301
Mutation Research, 52 (1978) 301--311 © Elsevier/North-Holland Biomedical Press
P E R T U R B A T I O N S IN SIMIAN VIRUS 40 DNA SYNTHESIS BY U L T R A V I O L E T LIGHT *
JON I. WILLIAMS and JAMES E. CLEAVER
Laboratory of Radiobiology, University of California, San Francisco, Calif. (U.S.A.) (Received 27 January 1978) (Revision received 26 June 1978) (Accepted 3 August 1978) Summary
Perturbations of Simian Virus 40 (SV40) DNA replication by ultraviolet (UV) light during the lytic cycle in permissive m o n k e y CV-1 cells resemble those seen in host cell DNA replication. Formation of Form I DNA molecules (i.e., completion of SV40 DNA synthesis) was more sensitive to UV irradiation than synthesis of replicative intermediates or Form II molecules, consistent with inhibition of DNA chain elongation. The observed amounts of [3H]thymidine incorporated in UV-irradiated molecules could be predicted on the assumption that pyrimidine dimers are responsible for blocking nascent DNA strand growth. The relative proportion of labeled Form I molecules in UV-irradiated cultures rapidly increased to near-control values with incubation after 20 or 40 J/m 2 of light (0.9--1.0 or 1.8--2.0 dimers per SV40 genome, respectively). This rapid increase and the failure of Form II molecules to •accumulate suggest that SV40 growing forks can rapidly bypass many dimers. Form II molecules formed after UV irradiation were not converted to linear (Form III) molecules by the dimer-specific T4 endonuclease V, suggesting either that there are no gaps opposite dimers in these molecules or that T4 endonuclease V cannot use Form II molecules as substrates.
Introduction
Physical and chemical agents that damage DNA also suppress normal semiconservative DNA synthesis [26] and may lead to mutagenesis and/or oncogenesis [32]. Ultraviolet (UV) light is one such agent that retards nascent DNA chain growth [9] and induces a high level of mutations [22]. The biochemical events that lead to mutations in mammalian cells after UV irradiation are poorly understood. Recent studies with the DNA t u m o r virus Simian Virus 40 (SV40) have utilized host-cell reactivation of UV-damaged virus as an * Supported by the U.S. Department of Energy.
302
/
FORM I
REPLICATIVE INTERMEDIATE FORMI I (Rl)
FORM 1
Fig. 1. R e p l i c a t i v e c y c l e of S V 4 0 D N A . S u p e r c o i l e d F o r m I m o l e c u l e s u n w i n d a n d b e g i n r e p l i c a t i o n of two daughter molecules. The replicative intermediates have partial supercoiled character. The c o m p l e t e d d a u g h t e r m o l e c u l e s ( F o r m II m o l e c u l e s ) c o n t a i n t r a n s i e n t s t r a n d b r e a k s t h a t are sealed to yield F o r m I m o l e c u l e s . P r o d u c t F o r m I m o l e c u l e s m a y r e e n t e r t h e r e p l i c a t i n g p o o l at l a t e r t i m e s .
analogue to cell survival curves in mammalian cells. The biochemical basis of host-cell reactivation is also poorly understood, although involvement of the host-cell DNA-repair systems is suspected [2]. Intracellular SV40 DNA molecules have been regarded as models of small mammalian cell replicons [10,11,13]. Several replicative forms of SV40 that represent a replicative cycle (Fig. 1) can be resolved by agarose gel electrophoresis. The mature, supercoiled SV40 Form I DNA molecules constantly enter and leave a replicating pool during viral DNA synthesis [1,21,23,30,33]. We have investigated the effects of UV irradiation on SV40 DNA synthesis during the period of maximum viral DNA synthesis to determine what perturbations occur in SV40 DNA replication and h o w the virus-host cell system responds to these perturbations. Materials and methods African green m o n k e y kidney CV-1 cells were donated by Dr. Joanne Leong (Dept. of Biochemistry, University of California, San Francisco) and grown in plastic Petri dishes (Falcon} in modified Eagle's medium (MEM, GIBCO, Grand Island, N.Y.) supplemented with 15% (v/v) fetal calf serum, 2 × 10-3M glutamine, penicillin and streptomycin (each 80 unit/ml) (GIBCO). SV40 viral stocks were prepared from a wild-type SV40 sample designated SV40-1 [7]. EcoR1 restriction enzyme (60 U/ml) was obtained from New England Biolabs (Beverly, Maine). Propidium iodide was bought from Calbiochem (La Jolla, Calif.). T4 endonuclease V, polyethylene glycol (PEG) fraction 2 [12], was a gift of Dr. E.C. Friedberg, Department of Pathology, Stanford University Medical Center, Stanford, Calif.
Preparation o f viral stocks and infection conditions SV40 stocks were prepared in confluent roller bottles of CV-1 cells as described by Cleaver and Weil [7]. The maintenance medium (MEM with 2% fetal calf serum) was changed every 3--4 days until more than 90% of all cells showed cytopathic effects. The medium was harvested, freeze-thawed three
303 times, sonicated for 15 sec (Heat Systems Ultrasonic) and centrifuged at 700 g for 5 min at 20 ° C. The supernatant containing 1--2 X 108 plaque-forming units (PFU)/ml, frozen in 50-ml volumes, was used as a crude viral stock without further purification and was stable for several weeks. Confluent CV-1 cultures were infected by removal of growth medium, addition of 0.20 ml of viral stock (5--10 PFU/cell) and incubation for 1 h at 37°C. Growth medium was then added and incubation continued at 37°C under a humidified 5% CO: atmosphere. Control cultures were mock-infected with 0.20 ml of saline A.
Irradia tio n Growth medium was removed from SV40-infected cultures and the cells were washed twice in Dulbecco's phosphate-buffered saline (PBS). A thin layer of PBS was left on the cells during irradiation. Cultures were irradiated with 254 nm UV light at an incident dose rate of 1.25 J/m2/sec, measured with a YSI-Kettering No. 65 radiometer. Labeling procedures Infected cultures were grown in [14C]thymidine (0.01 or 0.1 pCi/ml, 55 mCi/mmole) for 24 h after infection for uniform labeling of DNA with 14C. The 14C-labeling medium was then replaced with unlabeled medium and incubation continued for at least 16 h for depletion of the intracellular pools of exogenous [14C]thymidine. Mock-infected cultures were handled in an identical fashion. Cultures were then washed twice in PBS, irradiated with UV light, and labeled with [3H]thymidine (10.0 pCi/ml, 11 Ci/mmole) for 5 to 20 rain at 37°C. SV40 DNA was isolated either immediately or after growing for 1 or 3 h in MEM containing 5 × 10 -s M thymidine and 5 × 10 -s M deoxycytidine. SV40 DNA isolation and purification SV40 DNA was isolated from cell cultures by the m e t h o d of Hirt [15]. Hirt supernatants were dialyzed overnight against 40 volumes of T4 buffer (2 × 10 -2 M Tris--HC1, 10 -2 M Na2 EDTA, pH 7.78) and analyzed in 1.0% agarose tube gels by gel electrophoresis. Form II DNA was partially purified from some cultures by cesium chloride-propidium iodide (CsC1--PI2) equilibrium density centrifugation [17] for experiments with bacteriophage T4 endonuclease V. Agarose tube gel electrophoresis [1,33,34] Agarose tube gels were prepared as described by Tegtmeyer and Macasaet [33]. Gels were prerun at constant voltage (8--10 V/tube) for 30 min in gel buffer (3.6 × 10 -2 M Tris, 3 × 10-: M NaH:PO4, 10 -3 M Na2 EDTA, 0.2% sodium dodecyl sulfate [SDS], pH 7.60). A 100-pl DNA sample was mixed with an equal volume of tracking dye (0.001% bromphenol blue, 0.6% SDS, 30% [w/v] sucrose) and left for 5--15 min at 37°C before being added to the gels. The bromphenol blue marker moved at a rate of a b o u t 8 cm/h independent of gel concentration in the range 1.0--1.5%. Gels were cut in 3-mm slices after electrophoresis and individual slices placed in 10.0 ml of water miscible scintillation fluid (PCS, Amersham-Searle). The samples were left at room temperature for 2 days before counting in a Packard scintillation spectrometer.
304
EcoR1 restriction endonuclease treatment [25] SV40 DNA was dialyzed overnight against 500 volumes of EcoR1 buffer (10 -~ M Tris--HC1, 5 × 10 -2 M NaC1, 5 × 10 -3 M MgCl:, pH 7.50). An incubation mixture was prepared with 100 pl of dialyzed SV40 DNA plus 2.0 pl of E. coli t R N A (1 mg/ml) and 1.0 #l of EcoR1 enzyme (t>6000 unit/ml). The incubation mixture was incubated at 37°C for 1 h before the reaction was stopped by 25 pl of stopping solution (10 -1 M Tris--HC1, 10 -~ M Na2EDTA, pH 7.40) and 50 pl of tracking dye. Incubation mixtures were analyzed by 1.5% agarose tube gel electrophoresis. The position of Form III molecules was determined by comparison of DNA samples incubated with or without the EcoR1 enzyme. Cesium chloride--propidium iodide isopycnic gradient ultracen trifugation DNA samples in T4 buffer weighing 6.10 g were mixed with 5.75 g of CsC1 and 0.40 ml of PI2 (6 mg/ml) in a Beckman 50 Ti polyallomer centrifuge tube and centrifuged at 48 000 rpm for 41 h in a Beckman Ti rotor. The DNA bands were visualized in the dark by exposure to a bank of GE black lights and the upper DNA band (non-supercoiled DNA) was collected by pipette. Samples were extracted 4 times with isopropanol : H20 (9 : 1) for removal of PI2 and dialyzed overnight against 1 1 of T4 buffer before analysis with T4 endonuclease V. T4 endonuclease V assay [12] An incubation mixture was prepared with 25 pl of non-supercoiled SV40 DNA plus 100 pl of T4 buffer and either 5 pl of T4 endonuclease V (PEG fraction) or 5 pl of T4 buffer. The mixture was incubated for 60 min at 37°C before 50 pl of tracking dye was added and samples analyzed by 1.0% agarose tube gel electrophoresis. The activity of our T4 endonuclease V stock on double-stranded DNA was verified in parallel experiments on supercoiled SV40 DNA [38] and purified CV-1 DNA [39] from irradiated cultures; T4 endonuclease V recognizes dimers equally well in double-stranded or single-stranded DNA [24]. Results
SV40 DNA synthesis in control cultures The shortest pulse time used (5 min) gave a resolvable Form I peak in 1.0% agarose gel radioactivity profiles (Fig. 2) and incorporation proceeded linearly up to 20 min of labeling {Fig. 3), whereas Hirt supernatants from mockinfected cultures showed activity only in the top fraction even after 20 min of 3H-labeling (Fig. 2). Host-cell DNA was confined to the top fraction in all gels in the agarose concentration range 1.0--1.4%. Increasing the length of [3H]thymidine pulse increased total radioactivity in the broad replicative intermediate (RI) peak (R~ -~ 0.45--0.55 relative to Form I molecules) as well as the rapidly moving Form I peak (Fig. 2). A small peak of Form II DNA molecules electrophoresed slightly more slowly than Form I molecules (R~ = 0.75--0.85 relative to Form I molecules). These Form II molecules do not increase in number as rapidly as Form I molecules. The observed order of electrophoretic mobility
305
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Fig. 2. T r i t i u m r a d i o a c t i v i t y p r o f i l e s o f p u l s e - l a b e l e d S V 4 0 D N A i n 1 . 0 % a g a r o s e gels. T h e i d e n t i t y o f p e a k s a p p e a r i n g f o r t r i t i u m p u l s e s o f five ( 5 ' ) t o t w e n t y ( 2 0 ' ) r a i n w a s d e t e r m i n e d b y p u l s e - c h a s e e x p e r i ments. The radioactivity profile after a 20-min tritium pulse of the Hirt supernatant from a mock-infected CV-1 c u l t u r e (MI) y i e l d e d v e r y f e w c o u n t s in a p a r a l l e l gel. S o m e h o s t - c e l l D N A p e n e t r a t e d t h e first gel slice in all gels ( h c D N A ) a n d w a s n o t c o n s i d e r e d in e v a l u a t i o n o f d a t a . T o t a l 1 4 C p r e l a b e l c o u n t s in S V 4 0 DNA: 5', 1303 cpm; 10', 1554 cpm; 20', 1534 cpm. Fig. 3. E x t e n t o f [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n i n t o S V 4 0 D N A d u r i n g p u l s e - l a b e l i n g as a f u n c t i o n o f p u l s e l e n g t h . I n c o r p o r a t i o n is e x p r e s s e d as t h e 3 H / 1 4 C r a t i o t o c o r r e c t f o r v a r i a b l e size o f t h e S V 4 0 replic a t i n g p o o l in a given c u l t u r e . L i n e a r i n c o r p o r a t i o n w a s f o u n d w i t h i n 5 m i n o f i n i t i a t i o n o f p u l s e - l a b e l i n g .
(Form I > Form II/> RI) and Rf values agree with the work of previous investigators for 1.0% agarose gels [1,33,34]. The average increase in the 3H/14C ratio for labeled SV40 DNA summed over entire gel profiles was 13(+10)% after 60 min of post-tritium incubation ("chase") and 14(+22)% after 180 min of chase (Table 1). Thus the use of unlabeled thymidine and deoxycytidine in the chase medium efficiently terminated incorporation of [3H]thymidine into SV40 DNA daughter strands. The mean time for replication of an SV40 DNA molecule equals the time for the percentage of total tritium label in RI molecules to decrease to 50% during continuous labeling [23]. Table 1 summarizes the 3H/14C ratios observed in RI, Form II, and Form I molecules as well as the percentage of total 3H incorporated in these SV40 replicating forms as a function of time. The percentage of total label in RI molecules decreased to 50% in a b o u t 15 min (Table 1), indicating that this is the time for replication of the SV40 genome. S V 4 0 D N A synthesis in UV-irradiated cultures Exposure of SV40-infected cultures to UV light before pulse-labeling altered agarose tube gel radioactivity profiles (Fig. 4). The amount of [3H]thymidine in total SV40 DNA was suppressed to 61% of control at 20 J/m 2 and to 49% of control at 40 J / m 2 (Table 1); at these doses 1 and 2 dimers are induced per SV40 genome, respectively. The a m o u n t of label in Form I molecules was
TABLE 1
20 20 20
0 0 0
0 0 0
40 40 40
20 20 20
5 10 15
20 20 20
20 20 20
0 60 180
0 60 180
0 0 0
0 60 180
0 60 180
incubation after pulse labeling
Minutes of
57.7 ( 3 ) 4 9 . 4 (3) 2 0 . 6 (3)
69.6 (3) 3 1 . 4 (3) 4 0 . 1 (3)
27.3 (2) 68.3 (1) 82.4 (1)
218c(3) 160 c (3) 2 9 4 c (3)
963 c (3) 4 7 5 c (3) 3 1 1 e (3)
Replieative intermediate molecules a
10.2 12.4 5.8
7.2 9.7 10.6
1.9 2.6 2.9
103 167 102
74 73 154
Form II molecules
Mean 3H/14C ratios
AND PERCENTAGE
1.5 3.5 3.2
5.8 9.6 10.9
0.7 1.6 3.7
23 48 46
58 75 104 ± 7 ± 14 ± 10
± 12 ± 23 ± 49
5.2 ± 6.2 + 4.4 ±
9.8 ± 10.8 ± 12.3 ±
0.7 1.9 0.5
1.3 3.0 0.7
2.1 -+ 0 . 2 4.2 7.5
51 61 55
84 84 ~ 113
Total b
53 57 36 49 ( m e a n )
61 73 49 61 ( m e a n )
Percent of control ratio
58 35 25
43 16 16
63 61 54
47 21 14
35 15 10
Replicative intermediate molecules
P e r c e n t o f t o t a l 3 H label
3H LABEL FOR SV40 DNA WITH OR WITHOUT
Form I molecules
OF TOTAL
18 16 12
7 8 8
8 6 3
18 14 11
8 7 10
F o r m II molecules
UV IRRADIATION
BEFORE
24 49 63
50 76 76
29 33 43
35 65 75
57 78 80
Form I molecules
a N u m b e r of r e p l i c a t e c u l t u r e s in p a r e n t h e s e s . b E r r o r s are s t a n d a r d e r r o r s o f t h e m e a n a n d are r e p r e s e n t a t i v e o f t h e e r r o r s f o u n d f o r all v a l u e s in e a c h r o w . c M e a n 3 H / 1 4 C v a l u e s f o r e x p e r i m e n t a l p l a t e s l a b e l e d w i t h [ 14 C] t h y m i d i n e at a n a c t i v i t y o f a b o u t 0 . 0 1 ~ C i / m l ; all o t h e r p l a t e s w e r e l a b e l e d at 0.1 ~ C i / m l .
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Fig. 4. T r i t i u m r a d i o a c t i v i t y p r o f i l e s f o r S V 4 0 D N A p u l s e - l a b e l e d f o r 2 0 m i n and a n a l y z e d o n 1 . 0 % a g a r o s e t u b e gels b y e l e c t r o p h o r e s i s . E l e c t r o p h o r e s i s w a s f r o m l e f t t o r i g h t . A n a l y z e d S V 4 0 D N A r e c e i v e d ( A ) 0 J i m 2, (B) 2 0 J / m 2, a n d (C) 4 0 J / m 2 o f U V l i g h t b e f o r e i s o l a t i o n f r o m i n f e c t e d CV-1 cultures.
very sensitive to UV light exposure, whereas incorporation into RI and Form II molecules was less sensitive (Fig. 4). "Chasing" replicate cultures for 0, 1 or 3 h produced a rapid decline in RI molecules and a concurrent increase in Form I molecules (Table 1). The relative proportion and absolute amount of [3H]thymidine in Form II molecules declined little in 3 h (Table 1). The percentage of label entering Form II molecules relative to controls (Table 1) within 20 min after UV irradiation increased as expected if dimers define new termination sites and temporarily inhibit ring closure. However, Form II molecules did not accumulate in UV-irradiated SV40 DNA (Table 1), suggesting that ring closure occurs within 1 h of inhibition. A 10% increase in the relative proportion of label in RI after 3 h of chase was the only significant change in cultures exposed to 40 J/m 2 of UV light (Table 1). This suggests that few molecules are completely prevented from completing replication despite the presence of dimers ahead of the growing forks. Many dimers are therefore bypassed in a significantly shorter time than is required for actual dimer removal [38]. Because control cultures did n o t achieve a complete chase of label into Form I molecules, interpretation of the increase in RI label must take into account the reentrance of some labeled Form I molecules into the replicating pool [30,33]. This does n o t allow us to discriminate between (a) a small proportion of RI molecules absolutely blocked against further replication and (b) a proportion of RI molecules temporarily blocked and escaping blockage as some labeled Form I molecules reenter the replicating pool and are also temporarily blocked. Form H molecules carrying dimers The increased relative proportion of 3H-labeled Form II SV40 DNA molecules after UV exposure (Table 1) suggests that a significant fraction of these molecules may be blocked in the ligation-step replication. This block might
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Fig. 5. T r i t i u m r a d i o a c t i v i t y profiles for n o n - s u p e r c o i l e d S V 4 0 D N A isolated o n CsC1--PI 2 i s o p y c n i c g r a d i e n t s . S V 4 0 D N A w a s i s o l a t e d f r o m i n f e c t e d CV-1 c u l t u r e s e x p o s e d t o 0 J / m 2 (A, C) or 40 J / m 2 (B, D) of U V light, pulse-labeled 20 m i n w i t h [ 3 H ] t h y m i d i n e (10 # C i / m l , 11 C i / m m o l e ) , a n d e i t h e r e x t r a c t e d i m m e d i a t e l y (A, B) or i n c u b a t e d 60 rain in u n l a b e l e d m e d i u m b e f o r e e x t r a c t i o n (C, D). I s o l a t e d D N A w a s t r e a t e d 1 h w i t h T 4 b u f f e r (o) or a n excess o f T 4 e n d o n u c l e a s e V ( e ) b e f o r e analysis o n 1.5% agarose t u b e gels b y e l e c t r o p h o r e s i s . Positions o f R I , F o r m I a n d F o r m II m o l e c u l e s w e r e d e t e r m i n e d b y pulse a n d pulse-chase e x p e r i m e n t s ( n o t s h o w n ) . E l e c t r o p h o r e s i s was f r o m left to right.
be due to the presence of dimers opposite gaps. We tested an irradiated SV40 Form II DNA population for molecules carrying gaps opposite dimers by treating them with T4 endonuclease V and analyzing the DNA products for linearized (Form III) molecules. The position to which Form III molecules migrated was identified with SV40 DNA molecules treated with an excess of EcoR1 restriction endonuclease; the SV40 genome contains only one substrate site for this enzyme [25]. The T4 endonuclease failed to linearize a detectable fraction of Form II molecules made after UV irradiation (Fig. 5). In addition, the RI molecules that partially penetrated the gels were also unaffected by T4 endonuclease V treatment (Fig. 5). Discussion The major effect of UV irradiation on SV40 replication was to reduce the formation of mature Form I molecules. This may have occurred because UV p h o t o p r o d u c t s interfere with mammalian DNA-chain elongation [9]. At least three major events occur during SV40 replication, any of which could be affected by UV damage: (a) initiation of replication, (b) chain elongation and (c) termination with ligation to form supercoiled Form I molecules. Initiation was n o t photosensitive because the relative proportion of RI molecules did n o t decrease with increasing UV dose (Fig. 4, Table 1). Similarly, ligation was not photosensitive because the proportion of Form II molecules increased only slightly after irradiation and decreased with post-labeling incubation. Thus, DNA-chain elongation in SV40 molecules, as in mammalian DNA, appears to be retarded by UV photoproducts. If we assume that pyrimidine dimers are the lesions responsible for retard-
309 ing chain elongation, then it can be shown that the a m o u n t of [3H]thymidine incorporated into total SV40 DNA relative to controls should be approximately 0.77 at 20 J/m s and 0.57 at 40 J/m s . These values are similar to the experimental values of 0.61 and 0.49, respectively. The extent of inhibition of DNA replication is thus consistent with the hypothesis that dimers ahead of the growing fork retard chain growth. This size of newly made daughter DNA in replicating SV40 molecules after UV irradiation is also consistent with this hypothesis (A. Sarasin, unpublished observation). The pulse-chase experiments showed that DNA replication recovered in all RI molecules within 3 h of 20 J/m s UV light and in most RI molecules after 40 J/m s of UV light (Table 1). This process was shorter than the loss of T4 endonuclease V-sensitive sites in SV40 [38] and much shorter than dimer removal from mammalian cell DNA [5,39]. It is therefore likely that SV40 RI molecules overcome the UV-induced block to chain growth by bypassing dimers without removing them. The failure of RI or Form II molecules to accumulate during incubation after UV irradiation and pulse labeling suggests that daughter molecules with retarded growing forks replicate past most dimers within 60 min of irradiation. The likeliest mechanisms for such dimer bypass are recombination and "bypass replication." Molecular and genetic recombination have been detected at low levels in mammalian cells [16,20,27,36]. The recombination of multiple genetic markers in mixed infections with temperature-sensitive SV40 mutants has also been observed and is stimulated by UV irradiation of the virus before infection [8]. Such recombination may be induced by the presence of gaps at or near retarded growing forks; such gaps have been considered inducing events for production of infectious SV40 from transformed hamster cell lines [18,19,40, 41]. Recombination at retarded growing forks would lead to reassortment of dimers between parent and daughter DNA molecules and replication past the damage site. Dimers are detected in daughter molecules [ 37 ], b u t it is not clear whether they result from recombination or from elongation of daughter strands which initiated replication before UV irradiation and in which the dimers were produced directly by UV. An equally plausible mechanism has been suggested by recent observations of Radman et al. [28] that eukaryotic DNA polymerases can replicate past dimers in vitro with only a brief delay. Such "bypass replication" does n o t require recombination to circumvent dimers blocking replication. R u p p and Howard-Flanders [31] originally proposed that gaps are created in daughter DNA strands during replication past UV-induced damage sites. Attempts to detect gaps opposite dimers in mammalian DNA with purified dimer-specific endonucleases have been unsuccessful [4,24]. Two possible reasons for this lack of success are that (a) the structure of the substrate, consisting of doubleand single-stranded regions with a dimer opposite a gap in the vicinity of a growing fork, is refractory to endonucleolytic action by the T4 or M. luteus enzyme, or {b) the model is wrong and gaps are either never opposite dimers or are not opposite dimers long enough to be utilized as suitable enzyme substrates. A dimer-gap substrate in a Form II SV40 molecule offers a simpler conformation than that at an active mammalian cell replicon blocked by a dimer and might be cleaved by T4 endonuclease V. How-
310 ever, we found no evidence of nicking by T4 endonuclease V in DNA opposite the gap in Form II SV40 molecules, suggesting that a peculiar substrate conformation in the vicinity of a growing fork is not a sufficient explanation for the lack of enzyme action. We conclude that Clarkson and Hewitt's [4] modification of the Rupp and Howard-Flanders model best explains our data. Clarkson and Hewitt proposed that gaps are produced by gl'owing forks encountering dimers but that these gaps are filled before the next dimer is reached. This model is extended by the results of Radman et al. [28,29] to allow replication past the dimer as well as suppression of the 3'-+ 5' proofreading function recently detected in eukaryotic cells [29]. The biological sequelae of bypassing dimers are u n k n o w n b u t could involve replication errors that lead to mutagenic events. This explains both UV induction of mutations in SV40 [7] and the competition between SV40 DNA replication and repair which modifies observed levels of UV-induced mutagenesis [6]. Unexcised dimers could also cause functional changes such as early termination of transcription [3,14]. Inactivation of individual SV40 genomes due to low fluences of UV light may n o t cause observable changes in the SV40 infectious cycle b e y o n d delayed expression of plaque-forming ability [7] because only one surviving infectious particle is necessary to continue the lytic cycle. A complete infectious cycle yields about 100 infectious particles per cell [35], so one or more infectious virions per cell would be expected to be undamaged on the basis of Poi~son distributed dimers at UV fluences below 100 J/m 2 (4.6 pyrimidine dimers per SV40 genome) [38,39]. It remains to be shown what proportion of SV40 particles emerging from a UV-irradiated culture are infectious and/or mutated during a second lytic cycle. References 1 Aaij, C., a n d P. Borst, T h e gel e l e c t r o p h o r e s i s of D N A , B i o e h i m . B i o p h y s . A c t a , 269 ( 1 9 7 2 ) 1 9 2 - - 2 0 0 . 2 A b r a h a m s , P.J., a n d A.J. v a n d e r Eb, Host-ceU 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 S V 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 . 3 Berk, A.J., a n d P.A. S h a r p , U l t r a v i o l e t m a p p i n g of t h e a d e n o v i r u s 2 early p r o m o t e r s , Cell, 12 ( 1 9 7 7 ) 45--55. 4 Claxkson, J.M., a n d R . R . H e w i t t , Significance of d i m e r s t o t h e size of n e w l y s y n t h e s i z e d D N A in UVi r r a d i a t e d Chinese h a m s t e r o v a r y cells, B i o p h y s . J., 16 ( 1 9 7 6 ) 1 1 5 5 - - 1 1 6 4 . 5 Cleaver, J.E., R e p a i r processes for p h o t o c h e m i c a l d a m a g e in m a m m a l i a n cells, in: J.T. L e t t , H. A d l e r a n d M.R. Zelle (Eds.), A d v a n c e s in R a d i a t i o n Biology, Vol. 4, A c a d e m i c Press, N e w Y o r k , 1 9 7 4 , pp. 1--75. 6 Cleaver, J.E., Decline in m u t a t i o n f r e q u e n c y in t e m p e r a t u r e - s e n s i t i v e S V 4 0 viruses b e f o r e viral D N A s y n t h e s i s , M u t a t i o n Res., 44 ( 1 9 7 7 ) 2 9 1 - - 2 9 8 . 7 Cleaver, J . E . , a n d S. Well, U V - i n d u c e d r e v e r s i o n o f a t e m p e r a t u r e - s e n s i t i v e late m u t a n t of S i m i a n Virus 4 0 to a w i l d - t y p e p h e n o t y p e , J. Virol., 16 ( 1 9 7 5 ) 2 1 4 - - 2 1 6 . 8 D u b b s , D . R . , M. R a c h m e l e r a n d S. Kit, R e c o m b i n a t i o n b e t w e e n t e m p e r a t u r e - s e n s i t i v e m u t a n t s of S i m i a n virus 4 0 , V i r o l o g y , 57 ( 1 9 7 4 ) 1 6 1 - - 1 7 4 . 9 E d e n b e r g , H . J . , I n h i b i t i o n o f D N A r e p l i c a t i o n b y u l t r a v i o l e t light, Biophys. J., 16 ( 1 9 7 6 ) 8 4 9 - - 8 6 0 . 10 E d e n b e r g , H . J . , a n d J.A. H u b e r m a n , E u k a r y o t i c c h r o m o s o m e r e p l i c a t i o n , A n n . Rev. G e n e t . , 9 ( 1 9 7 5 ) 245--284. 11 F a r e e d , G.C., C.F. G a r o n a n d N.P. S a l z m a n , Origin a n d d i r e c t i o n o f S i m i a n Virus 4 0 d e o x y r i b o n u c l e i c acid r e p l i c a t i o n , J. Virol., 10 ( 1 9 7 2 ) 4 8 4 - - 4 9 1 . 12 F r i e d b e r g , E.C., a n d J.J. K i n g ; D a r k r e p a i r o f u l t r a v i o l e t - i r r a d i a t e d d e o x y r i b o n u c l e i c acid b y b a c t e r i o p h a g e T 4 : p u r i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f a d i m e r - s p e e i f i c p h a g e - i n d u c e d e n d o n u c l e a s e , J. Bacteriol., 106 ( 1 9 7 1 ) 5 0 0 - - 5 0 7 . 13 G r i f f i t h , J.D., C h r o m a t i n s t r u c t u r e : d e d u c e d f r o m a m i n i c h r o m o s o m e , Science, 187 ( 1 9 7 5 ) 1 2 0 2 - 1203.
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Mutation Research, 52 (1978) 301--311 © Elsevier/North-Holland Biomedical Press
P E R T U R B A T I O N S IN SIMIAN VIRUS 40 DNA SYNTHESIS BY U L T R A V I O L E T LIGHT *
JON I. WILLIAMS and JAMES E. CLEAVER
Laboratory of Radiobiology, University of California, San Francisco, Calif. (U.S.A.) (Received 27 January 1978) (Revision received 26 June 1978) (Accepted 3 August 1978) Summary
Perturbations of Simian Virus 40 (SV40) DNA replication by ultraviolet (UV) light during the lytic cycle in permissive m o n k e y CV-1 cells resemble those seen in host cell DNA replication. Formation of Form I DNA molecules (i.e., completion of SV40 DNA synthesis) was more sensitive to UV irradiation than synthesis of replicative intermediates or Form II molecules, consistent with inhibition of DNA chain elongation. The observed amounts of [3H]thymidine incorporated in UV-irradiated molecules could be predicted on the assumption that pyrimidine dimers are responsible for blocking nascent DNA strand growth. The relative proportion of labeled Form I molecules in UV-irradiated cultures rapidly increased to near-control values with incubation after 20 or 40 J/m 2 of light (0.9--1.0 or 1.8--2.0 dimers per SV40 genome, respectively). This rapid increase and the failure of Form II molecules to •accumulate suggest that SV40 growing forks can rapidly bypass many dimers. Form II molecules formed after UV irradiation were not converted to linear (Form III) molecules by the dimer-specific T4 endonuclease V, suggesting either that there are no gaps opposite dimers in these molecules or that T4 endonuclease V cannot use Form II molecules as substrates.
Introduction
Physical and chemical agents that damage DNA also suppress normal semiconservative DNA synthesis [26] and may lead to mutagenesis and/or oncogenesis [32]. Ultraviolet (UV) light is one such agent that retards nascent DNA chain growth [9] and induces a high level of mutations [22]. The biochemical events that lead to mutations in mammalian cells after UV irradiation are poorly understood. Recent studies with the DNA t u m o r virus Simian Virus 40 (SV40) have utilized host-cell reactivation of UV-damaged virus as an * Supported by the U.S. Department of Energy.
302
/
FORM I
REPLICATIVE INTERMEDIATE FORMI I (Rl)
FORM 1
Fig. 1. R e p l i c a t i v e c y c l e of S V 4 0 D N A . S u p e r c o i l e d F o r m I m o l e c u l e s u n w i n d a n d b e g i n r e p l i c a t i o n of two daughter molecules. The replicative intermediates have partial supercoiled character. The c o m p l e t e d d a u g h t e r m o l e c u l e s ( F o r m II m o l e c u l e s ) c o n t a i n t r a n s i e n t s t r a n d b r e a k s t h a t are sealed to yield F o r m I m o l e c u l e s . P r o d u c t F o r m I m o l e c u l e s m a y r e e n t e r t h e r e p l i c a t i n g p o o l at l a t e r t i m e s .
analogue to cell survival curves in mammalian cells. The biochemical basis of host-cell reactivation is also poorly understood, although involvement of the host-cell DNA-repair systems is suspected [2]. Intracellular SV40 DNA molecules have been regarded as models of small mammalian cell replicons [10,11,13]. Several replicative forms of SV40 that represent a replicative cycle (Fig. 1) can be resolved by agarose gel electrophoresis. The mature, supercoiled SV40 Form I DNA molecules constantly enter and leave a replicating pool during viral DNA synthesis [1,21,23,30,33]. We have investigated the effects of UV irradiation on SV40 DNA synthesis during the period of maximum viral DNA synthesis to determine what perturbations occur in SV40 DNA replication and h o w the virus-host cell system responds to these perturbations. Materials and methods African green m o n k e y kidney CV-1 cells were donated by Dr. Joanne Leong (Dept. of Biochemistry, University of California, San Francisco) and grown in plastic Petri dishes (Falcon} in modified Eagle's medium (MEM, GIBCO, Grand Island, N.Y.) supplemented with 15% (v/v) fetal calf serum, 2 × 10-3M glutamine, penicillin and streptomycin (each 80 unit/ml) (GIBCO). SV40 viral stocks were prepared from a wild-type SV40 sample designated SV40-1 [7]. EcoR1 restriction enzyme (60 U/ml) was obtained from New England Biolabs (Beverly, Maine). Propidium iodide was bought from Calbiochem (La Jolla, Calif.). T4 endonuclease V, polyethylene glycol (PEG) fraction 2 [12], was a gift of Dr. E.C. Friedberg, Department of Pathology, Stanford University Medical Center, Stanford, Calif.
Preparation o f viral stocks and infection conditions SV40 stocks were prepared in confluent roller bottles of CV-1 cells as described by Cleaver and Weil [7]. The maintenance medium (MEM with 2% fetal calf serum) was changed every 3--4 days until more than 90% of all cells showed cytopathic effects. The medium was harvested, freeze-thawed three
303 times, sonicated for 15 sec (Heat Systems Ultrasonic) and centrifuged at 700 g for 5 min at 20 ° C. The supernatant containing 1--2 X 108 plaque-forming units (PFU)/ml, frozen in 50-ml volumes, was used as a crude viral stock without further purification and was stable for several weeks. Confluent CV-1 cultures were infected by removal of growth medium, addition of 0.20 ml of viral stock (5--10 PFU/cell) and incubation for 1 h at 37°C. Growth medium was then added and incubation continued at 37°C under a humidified 5% CO: atmosphere. Control cultures were mock-infected with 0.20 ml of saline A.
Irradia tio n Growth medium was removed from SV40-infected cultures and the cells were washed twice in Dulbecco's phosphate-buffered saline (PBS). A thin layer of PBS was left on the cells during irradiation. Cultures were irradiated with 254 nm UV light at an incident dose rate of 1.25 J/m2/sec, measured with a YSI-Kettering No. 65 radiometer. Labeling procedures Infected cultures were grown in [14C]thymidine (0.01 or 0.1 pCi/ml, 55 mCi/mmole) for 24 h after infection for uniform labeling of DNA with 14C. The 14C-labeling medium was then replaced with unlabeled medium and incubation continued for at least 16 h for depletion of the intracellular pools of exogenous [14C]thymidine. Mock-infected cultures were handled in an identical fashion. Cultures were then washed twice in PBS, irradiated with UV light, and labeled with [3H]thymidine (10.0 pCi/ml, 11 Ci/mmole) for 5 to 20 rain at 37°C. SV40 DNA was isolated either immediately or after growing for 1 or 3 h in MEM containing 5 × 10 -s M thymidine and 5 × 10 -s M deoxycytidine. SV40 DNA isolation and purification SV40 DNA was isolated from cell cultures by the m e t h o d of Hirt [15]. Hirt supernatants were dialyzed overnight against 40 volumes of T4 buffer (2 × 10 -2 M Tris--HC1, 10 -2 M Na2 EDTA, pH 7.78) and analyzed in 1.0% agarose tube gels by gel electrophoresis. Form II DNA was partially purified from some cultures by cesium chloride-propidium iodide (CsC1--PI2) equilibrium density centrifugation [17] for experiments with bacteriophage T4 endonuclease V. Agarose tube gel electrophoresis [1,33,34] Agarose tube gels were prepared as described by Tegtmeyer and Macasaet [33]. Gels were prerun at constant voltage (8--10 V/tube) for 30 min in gel buffer (3.6 × 10 -2 M Tris, 3 × 10-: M NaH:PO4, 10 -3 M Na2 EDTA, 0.2% sodium dodecyl sulfate [SDS], pH 7.60). A 100-pl DNA sample was mixed with an equal volume of tracking dye (0.001% bromphenol blue, 0.6% SDS, 30% [w/v] sucrose) and left for 5--15 min at 37°C before being added to the gels. The bromphenol blue marker moved at a rate of a b o u t 8 cm/h independent of gel concentration in the range 1.0--1.5%. Gels were cut in 3-mm slices after electrophoresis and individual slices placed in 10.0 ml of water miscible scintillation fluid (PCS, Amersham-Searle). The samples were left at room temperature for 2 days before counting in a Packard scintillation spectrometer.
304
EcoR1 restriction endonuclease treatment [25] SV40 DNA was dialyzed overnight against 500 volumes of EcoR1 buffer (10 -~ M Tris--HC1, 5 × 10 -2 M NaC1, 5 × 10 -3 M MgCl:, pH 7.50). An incubation mixture was prepared with 100 pl of dialyzed SV40 DNA plus 2.0 pl of E. coli t R N A (1 mg/ml) and 1.0 #l of EcoR1 enzyme (t>6000 unit/ml). The incubation mixture was incubated at 37°C for 1 h before the reaction was stopped by 25 pl of stopping solution (10 -1 M Tris--HC1, 10 -~ M Na2EDTA, pH 7.40) and 50 pl of tracking dye. Incubation mixtures were analyzed by 1.5% agarose tube gel electrophoresis. The position of Form III molecules was determined by comparison of DNA samples incubated with or without the EcoR1 enzyme. Cesium chloride--propidium iodide isopycnic gradient ultracen trifugation DNA samples in T4 buffer weighing 6.10 g were mixed with 5.75 g of CsC1 and 0.40 ml of PI2 (6 mg/ml) in a Beckman 50 Ti polyallomer centrifuge tube and centrifuged at 48 000 rpm for 41 h in a Beckman Ti rotor. The DNA bands were visualized in the dark by exposure to a bank of GE black lights and the upper DNA band (non-supercoiled DNA) was collected by pipette. Samples were extracted 4 times with isopropanol : H20 (9 : 1) for removal of PI2 and dialyzed overnight against 1 1 of T4 buffer before analysis with T4 endonuclease V. T4 endonuclease V assay [12] An incubation mixture was prepared with 25 pl of non-supercoiled SV40 DNA plus 100 pl of T4 buffer and either 5 pl of T4 endonuclease V (PEG fraction) or 5 pl of T4 buffer. The mixture was incubated for 60 min at 37°C before 50 pl of tracking dye was added and samples analyzed by 1.0% agarose tube gel electrophoresis. The activity of our T4 endonuclease V stock on double-stranded DNA was verified in parallel experiments on supercoiled SV40 DNA [38] and purified CV-1 DNA [39] from irradiated cultures; T4 endonuclease V recognizes dimers equally well in double-stranded or single-stranded DNA [24]. Results
SV40 DNA synthesis in control cultures The shortest pulse time used (5 min) gave a resolvable Form I peak in 1.0% agarose gel radioactivity profiles (Fig. 2) and incorporation proceeded linearly up to 20 min of labeling {Fig. 3), whereas Hirt supernatants from mockinfected cultures showed activity only in the top fraction even after 20 min of 3H-labeling (Fig. 2). Host-cell DNA was confined to the top fraction in all gels in the agarose concentration range 1.0--1.4%. Increasing the length of [3H]thymidine pulse increased total radioactivity in the broad replicative intermediate (RI) peak (R~ -~ 0.45--0.55 relative to Form I molecules) as well as the rapidly moving Form I peak (Fig. 2). A small peak of Form II DNA molecules electrophoresed slightly more slowly than Form I molecules (R~ = 0.75--0.85 relative to Form I molecules). These Form II molecules do not increase in number as rapidly as Form I molecules. The observed order of electrophoretic mobility
305
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Fig. 2. T r i t i u m r a d i o a c t i v i t y p r o f i l e s o f p u l s e - l a b e l e d S V 4 0 D N A i n 1 . 0 % a g a r o s e gels. T h e i d e n t i t y o f p e a k s a p p e a r i n g f o r t r i t i u m p u l s e s o f five ( 5 ' ) t o t w e n t y ( 2 0 ' ) r a i n w a s d e t e r m i n e d b y p u l s e - c h a s e e x p e r i ments. The radioactivity profile after a 20-min tritium pulse of the Hirt supernatant from a mock-infected CV-1 c u l t u r e (MI) y i e l d e d v e r y f e w c o u n t s in a p a r a l l e l gel. S o m e h o s t - c e l l D N A p e n e t r a t e d t h e first gel slice in all gels ( h c D N A ) a n d w a s n o t c o n s i d e r e d in e v a l u a t i o n o f d a t a . T o t a l 1 4 C p r e l a b e l c o u n t s in S V 4 0 DNA: 5', 1303 cpm; 10', 1554 cpm; 20', 1534 cpm. Fig. 3. E x t e n t o f [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n i n t o S V 4 0 D N A d u r i n g p u l s e - l a b e l i n g as a f u n c t i o n o f p u l s e l e n g t h . I n c o r p o r a t i o n is e x p r e s s e d as t h e 3 H / 1 4 C r a t i o t o c o r r e c t f o r v a r i a b l e size o f t h e S V 4 0 replic a t i n g p o o l in a given c u l t u r e . L i n e a r i n c o r p o r a t i o n w a s f o u n d w i t h i n 5 m i n o f i n i t i a t i o n o f p u l s e - l a b e l i n g .
(Form I > Form II/> RI) and Rf values agree with the work of previous investigators for 1.0% agarose gels [1,33,34]. The average increase in the 3H/14C ratio for labeled SV40 DNA summed over entire gel profiles was 13(+10)% after 60 min of post-tritium incubation ("chase") and 14(+22)% after 180 min of chase (Table 1). Thus the use of unlabeled thymidine and deoxycytidine in the chase medium efficiently terminated incorporation of [3H]thymidine into SV40 DNA daughter strands. The mean time for replication of an SV40 DNA molecule equals the time for the percentage of total tritium label in RI molecules to decrease to 50% during continuous labeling [23]. Table 1 summarizes the 3H/14C ratios observed in RI, Form II, and Form I molecules as well as the percentage of total 3H incorporated in these SV40 replicating forms as a function of time. The percentage of total label in RI molecules decreased to 50% in a b o u t 15 min (Table 1), indicating that this is the time for replication of the SV40 genome. S V 4 0 D N A synthesis in UV-irradiated cultures Exposure of SV40-infected cultures to UV light before pulse-labeling altered agarose tube gel radioactivity profiles (Fig. 4). The amount of [3H]thymidine in total SV40 DNA was suppressed to 61% of control at 20 J/m 2 and to 49% of control at 40 J / m 2 (Table 1); at these doses 1 and 2 dimers are induced per SV40 genome, respectively. The a m o u n t of label in Form I molecules was
TABLE 1
20 20 20
0 0 0
0 0 0
40 40 40
20 20 20
5 10 15
20 20 20
20 20 20
0 60 180
0 60 180
0 0 0
0 60 180
0 60 180
incubation after pulse labeling
Minutes of
57.7 ( 3 ) 4 9 . 4 (3) 2 0 . 6 (3)
69.6 (3) 3 1 . 4 (3) 4 0 . 1 (3)
27.3 (2) 68.3 (1) 82.4 (1)
218c(3) 160 c (3) 2 9 4 c (3)
963 c (3) 4 7 5 c (3) 3 1 1 e (3)
Replieative intermediate molecules a
10.2 12.4 5.8
7.2 9.7 10.6
1.9 2.6 2.9
103 167 102
74 73 154
Form II molecules
Mean 3H/14C ratios
AND PERCENTAGE
1.5 3.5 3.2
5.8 9.6 10.9
0.7 1.6 3.7
23 48 46
58 75 104 ± 7 ± 14 ± 10
± 12 ± 23 ± 49
5.2 ± 6.2 + 4.4 ±
9.8 ± 10.8 ± 12.3 ±
0.7 1.9 0.5
1.3 3.0 0.7
2.1 -+ 0 . 2 4.2 7.5
51 61 55
84 84 ~ 113
Total b
53 57 36 49 ( m e a n )
61 73 49 61 ( m e a n )
Percent of control ratio
58 35 25
43 16 16
63 61 54
47 21 14
35 15 10
Replicative intermediate molecules
P e r c e n t o f t o t a l 3 H label
3H LABEL FOR SV40 DNA WITH OR WITHOUT
Form I molecules
OF TOTAL
18 16 12
7 8 8
8 6 3
18 14 11
8 7 10
F o r m II molecules
UV IRRADIATION
BEFORE
24 49 63
50 76 76
29 33 43
35 65 75
57 78 80
Form I molecules
a N u m b e r of r e p l i c a t e c u l t u r e s in p a r e n t h e s e s . b E r r o r s are s t a n d a r d e r r o r s o f t h e m e a n a n d are r e p r e s e n t a t i v e o f t h e e r r o r s f o u n d f o r all v a l u e s in e a c h r o w . c M e a n 3 H / 1 4 C v a l u e s f o r e x p e r i m e n t a l p l a t e s l a b e l e d w i t h [ 14 C] t h y m i d i n e at a n a c t i v i t y o f a b o u t 0 . 0 1 ~ C i / m l ; all o t h e r p l a t e s w e r e l a b e l e d at 0.1 ~ C i / m l .
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Minutes
PULSE LABELING
SUMMARY OF MEAN 3H/14C RATIOS
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Fig. 4. T r i t i u m r a d i o a c t i v i t y p r o f i l e s f o r S V 4 0 D N A p u l s e - l a b e l e d f o r 2 0 m i n and a n a l y z e d o n 1 . 0 % a g a r o s e t u b e gels b y e l e c t r o p h o r e s i s . E l e c t r o p h o r e s i s w a s f r o m l e f t t o r i g h t . A n a l y z e d S V 4 0 D N A r e c e i v e d ( A ) 0 J i m 2, (B) 2 0 J / m 2, a n d (C) 4 0 J / m 2 o f U V l i g h t b e f o r e i s o l a t i o n f r o m i n f e c t e d CV-1 cultures.
very sensitive to UV light exposure, whereas incorporation into RI and Form II molecules was less sensitive (Fig. 4). "Chasing" replicate cultures for 0, 1 or 3 h produced a rapid decline in RI molecules and a concurrent increase in Form I molecules (Table 1). The relative proportion and absolute amount of [3H]thymidine in Form II molecules declined little in 3 h (Table 1). The percentage of label entering Form II molecules relative to controls (Table 1) within 20 min after UV irradiation increased as expected if dimers define new termination sites and temporarily inhibit ring closure. However, Form II molecules did not accumulate in UV-irradiated SV40 DNA (Table 1), suggesting that ring closure occurs within 1 h of inhibition. A 10% increase in the relative proportion of label in RI after 3 h of chase was the only significant change in cultures exposed to 40 J/m 2 of UV light (Table 1). This suggests that few molecules are completely prevented from completing replication despite the presence of dimers ahead of the growing forks. Many dimers are therefore bypassed in a significantly shorter time than is required for actual dimer removal [38]. Because control cultures did n o t achieve a complete chase of label into Form I molecules, interpretation of the increase in RI label must take into account the reentrance of some labeled Form I molecules into the replicating pool [30,33]. This does n o t allow us to discriminate between (a) a small proportion of RI molecules absolutely blocked against further replication and (b) a proportion of RI molecules temporarily blocked and escaping blockage as some labeled Form I molecules reenter the replicating pool and are also temporarily blocked. Form H molecules carrying dimers The increased relative proportion of 3H-labeled Form II SV40 DNA molecules after UV exposure (Table 1) suggests that a significant fraction of these molecules may be blocked in the ligation-step replication. This block might
308
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Fig. 5. T r i t i u m r a d i o a c t i v i t y profiles for n o n - s u p e r c o i l e d S V 4 0 D N A isolated o n CsC1--PI 2 i s o p y c n i c g r a d i e n t s . S V 4 0 D N A w a s i s o l a t e d f r o m i n f e c t e d CV-1 c u l t u r e s e x p o s e d t o 0 J / m 2 (A, C) or 40 J / m 2 (B, D) of U V light, pulse-labeled 20 m i n w i t h [ 3 H ] t h y m i d i n e (10 # C i / m l , 11 C i / m m o l e ) , a n d e i t h e r e x t r a c t e d i m m e d i a t e l y (A, B) or i n c u b a t e d 60 rain in u n l a b e l e d m e d i u m b e f o r e e x t r a c t i o n (C, D). I s o l a t e d D N A w a s t r e a t e d 1 h w i t h T 4 b u f f e r (o) or a n excess o f T 4 e n d o n u c l e a s e V ( e ) b e f o r e analysis o n 1.5% agarose t u b e gels b y e l e c t r o p h o r e s i s . Positions o f R I , F o r m I a n d F o r m II m o l e c u l e s w e r e d e t e r m i n e d b y pulse a n d pulse-chase e x p e r i m e n t s ( n o t s h o w n ) . E l e c t r o p h o r e s i s was f r o m left to right.
be due to the presence of dimers opposite gaps. We tested an irradiated SV40 Form II DNA population for molecules carrying gaps opposite dimers by treating them with T4 endonuclease V and analyzing the DNA products for linearized (Form III) molecules. The position to which Form III molecules migrated was identified with SV40 DNA molecules treated with an excess of EcoR1 restriction endonuclease; the SV40 genome contains only one substrate site for this enzyme [25]. The T4 endonuclease failed to linearize a detectable fraction of Form II molecules made after UV irradiation (Fig. 5). In addition, the RI molecules that partially penetrated the gels were also unaffected by T4 endonuclease V treatment (Fig. 5). Discussion The major effect of UV irradiation on SV40 replication was to reduce the formation of mature Form I molecules. This may have occurred because UV p h o t o p r o d u c t s interfere with mammalian DNA-chain elongation [9]. At least three major events occur during SV40 replication, any of which could be affected by UV damage: (a) initiation of replication, (b) chain elongation and (c) termination with ligation to form supercoiled Form I molecules. Initiation was n o t photosensitive because the relative proportion of RI molecules did n o t decrease with increasing UV dose (Fig. 4, Table 1). Similarly, ligation was not photosensitive because the proportion of Form II molecules increased only slightly after irradiation and decreased with post-labeling incubation. Thus, DNA-chain elongation in SV40 molecules, as in mammalian DNA, appears to be retarded by UV photoproducts. If we assume that pyrimidine dimers are the lesions responsible for retard-
309 ing chain elongation, then it can be shown that the a m o u n t of [3H]thymidine incorporated into total SV40 DNA relative to controls should be approximately 0.77 at 20 J/m s and 0.57 at 40 J/m s . These values are similar to the experimental values of 0.61 and 0.49, respectively. The extent of inhibition of DNA replication is thus consistent with the hypothesis that dimers ahead of the growing fork retard chain growth. This size of newly made daughter DNA in replicating SV40 molecules after UV irradiation is also consistent with this hypothesis (A. Sarasin, unpublished observation). The pulse-chase experiments showed that DNA replication recovered in all RI molecules within 3 h of 20 J/m s UV light and in most RI molecules after 40 J/m s of UV light (Table 1). This process was shorter than the loss of T4 endonuclease V-sensitive sites in SV40 [38] and much shorter than dimer removal from mammalian cell DNA [5,39]. It is therefore likely that SV40 RI molecules overcome the UV-induced block to chain growth by bypassing dimers without removing them. The failure of RI or Form II molecules to accumulate during incubation after UV irradiation and pulse labeling suggests that daughter molecules with retarded growing forks replicate past most dimers within 60 min of irradiation. The likeliest mechanisms for such dimer bypass are recombination and "bypass replication." Molecular and genetic recombination have been detected at low levels in mammalian cells [16,20,27,36]. The recombination of multiple genetic markers in mixed infections with temperature-sensitive SV40 mutants has also been observed and is stimulated by UV irradiation of the virus before infection [8]. Such recombination may be induced by the presence of gaps at or near retarded growing forks; such gaps have been considered inducing events for production of infectious SV40 from transformed hamster cell lines [18,19,40, 41]. Recombination at retarded growing forks would lead to reassortment of dimers between parent and daughter DNA molecules and replication past the damage site. Dimers are detected in daughter molecules [ 37 ], b u t it is not clear whether they result from recombination or from elongation of daughter strands which initiated replication before UV irradiation and in which the dimers were produced directly by UV. An equally plausible mechanism has been suggested by recent observations of Radman et al. [28] that eukaryotic DNA polymerases can replicate past dimers in vitro with only a brief delay. Such "bypass replication" does n o t require recombination to circumvent dimers blocking replication. R u p p and Howard-Flanders [31] originally proposed that gaps are created in daughter DNA strands during replication past UV-induced damage sites. Attempts to detect gaps opposite dimers in mammalian DNA with purified dimer-specific endonucleases have been unsuccessful [4,24]. Two possible reasons for this lack of success are that (a) the structure of the substrate, consisting of doubleand single-stranded regions with a dimer opposite a gap in the vicinity of a growing fork, is refractory to endonucleolytic action by the T4 or M. luteus enzyme, or {b) the model is wrong and gaps are either never opposite dimers or are not opposite dimers long enough to be utilized as suitable enzyme substrates. A dimer-gap substrate in a Form II SV40 molecule offers a simpler conformation than that at an active mammalian cell replicon blocked by a dimer and might be cleaved by T4 endonuclease V. How-
310 ever, we found no evidence of nicking by T4 endonuclease V in DNA opposite the gap in Form II SV40 molecules, suggesting that a peculiar substrate conformation in the vicinity of a growing fork is not a sufficient explanation for the lack of enzyme action. We conclude that Clarkson and Hewitt's [4] modification of the Rupp and Howard-Flanders model best explains our data. Clarkson and Hewitt proposed that gaps are produced by gl'owing forks encountering dimers but that these gaps are filled before the next dimer is reached. This model is extended by the results of Radman et al. [28,29] to allow replication past the dimer as well as suppression of the 3'-+ 5' proofreading function recently detected in eukaryotic cells [29]. The biological sequelae of bypassing dimers are u n k n o w n b u t could involve replication errors that lead to mutagenic events. This explains both UV induction of mutations in SV40 [7] and the competition between SV40 DNA replication and repair which modifies observed levels of UV-induced mutagenesis [6]. Unexcised dimers could also cause functional changes such as early termination of transcription [3,14]. Inactivation of individual SV40 genomes due to low fluences of UV light may n o t cause observable changes in the SV40 infectious cycle b e y o n d delayed expression of plaque-forming ability [7] because only one surviving infectious particle is necessary to continue the lytic cycle. A complete infectious cycle yields about 100 infectious particles per cell [35], so one or more infectious virions per cell would be expected to be undamaged on the basis of Poi~son distributed dimers at UV fluences below 100 J/m 2 (4.6 pyrimidine dimers per SV40 genome) [38,39]. It remains to be shown what proportion of SV40 particles emerging from a UV-irradiated culture are infectious and/or mutated during a second lytic cycle. References 1 Aaij, C., a n d P. Borst, T h e gel e l e c t r o p h o r e s i s of D N A , B i o e h i m . B i o p h y s . A c t a , 269 ( 1 9 7 2 ) 1 9 2 - - 2 0 0 . 2 A b r a h a m s , P.J., a n d A.J. v a n d e r Eb, Host-ceU 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 S V 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 . 3 Berk, A.J., a n d P.A. S h a r p , U l t r a v i o l e t m a p p i n g of t h e a d e n o v i r u s 2 early p r o m o t e r s , Cell, 12 ( 1 9 7 7 ) 45--55. 4 Claxkson, J.M., a n d R . R . H e w i t t , Significance of d i m e r s t o t h e size of n e w l y s y n t h e s i z e d D N A in UVi r r a d i a t e d Chinese h a m s t e r o v a r y cells, B i o p h y s . J., 16 ( 1 9 7 6 ) 1 1 5 5 - - 1 1 6 4 . 5 Cleaver, J.E., R e p a i r processes for p h o t o c h e m i c a l d a m a g e in m a m m a l i a n cells, in: J.T. L e t t , H. A d l e r a n d M.R. Zelle (Eds.), A d v a n c e s in R a d i a t i o n Biology, Vol. 4, A c a d e m i c Press, N e w Y o r k , 1 9 7 4 , pp. 1--75. 6 Cleaver, J.E., Decline in m u t a t i o n f r e q u e n c y in t e m p e r a t u r e - s e n s i t i v e S V 4 0 viruses b e f o r e viral D N A s y n t h e s i s , M u t a t i o n Res., 44 ( 1 9 7 7 ) 2 9 1 - - 2 9 8 . 7 Cleaver, J . E . , a n d S. Well, U V - i n d u c e d r e v e r s i o n o f a t e m p e r a t u r e - s e n s i t i v e late m u t a n t of S i m i a n Virus 4 0 to a w i l d - t y p e p h e n o t y p e , J. Virol., 16 ( 1 9 7 5 ) 2 1 4 - - 2 1 6 . 8 D u b b s , D . R . , M. R a c h m e l e r a n d S. Kit, R e c o m b i n a t i o n b e t w e e n t e m p e r a t u r e - s e n s i t i v e m u t a n t s of S i m i a n virus 4 0 , V i r o l o g y , 57 ( 1 9 7 4 ) 1 6 1 - - 1 7 4 . 9 E d e n b e r g , H . J . , I n h i b i t i o n o f D N A r e p l i c a t i o n b y u l t r a v i o l e t light, Biophys. J., 16 ( 1 9 7 6 ) 8 4 9 - - 8 6 0 . 10 E d e n b e r g , H . J . , a n d J.A. H u b e r m a n , E u k a r y o t i c c h r o m o s o m e r e p l i c a t i o n , A n n . Rev. G e n e t . , 9 ( 1 9 7 5 ) 245--284. 11 F a r e e d , G.C., C.F. G a r o n a n d N.P. S a l z m a n , Origin a n d d i r e c t i o n o f S i m i a n Virus 4 0 d e o x y r i b o n u c l e i c acid r e p l i c a t i o n , J. Virol., 10 ( 1 9 7 2 ) 4 8 4 - - 4 9 1 . 12 F r i e d b e r g , E.C., a n d J.J. K i n g ; D a r k r e p a i r o f u l t r a v i o l e t - i r r a d i a t e d d e o x y r i b o n u c l e i c acid b y b a c t e r i o p h a g e T 4 : p u r i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f a d i m e r - s p e e i f i c p h a g e - i n d u c e d e n d o n u c l e a s e , J. Bacteriol., 106 ( 1 9 7 1 ) 5 0 0 - - 5 0 7 . 13 G r i f f i t h , J.D., C h r o m a t i n s t r u c t u r e : d e d u c e d f r o m a m i n i c h r o m o s o m e , Science, 187 ( 1 9 7 5 ) 1 2 0 2 - 1203.
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