401
Mutation Research, 62 (1979) 401--405 © Elsevier/North-Holland Biomedical Press
HOST-CELL REACTIVATION OF UV-IRRADIATED A D E N O V I R U S IN COCKAYNE SYNDROME
D.I. HOAR and F. DAVIS Department of Medical Genetics, University of Toronto, Toronto, Ont. M5S 1A8 (Canada) (Received 30 May 1979) (Accepted 14 June 1979)
Summary Measurements of the host-cell reactivation (HCR) of mutagen-treated virus provides a very sensitive tool for detecting abnormal DNA repair. The best example of the utility of HCR studies in the examination of the DNA-repair capacity of human cells has come from studies of cells from the UV-sensitive repair-deficient xeroderma pigmentosum (XP) patients. We have examined the HCR of UV-treated adenovirus t y p e 5 (Ad5) and type 2 (Ad2) in cells from patients with Cockayne syndrome (CS), another sunsensitive syndrome whose cells also exhibits UV-sensitivity in culture. Comparisons with obligate heterozygotes and normal controls failed to reveal an abnormality in the HCR capacity of the CS cells. As the abnormality in DNA metabolism in CS appears to be in a late step in excision repair, a bypass mechanism may exist in these cells for circumventing the defect in the repair of viral DNA.
In microbial and human somatic cells, host-cell reactivation (HCR) of mutagen-treated virus has proven to be a sensitive method for revealing abnormalities in host DNA-repair functions. The utilization of a host UV-excision-repair system by infecting UV-irradiated virus can be readily demonstrated when an excision-deficient host is employed. Thus the classical xeroderma pigmentosum (XP) patients' cells that are deficient in UV excision repair [ 5,12] are also deficient in the HCR of Adenovirus 2(Ad2) [6] Herpes Simplex Virus (HSV) type 1 [9] or SV40 DNA [1]. These findings suggest that Ad2, SV40, and HSV utilize at least some of the same repair functions as the host. This deficiency in HCR closely parallels the sensitivity of the cultured XP cells to mutagens causing damage repaired by the "long patch repair" system [11]. Cockayne Syndrome (CS) is a rare recessive genetic disorder with some characteristics similar to XP [3]. Of concern here is the observation that CS cells in culture exhibit increased UV-light sensitivity [2,4,13] and appear to have a
402 defect in DNA repair [8]. As defective repair would be predicted to decreased HCR, these studies were undertaken to examine whether or not the cellular UV-sensitivity might alter the HCR of Ad5 in CS. As a normal HCR response was observed, it would appear that the defect in DNA repair present in CS is not required or can be bypassed in the HCR of UV-irradiated adenovirus type 5. Materials and methods Cell strains and virus stocks The CS and CS heterozygous parent strains used in these studies are presented in Table 1. The cell strain XP7BE was obtained from Dr. M. Paterson. The CS cell strains are 2--3-fold more UV-sensitive than controls while the CS heterozygotes show a UV-sensitivity identical to our control cell strains. The control cell strains C57CTO and C192CTO as well as the cell-culture conditions and medium have been previously described [8]. Adenovirus type 5 ( A d 5 ) w a s obtained from Dr. D. Spandidos and the adenovirus type 2 from Dr. A. Rainbow. Virus lysates were prepared by freeze-thawing cultures of BHK21 cells 7--10 days post-infection at low multiplicity. The cellular debris was removed by centrifugation and the cleared lysates stored at 4 ° C. For higher titre lysates, infected cells were harvested and concentrated before disruption by a combination of freeze-thawing and sonnication. These lysates were then centrifuged to equilibrium in a CsC1 gradient according to the m e t h o d of Girvitz and Rainbow [71. Plaque assay Freshly confluent monolayers in 60-mm petri dishes (Lux, contur) were washed once with PBS [8] and 0.2 ml of each virus dilution (diluted in PBS) was placed in the centre of 3--6 monolayers. Following 2 h for adsorption at room temperature with frequent rocking to distribute the virus evenly, the plates were overlaid with 8 ml of s-medium [15] containing 1.0% agar noble (Difco), 5% fetal calf serum (FCS) and 25 mM MgC12. 5 days later a second identical overlay of 8 ml was made. After 10--14 days, plaques were visualized TABLE 1 SKIN FIBROBLAST CELL STRAINS Ceil s t r a i n a
Sex
Age (yr) b
Genotype
Relationship
CSH 3 WI CS 10 WI CS 2 7 8 C T O c CS 6 1 8 C T O CSH 6 9 5 C T O CS 6 9 8 C T O
F M M M F F
32 4 24 4 45 8
Heterozygote Cockayne Cockayne Cockayne Heterozygote Cockayne
p a r e n t o f CSIOWI b r o t h e r of 6 9 8 m o t h e r of 278 a n d 6 9 8 sister of 278
a Cell strain n o m e n c l a t u r e : CS, C o c k a y n e S y n d r o m e ; CSH, CS h e t e r o z y g o t e ; WI, Winnipeg, M a n i t o b a Canada; CTO, Canada Toronto Ontario. b A g e at t i m e o f b i o p s y . c This strain n o w available f r o m T h e H u m a n G e n e t i c M u t a n t Cell R e p o s i t o r y , C a m d e n NJ as GM 2 8 3 8 .
403
~\
Z
....%;
II
°t'::~ \
Y:
X.
•
10 ; ' ~
i 10sk
2 u v Dose JM ~ × 10 ~
Fig. 1. HCR. o f A d 5 virus in CS (5 E x p t s . ) star, X P T B E (1 E x p t . ) . t h e a v e r a g e f o r all CSH. T h e i n s e r t and the a v e r a g e of all CS a n d CSH
5
10
U V D o s e J M 2 x 10 2
a n d CSH. O p e n s y m b o l s CSH (4 E x p t s . ) , closed s y m b o l s CS p a t i e n t s T h e d a s h e d line is t h e a v e r a g e survival f o r all CS a n d the d o t t e d line is p r o v i d e s a c o m p a r i s o n b e t w e e n t h e H C R d e f e c t i v e strain X P 7 B E (stars) strains ( o p e n circles).
Fig. 2. C o m p a r i s o n of H C R o f A d 5 a n d A d 2 in CS a n d CSH. Solid triangles, CSH6915CTO a n d A d 2 ; solid circles, C S 2 7 8 C T O a n d A d 2 ; o p e n triangles a n d e r r o r bars, a v e r a g e + s t a n d a r d e r r o r o f t h e m e a n f o r all CSH w i t h A d 5 ; o p e n circles a n d ' e r r o r b a r s , a v e r a g e ± s t a n d a r d e r r o r of t h e m e a n f o r all CS E x p t s . w i t h Ad5.
by first fixing plates for 2--16 h by flooding the plates with 4% formaldehyde in PBS, removing the agar carefully, and finally staining with 0.8% methylene blue in 70% isopropanol. All counting was performed using a dissecting microscope at 8X magnification. Virus irradiation
For routine HCR studies virus lysates of approx. 107 plaque-forming units/ ml (pfu) (assayed on human diploid fibroblast) were diluted 1/100 in PBS and UV irradiated in 2-ml lots in 60-mm petri dishes at room temperature. Diluted lysates were rocked constantly at room temperature during irradiation at a dose rate of 10 Jm -2 sec -1 (measured with a Black Ray meter model J225; Ultra Violet products, San Gabriel, CA), by a source consisting of 2 G.E. G4T4/1 germicidal tubes. Immediately following irradiation, lysates were diluted and plated as indicated above under subdued lighting. With the cell strain XP7BE it was necessary to start with lysates containing in excess of 10 l° pfu/ml to recover plaques at higher UV doses. Lysates diluted identically were ~-irradiated by placing plastic tubes in ice and exposing them to a 137Cs source. The dose rate was 104 rad/min.
404
TABLE 2 ~-RAY INACTIVATION Cell s t r a i n
Relative survival a
CS10 Wl CS61SCT0 CS69SCTO CSH3Wl CSH695CTO
0.21 0.22 b 0.27 0.19 0.27
a Survival a f t e r 3 k r a d o f "y-radiation r e l a t i v e to u n i r r a d i a t e d . b Average of 2 Expts.
Results and discussion Studies on UV-sensitivity of CS patients and heterozygotes have shown that although CS patients are 2--3-fold more UV-sensitive, their heterozygous parents have in all respects a normal UV-sensitivity and do not show any manifestations of disease [8]. The data presented in Fig. I indicates no significant difference in the HCR of Ad5 in CS and heterozygous parent cells. In 5 independent experiments, we have found 4 unrelated CS patients and 3 unrelated heterozygotes to show normal HCR when compared to 2 normal control cell strains. The control values are not plotted for the sake of clarity. The cell strain XP7BE from complementation group A was included as a HCR deficient control and clearly demonstrates decreased HCR (Fig. 1 insert). As the majority of published HCR studies have been undertaken with Ad2, we conducted one experiment with Ad2 that included 2 CS patients, one heterozygous parent and one control. Fig. 2 shows the data obtained for this experim e n t with Ad2 and also includes the average values + the standard error of the mean of all Ad5 experiments. The results presented here for Ad2 are directly comparable to those published previously by Day [6] and as the CS heterozygote and CS patient data are nearly identical to published normal HCR values, we feel it is justified to conclude that CS shows a normal UV HCR response. As 7-ray HCR deficiency has been reported in cells from patients with another premature aging syndrome, i.e. Progeria [ 10], we performed two HCR experiments where the Ad5 virus had received 3000 rad of 7-rays. In Table 2 the relative survival values are given for 3 CS patients (one repeated twice) and two heterozygotes. The survivals of the patients bracket those obtained for the two heterozygotes suggesting the 7-HCR response is also normal. In light of the observation that defective excision repair usually results in a reduction in HCR, these findings with CS are curious. We have observed an abnormality in DNA repair in CS that can best be interpreted as a defect in the rate of the final closure (ligation) of excision-repair patches [8]. As mammalian cells contain 2 DNA ligase activities [14] it is possible that the lesion leading to UV-sensitivity in CS is repaired adequately in the infecting viral genome by the second enzyme. If this were the case, a decreased HCR response might n o t be expected.
Acknowledgements This work was supported by MRC of Canada grants MA4998 and MT4734.
405
References 1 A b r a h a m s , P.J., and A.J. v a n d e r Eb, Host-cell r e a c t i v a t i o n of u l t r a v i o l e t - i r r a d i a t e d SV 4 0 D N A in five c o m p l e m e n t a t i o n g r o u p s of x e r o d e r m a p i g m e n t o s u m , M u t a t i o n Res., 35 ( 1 9 7 6 ) 1 3 - - 2 2 . 2 A n d r e w s , A . D . , F.W. Y o d c r , S.F. B a r r e t t and J.H. R o b b i n s , C o c k a y n e ' s S y n d r o m e fibroblasts have d e c r e a s e d c o l o n y f o r m i n g ability b u t n o r m a l r a t e s of u n s c h e d u l e d D N A s y n t h e s i s a f t e r u l t r a v i o l e t i r r a d i a t i o n , Clin, Res., 24 ( 1 9 7 6 ) 6 2 4 A . 3 B e r g s m a , D., Birth D e f e c t s Atlas a n d C o m p e n d i u m , Williams a n d Wilkins, B a l t i m o r e , 1 9 7 3 . 4 Chu, E . H . Y . , R.D. S c h m i c k e l , M.H. W a d e , C.C. C h a n g a n d I . E . T r o s k o , U l t r a v i o l e t light sensitivity and d e f e c t in D N A r e p a i r in f i b r o b l a s t s d e r i v e d f r o m t w o p a t i e n t s witb C o c k a y n e s y n d r o m e , A m . J. H u m . G e n e t . , 27 ( 1 9 7 5 ) 2 6 A . 5 Cleaver, I . E . , D N A r e p a i r a n d r a d i a t i o n sensitivity in h u m a n [ x e r o d e r m a p i g m e n t o s u m ] cells, Int. J. R a d i a t . Biol., 18 ( 1 9 7 0 ) 5 5 7 - - 5 6 5 . 6 D a y III, R.S., Studies o n the r e p a i r o f A d e n o v i r u s 2 b y h u m a n fibroblasts using n o r m a l x e r o d e r m a p i g m e n t o s u m a n d x e r n d e r m a p i g m e n t o s u m h e t e r o z y g o u s strains, C a n c e r Res., 34 ( 1 9 7 4 ) 1 9 6 5 - - 1 9 7 0 . 7 Girvitz, S.C., an,J A.J. R a i n b o w , U l t r a v i o l e t t r a n s c r i p t i o n a l u n i t m a p p i n g for the late genes in A d e n o virus T y p e 2, V i r o l o g y , 84 ( 1 9 7 8 ) 7 5 - - 8 6 . 8 H o a r , D.I., and C. W a g h o r n e , D N A r e p a i r in C o c k a y n e S y n d r o m e , A m . J. H u m . G e n e t . , 30 ( 1 9 7 8 ) 590--601. 9 L y t l e , C.D., R.S. D a y IIl, K.B. H e l l m a n a n d L.E. B o c k s t a h l e r , I n f e c t i o n o f U V - i r r a d i a t e d x e r o d e r m a p i g m e n t o s u m f i b r o b l a s t s b y H e r p e s s i m p l e x vii'us: s t u d y of c a p a c i t y a n d Weigle r e a c t i v a t i o n , M u t a t i o n Res., 36 ( 1 9 7 6 ) 2 5 7 - - 2 6 4 . 10 R a i n b o w , A.J., a n d M. H o w e s , D e c r e a s e d repair o f g a m m a ray d a m a g e d D N A in Progeria, B i o e h e m . Biophys. Res. C o m m u n . , 74 ( 1 9 7 7 ) 7 1 4 - - 7 1 9 . 11 R e g a n , I . D . , and R.B. S e t l o w , T w o f o r m s of r e p a i r in the D N A of h u m a n cells d a m a g e d b y c h e m i c a l c a r c i n o g e n s a n d m u t a g e n s , C a n c e r Res., 34 ( 1 9 7 4 ) 3 3 1 8 - - 3 3 2 5 . 12 R o b b i n s , J . H . , K.H. K r a e m e r , M.A. L u t z n e r , B.W. F e s t o f f a n d H.G. C o o n , A n i n h e r i t e d disease w i t h sun sensitivity m u l t i p l e c u t a n e o u s n e o p l a s m s a n d a b n o r m a l D N A repair, A n n . I n t e r n . Med., 80 ( 1 9 7 4 ) 221--248. 13 S c h m i c k e l , R.D., Chu E h y , I . E . T r o s k o a n d C.C. C h a n g , C o c k a y n e s y n d r o m e : a cellular sensitivity to u l t r a v i o l e t light, Pediatrics, 60 ( 1 9 7 7 ) 1 3 5 - - 1 3 9 . 14 S o d e r h a l l , S., a n d T. L i n d a h l , M a m m a l i a n D N A ligases, serological e v i d e n c e for t w o s e p a r a t e e n z y m e s , J. Biol. C h e m . , 2 5 0 ( 1 9 7 5 ) 8 4 3 8 - - 8 4 4 4 . 15 S t a n n e r s , C.P., G.L. Eliceiri and H. G r e e n , T w o t y p e s of r i b o s o m e s in m o u s e - h a m s t e r h y b r i d cells, Nature (London) 230 (1971) 52--54.
Mutation Research, 62 (1979) 401--405 © Elsevier/North-Holland Biomedical Press
HOST-CELL REACTIVATION OF UV-IRRADIATED A D E N O V I R U S IN COCKAYNE SYNDROME
D.I. HOAR and F. DAVIS Department of Medical Genetics, University of Toronto, Toronto, Ont. M5S 1A8 (Canada) (Received 30 May 1979) (Accepted 14 June 1979)
Summary Measurements of the host-cell reactivation (HCR) of mutagen-treated virus provides a very sensitive tool for detecting abnormal DNA repair. The best example of the utility of HCR studies in the examination of the DNA-repair capacity of human cells has come from studies of cells from the UV-sensitive repair-deficient xeroderma pigmentosum (XP) patients. We have examined the HCR of UV-treated adenovirus t y p e 5 (Ad5) and type 2 (Ad2) in cells from patients with Cockayne syndrome (CS), another sunsensitive syndrome whose cells also exhibits UV-sensitivity in culture. Comparisons with obligate heterozygotes and normal controls failed to reveal an abnormality in the HCR capacity of the CS cells. As the abnormality in DNA metabolism in CS appears to be in a late step in excision repair, a bypass mechanism may exist in these cells for circumventing the defect in the repair of viral DNA.
In microbial and human somatic cells, host-cell reactivation (HCR) of mutagen-treated virus has proven to be a sensitive method for revealing abnormalities in host DNA-repair functions. The utilization of a host UV-excision-repair system by infecting UV-irradiated virus can be readily demonstrated when an excision-deficient host is employed. Thus the classical xeroderma pigmentosum (XP) patients' cells that are deficient in UV excision repair [ 5,12] are also deficient in the HCR of Adenovirus 2(Ad2) [6] Herpes Simplex Virus (HSV) type 1 [9] or SV40 DNA [1]. These findings suggest that Ad2, SV40, and HSV utilize at least some of the same repair functions as the host. This deficiency in HCR closely parallels the sensitivity of the cultured XP cells to mutagens causing damage repaired by the "long patch repair" system [11]. Cockayne Syndrome (CS) is a rare recessive genetic disorder with some characteristics similar to XP [3]. Of concern here is the observation that CS cells in culture exhibit increased UV-light sensitivity [2,4,13] and appear to have a
402 defect in DNA repair [8]. As defective repair would be predicted to decreased HCR, these studies were undertaken to examine whether or not the cellular UV-sensitivity might alter the HCR of Ad5 in CS. As a normal HCR response was observed, it would appear that the defect in DNA repair present in CS is not required or can be bypassed in the HCR of UV-irradiated adenovirus type 5. Materials and methods Cell strains and virus stocks The CS and CS heterozygous parent strains used in these studies are presented in Table 1. The cell strain XP7BE was obtained from Dr. M. Paterson. The CS cell strains are 2--3-fold more UV-sensitive than controls while the CS heterozygotes show a UV-sensitivity identical to our control cell strains. The control cell strains C57CTO and C192CTO as well as the cell-culture conditions and medium have been previously described [8]. Adenovirus type 5 ( A d 5 ) w a s obtained from Dr. D. Spandidos and the adenovirus type 2 from Dr. A. Rainbow. Virus lysates were prepared by freeze-thawing cultures of BHK21 cells 7--10 days post-infection at low multiplicity. The cellular debris was removed by centrifugation and the cleared lysates stored at 4 ° C. For higher titre lysates, infected cells were harvested and concentrated before disruption by a combination of freeze-thawing and sonnication. These lysates were then centrifuged to equilibrium in a CsC1 gradient according to the m e t h o d of Girvitz and Rainbow [71. Plaque assay Freshly confluent monolayers in 60-mm petri dishes (Lux, contur) were washed once with PBS [8] and 0.2 ml of each virus dilution (diluted in PBS) was placed in the centre of 3--6 monolayers. Following 2 h for adsorption at room temperature with frequent rocking to distribute the virus evenly, the plates were overlaid with 8 ml of s-medium [15] containing 1.0% agar noble (Difco), 5% fetal calf serum (FCS) and 25 mM MgC12. 5 days later a second identical overlay of 8 ml was made. After 10--14 days, plaques were visualized TABLE 1 SKIN FIBROBLAST CELL STRAINS Ceil s t r a i n a
Sex
Age (yr) b
Genotype
Relationship
CSH 3 WI CS 10 WI CS 2 7 8 C T O c CS 6 1 8 C T O CSH 6 9 5 C T O CS 6 9 8 C T O
F M M M F F
32 4 24 4 45 8
Heterozygote Cockayne Cockayne Cockayne Heterozygote Cockayne
p a r e n t o f CSIOWI b r o t h e r of 6 9 8 m o t h e r of 278 a n d 6 9 8 sister of 278
a Cell strain n o m e n c l a t u r e : CS, C o c k a y n e S y n d r o m e ; CSH, CS h e t e r o z y g o t e ; WI, Winnipeg, M a n i t o b a Canada; CTO, Canada Toronto Ontario. b A g e at t i m e o f b i o p s y . c This strain n o w available f r o m T h e H u m a n G e n e t i c M u t a n t Cell R e p o s i t o r y , C a m d e n NJ as GM 2 8 3 8 .
403
~\
Z
....%;
II
°t'::~ \
Y:
X.
•
10 ; ' ~
i 10sk
2 u v Dose JM ~ × 10 ~
Fig. 1. HCR. o f A d 5 virus in CS (5 E x p t s . ) star, X P T B E (1 E x p t . ) . t h e a v e r a g e f o r all CSH. T h e i n s e r t and the a v e r a g e of all CS a n d CSH
5
10
U V D o s e J M 2 x 10 2
a n d CSH. O p e n s y m b o l s CSH (4 E x p t s . ) , closed s y m b o l s CS p a t i e n t s T h e d a s h e d line is t h e a v e r a g e survival f o r all CS a n d the d o t t e d line is p r o v i d e s a c o m p a r i s o n b e t w e e n t h e H C R d e f e c t i v e strain X P 7 B E (stars) strains ( o p e n circles).
Fig. 2. C o m p a r i s o n of H C R o f A d 5 a n d A d 2 in CS a n d CSH. Solid triangles, CSH6915CTO a n d A d 2 ; solid circles, C S 2 7 8 C T O a n d A d 2 ; o p e n triangles a n d e r r o r bars, a v e r a g e + s t a n d a r d e r r o r o f t h e m e a n f o r all CSH w i t h A d 5 ; o p e n circles a n d ' e r r o r b a r s , a v e r a g e ± s t a n d a r d e r r o r of t h e m e a n f o r all CS E x p t s . w i t h Ad5.
by first fixing plates for 2--16 h by flooding the plates with 4% formaldehyde in PBS, removing the agar carefully, and finally staining with 0.8% methylene blue in 70% isopropanol. All counting was performed using a dissecting microscope at 8X magnification. Virus irradiation
For routine HCR studies virus lysates of approx. 107 plaque-forming units/ ml (pfu) (assayed on human diploid fibroblast) were diluted 1/100 in PBS and UV irradiated in 2-ml lots in 60-mm petri dishes at room temperature. Diluted lysates were rocked constantly at room temperature during irradiation at a dose rate of 10 Jm -2 sec -1 (measured with a Black Ray meter model J225; Ultra Violet products, San Gabriel, CA), by a source consisting of 2 G.E. G4T4/1 germicidal tubes. Immediately following irradiation, lysates were diluted and plated as indicated above under subdued lighting. With the cell strain XP7BE it was necessary to start with lysates containing in excess of 10 l° pfu/ml to recover plaques at higher UV doses. Lysates diluted identically were ~-irradiated by placing plastic tubes in ice and exposing them to a 137Cs source. The dose rate was 104 rad/min.
404
TABLE 2 ~-RAY INACTIVATION Cell s t r a i n
Relative survival a
CS10 Wl CS61SCT0 CS69SCTO CSH3Wl CSH695CTO
0.21 0.22 b 0.27 0.19 0.27
a Survival a f t e r 3 k r a d o f "y-radiation r e l a t i v e to u n i r r a d i a t e d . b Average of 2 Expts.
Results and discussion Studies on UV-sensitivity of CS patients and heterozygotes have shown that although CS patients are 2--3-fold more UV-sensitive, their heterozygous parents have in all respects a normal UV-sensitivity and do not show any manifestations of disease [8]. The data presented in Fig. I indicates no significant difference in the HCR of Ad5 in CS and heterozygous parent cells. In 5 independent experiments, we have found 4 unrelated CS patients and 3 unrelated heterozygotes to show normal HCR when compared to 2 normal control cell strains. The control values are not plotted for the sake of clarity. The cell strain XP7BE from complementation group A was included as a HCR deficient control and clearly demonstrates decreased HCR (Fig. 1 insert). As the majority of published HCR studies have been undertaken with Ad2, we conducted one experiment with Ad2 that included 2 CS patients, one heterozygous parent and one control. Fig. 2 shows the data obtained for this experim e n t with Ad2 and also includes the average values + the standard error of the mean of all Ad5 experiments. The results presented here for Ad2 are directly comparable to those published previously by Day [6] and as the CS heterozygote and CS patient data are nearly identical to published normal HCR values, we feel it is justified to conclude that CS shows a normal UV HCR response. As 7-ray HCR deficiency has been reported in cells from patients with another premature aging syndrome, i.e. Progeria [ 10], we performed two HCR experiments where the Ad5 virus had received 3000 rad of 7-rays. In Table 2 the relative survival values are given for 3 CS patients (one repeated twice) and two heterozygotes. The survivals of the patients bracket those obtained for the two heterozygotes suggesting the 7-HCR response is also normal. In light of the observation that defective excision repair usually results in a reduction in HCR, these findings with CS are curious. We have observed an abnormality in DNA repair in CS that can best be interpreted as a defect in the rate of the final closure (ligation) of excision-repair patches [8]. As mammalian cells contain 2 DNA ligase activities [14] it is possible that the lesion leading to UV-sensitivity in CS is repaired adequately in the infecting viral genome by the second enzyme. If this were the case, a decreased HCR response might n o t be expected.
Acknowledgements This work was supported by MRC of Canada grants MA4998 and MT4734.
405
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