mechanism may constitute a major
repair pathway in multiply infected cells, capable of acting on
many different types of lesions. The possibility that herpes
viruses are repaired by genetic recombination also suggests
that a similar mechanism may exist in human cells, made
possible by the diploid nature of these cells.
ACKNOWLEDGMENTS
The authors thank Dr. D. W. Mount for many helpful discussions. R. E. Almy
for excellent technical assistance. Drs. P. Shaffer and D. Coen for the generous
gift of the herpes mutants, and Dr. P. Henson for kindly providing the TC-7 cells.
REFERENCES
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exposed to near ultraviolet light in the presence of 4,5',8-trimethylpsoralen.
Biochim. Biophys. Acta. 33Õ. 181-193, 1973.
2. Benyesh-Melnick,
M., Schaffer, P. A., Courtney, R. J., Esparza, J., and
Kimura, S. Viral gene functions expressed and detected by temperaturesensitive mutants of herpes simplex virus. Cold Spring Harbor Symp. Quant.
Biol.,34. 731-746, 1974.
3. Chartrand. P., Crumpacker, C. S., Schaffer, P. A., and Wilkie, N. M. Physical
and genetic analysis of the herpes simplex virus DNA polymerase locus.
Virology, 103: 311-326, 1980.
4. Cole, R. S. Psoralen monoadducts and interstrand cross-links in DNA.
Biochim. Biophys. Acta, 254. 30-39, 1971.
5. Cole, R. S. Inactivation of Escherichia coli. F' episomes at transfer, and
bacteriophage lambda by psoralen plus 360-nm light: significance of deoxyribonucleic acid cross-links. J. Bacteriol., »07:846-852. 1971.
6. Cole, R. S. Repair of DNA containing ¡nterstrand crosslinks in Escherichia
coli: sequential excision and recombination. Proc. Nati. Acad. Sei. U. S. A.,
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CANCER
RESEARCH
VOL.
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41
Repair of Psoralen-treated DNA by Genetic Recombination in
Human Cells Infected with Herpes Simplex Virus
Jennifer D. Hall and Karen Scherer
Cancer Res 1981;41:5033-5038.
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