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Postreplication repair of DNA in chick cells: Studies using photoreactivation
179
Biochimica et Biophysica Acta, 402 (1975) 179--187 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98372
POSTREPLICATION R E P A I R OF DNA IN CHICK CELLS: STUDIES USING PHOTOREACTIVATION
A.R. LEHMANN and S. STEVENS
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BNI 9QG (U.K.) (Received February 26th, 1975)
Summary During replication of DNA after ultraviolet irradiation, gaps are left in the newly-synthesized DNA strands in both bacterial and animal cells and these gaps are subsequently sealed by a process known as postreplication repair. In order to test whether it is the ultraviolet-induced pyrimidine dimers which are responsible for the production of these daughter-strand gaps in animal cells, we have used chick e m b r y o fibroblasts. In these cells the pyrimidine dimers are photoreactivable, i.e. they can be split b y an enzymatic process dependent on visible or near ultraviolet light. Our results indicate that chick cells possess a postreplication repair system similar to that in mammalian cells; gaps are produced in the newly-synthesized strands and then filled in. If the ultravioletirradiated cells are first photoreactivated to remove most of the dimers, the number of daughter-strand gaps produced is much less than without photoreactivation. This suggests that the dimers are indeed responsible for the formation of many of the gaps in the newly-synthesized DNA. Ultraviolet light also inhibits the overall rate of DNA synthesis. This inhibition is, however, only partly overcome by photoreactivation.
Introduction
Postreplication repair of DNA in damaged cells refers to the process whereby lesions remaining in the DNA of the cells are circumvented during or after replication (reviewed in ref. 1). As with bacteria [2], experiments using alkaline sucrose gradients have shown that gaps are left in the daughter strands of DNA synthesized in ultraviolet-irradiated cells derived from a variety of species of mammals, and these gaps are sealed on further incubation [3--8]. Experiments with Escherichia coli have provided good indirect evidence suggesting that the gaps in the daughter strands are opposite the pyrimidine dimers in the parental strands [9--11]. These experiments made use of the process of
180 photoreactivation (the enzymic splitting of the pyrimidine dimers in situ in the presence of visible or near ultraviolet light (reviewed in refs 12 and 13}). Previous attempts to demonstrate photoreactivation in vivo in placental mammals have been unsuccessful [12,14,15]; only very recently has a successful demonstration been reported [16]. Thus, the only evidence to suggest that daughter-strand gaps might be opposite the pyrimidine dimers in ultravioletirradiated animal cells, is a very approximate correspondence in some cell lines between the average interdimer distance in the parental strands, and the average size of the newly-synthesized pieces of DNA [6,8,17]. For technical reasons, neither of these measurements can be made accurately so that this comparison is a rather crude approximation. In order to investigate this problem in a more satisfactory way we have studied postreplication repair in chick e m b r y o fibroblasts. Cook and McGrath [18] showed that extracts from chick embryos contained photoreactivating enzyme, and Pfefferkorn and Coady [19] and more recently Paterson et al. [20] showed that in the presence of visible light, pyrimidine dimers (measured directly or as sites susceptible to an ultravioletspecific endonuclease) were rapidly lost from the DNA of ultraviolet-irradiated chick embryo cells during incubation at 37°C. In contrast only a very small fraction of the dimers were removed during prolonged incubation periods in the dark. This suggests that these cells can efficiently remove dimers from their DNA by photoreactivation but only to a very limited extent b y excision-repair. Since these cells do have an efficient photoreactivation system, we have been able to carry o u t experiments similar to those with E. coli [10]. Our results have provided information on the role of the pyrimidine dimer in interrupting DNA synthesis in these cells after ultraviolet irradiation. While this manuscript was in preparation, a report appeared of similar work by Buhl et al. [21] using a marsupial (Potorous tridactylis) cell line. Materials and methods
Cell culture 12
181 the petri dishes put into sealed containers. These were placed on a sheet of perspex, 6 mm thick, which lay on two Osram 20 W fluorescent tubes, in a warm r o o m (37°C) and incubated for 2 h. Control cells were treated identically b u t incubated in the dark.
Measurement of pyrimidine dimers 0.5 • 106--1.0 • 106 cells were seeded on 9 cm petri dishes and incubated for 16--24 h with 5 pCi/ml [ 3H]thymidine, 10 -~ M unlabelled thymidine. The cells were ultraviolet-irradiated and photoreactivated if required; (a) Direct measurement. The cells were trypsinized and DNA was extracted by a modified Marmur [22] procedure. The percentage of radioactivity in pyrimidine dimers was determined as described b y Carrier and Setlow [ 2 3 ] , with the modification using thin-layer chromatography (Polygram - CEL300 Camlab, Cambridge) [24]. (b) Measurement of ultraviolet-endonuclease-susceptible sites. The method used was essentially the same as described b y Wilkins [ 2 5 ] . The cells were scraped off the plates into 1 ml EDTA in buffered saline (0.2 g/l), spun down and resuspended in 0.15 ml 10% sucrose in 0.1 M Tris • HC1 (pH 7.6) for 10 rain at 4°C. 1.4 ml H2 O were added and the cells left for a further 10 rain. To 80 gl were added 10 pl of buffer (0.5 M Tris • HC1/0.1 M mercaptoethanol/ 0.01 M EDTA, 60 pg/ml DNA, pH 8.0). The cells were vortexed for 10 s and then 20 pl of purified ultraviolet-endonuclease from Micrococcus luteus (generously supplied by Dr R.A. Oosterbaan, Medical Biological Laboratory, TNO, Rijswijk, Netherlands) were added. After 20 rain the cells were chilled, exposed to 2 krad 7-irradiation (see below), lysed on top of alkaline sucrose gradients and the DNA centrifuged and analysed as described previously [26].
Post-replication repair 2 • 105 Cells were incubated for 16--24 h in 5 cm petri dishes, ultravioletirradiated and photoreactivated if required. All subsequent procedures t o o k place in the dark. The newly-synthesized DNA was labelled with 17 pCi/ml [ all] thymidine (20--25 Ci/mmol) for various periods of time, such that the amount of DNA labelled in cultures treated differently was approximately the same (This avoids labelling artefacts [6,27] ). In pulse-chase experiments, the radioactive medium was removed and the cells washed, and then incubated in fresh medium containing 10 -s M thymidine, 10 -s M deoxycytidine for various periods. In some experiments caffeine was present at 0.3 mg/ml during the pulse and chase periods. The cells were then scraped off the dishes with a piece of silicone rubber into 0.3 ml buffered saline containing 0.2 g/l sodium EDTA and exposed to 2 krad of 7-irradiation to overcome entanglement problems during subsequent centrifugation [6]. Approx. 2 • 104 cells were lysed on t o p of alkaline sucrose gradients, which were centrifuged and analysed, as described previously [26].
Thymidine incorporation experiments 2 • l 0 s cells were incubated for 16--24 h with 0.05 p Ci/ml [~4C] thymidine (62 Ci/mol). The medium was removed, the cells irradiated and photoreactivated as required. The medium was then replaced with fresh medium contain-
182 ing 5 #Ci/ml [ 3H] thymidine {20--25 Ci/mmol) and incubated for 1 - 5 h. The medium was removed, the cells washed and scraped off the plates into 0.3 ml EDTA solution (see above). Duplicate 0.1-ml samples were taken onto Whatman 3MM paper squares, which had been previously soaked in sodium dodecyl sulphate and dried. Two drops of sodium dodecyl sulphate were added to complete cell lysis, and the papers were subsequently washed 3 times in 5% trichloracetic acid, twice in ethanol, dried and counted in an Intertechnique SL40 liquid scintillation counter. The 3H/~4C ratio gave a measure of the a m o u n t of 3H incorporated per cell. In some experiments, before pulse-labelling, fluorodeoxyuridine was added to a concentration of 10 '~ M for 15 min at 37°C in the dark. The medium was then replaced with fresh medium containing 10 -6 M fluorodeoxyuridine, 2.5 pM thymidine, 10 pCi/ml [~H] thymidine and the cells incubated for 1.5 h. Results
1. Post replication repair in chick cells Fig. 1 shows sucrose gradient profiles of newly-synthesized DNA from unirradiated and ultraviolet-irradiated chick cells. In unirradiated cells, DNA synthesized in a short pulse-label was heterogeneous in size {Fig. la} and on subsequent incubation for 1.5 h it was converted to high molecular weight (Fig. lb). This was presumably the result of the normal processes of chain elongation and ligation of adjacent replicating units. After an ultraviolet fluence of 12.5 J • m -2 the newly-synthesized DNA was much smaller than in unirradiated cells 8 6 /., 2 " ] "~ o
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183 (Fig. lc) indicating the presence of daughter-strand gaps. After a further incubation for 3 h and 6 h these gaps were sealed and high molecular weight DNA was produced (Figs l d , e). With a higher ultraviolet fluence (Fig. l f ) newly synthesized DNA was even smaller and it then took some 6 h until high molecular weight DNA was produced (Figs lg, h). These characteristics of postreplication repair in chick cells are similar to those of cells from various mammalian species (e.g. see refs 6,8). Caffeine inhibits postreplication repair in many rodent cell lines [28] but not in normal human cell lines [29]. Under comparable conditions it did not inhibit this process in the chick cells (results not shown}.
2. Photoreactivation The results in Table I show that during a 2 h incubation of the chick cells at 37 °C in medium in the presence of visible light, pyrimidine dimers were lost from the DNA. After an ultraviolet fluence of 12.5 J • m -2 almost all the dimers disappeared whereas after 25.0 J • m -2 about 30% remained. Similar results were obtained whether the dimers were measured directly or by the indirect, but more sensitive technique of measuring the number of sites in the DNA (presumed to be pyrimidine dimers) which are susceptible to the ultravioletspecific endonuclease from M. luteus [25] (Table I). These findings are in accordance with the results of Paterson et al. [20]. 3. Effect o f photoreactivation on size of newly-synthesized DNA As shown in Fig. 1 the size of DNA synthesized in ultraviolet-irradiated cells was smaller than that in unirradiated cells. Very similar profiles were obtained if the cells were incubated for 2 h in the dark after irradiation prior to pulse-labelling (Fig. 2). In contrast, if the cells were incubated for 2 h in the light, and then pulse-labeled, the size of newly-synthesized DNA was bigger than that from the dark-incubated cells, and approached that of the unirradiated cells (Fig. 2). Thus after photoreactivation the newly-synthesized DNA contained fewer gaps than the DNA from cells which had been ultraviolet-irradiated but not photoreactivated. The profiles from unirradiated cells which were subsequently incubated in the dark or in the light were not significantly different. TABLE I P H O T O R E A C T I V A T I O N (PR) O F D I M E R S Ceils l a b e l l e d o v e r n i g h t w e r e u l t r a v i o l e t - i r r a d i a t e d a n d i n c u b a t e d for 2 h in t h e d a r k o r light, a n d t h e p y r i m i d i n e d i m e r s w e r e m e a s u r e d e i t h e r d i r e c t l y [ 2 3 ] ( c o l u m n s 3, 4, 5) or as u l t r a v i o l e t - e n d o n u c l e a s e s u s c e p t i b l e sites { 2 5 ] ( c o l u m n s 6, 7). Ultraviolet exposure (J • m -2)
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184 4. Inhibition o f D N A synthesis by ultraviolet light and the effect o f photoreactivation As in mammalian systems [30] ultraviolet-light inhibited DNA synthesis in the chick cells, as measured b y incorporation of [3 HI thymidine into DNA. The kinetics of this inhibition are shown in Fig. 3 (lower curve). Photoreactivation prior to measuring the rate of thymidine incorporation decreased this inhibition. However, after fluences of 12.5 J • m -2 and 25.0 J • m -2, 93% and 70% of the pyrimidine dimers were removed from the DNA by photoreactivation (Table I) but the inhibition of thymidine incorporation was relieved by photoreactivation to a much smaller extent (Fig. 3). In order to check whether the thymidine incorporation data of Fig. 3 were a true measure of DNA synthesis rather than the result of changes in pool sizes, nucleoside transport rates etc., these experiments were repeated in the presence of 1 pM fluorodeoxyuridine and 3 pM thymidine. Under these conditions endogenous synthesis of thymidine monophosphate is inhibited, so that exogenous thymidine is the sole source of thymidine nucleotides. Essentially the same results were obtained under these conditions as those shown in Fig. 3. UV E x p o s ~ e ~Jm-~) I00 r
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Fig. 2. E f f e c t of p h o t o r e a c t i v a t i o n o n size o f n e w l y - s y n t h e s i z e d D N A . Cells w e r e u n i r r a d i a t e d , i n c u b a t e d for 2 h in t h e d a r k ( ) a n d pulse-labelled for 25 rain, o r u l t r a v i o l e t - i r r a d i a t e d ( 1 2 . 5 J . m -2) a n d i n c u b a t e d for 2 h in t h e d a r k ( . . . . . . ) o r light ( . . . . . . ) a n d pulse-labelled f o r 60 or 40 rain r e s p e c t i v e l y w i t h [ 3HI t h y m i d i n e . C e n t r i f u g a t i o n in alkaline s u c r o s e g r a d i e n t s a t 3 8 0 0 0 r e v . / m i n f o r 70 rain. Sedim e n t a t i o n f r o m r i g h t to left. Fig. 3. I n h i b i t i o n o f D N A S y n t h e s i s b y u l t r a v i o l e t light. Cells labelled o v e r n i g h t w i t h [ ! 4 C ] t h y m i d i n e were u l t r a v i o l e t - i r r a d i a t e d , i n c u b a t e d f o r 2 h in t h e d a r k (o o) o r light (o o) a n d p u i s e - l a b e n e d for 1.5 h with [ 3HI t h y m i d i n e . T h e cells w e r e s c r a p e d o f f t h e p l a t e s a n d t h e a m o u n t o f acid-insoluble 3H a n d 14C r a d i o a c t i v i t y d e t e r m i n e d . T h e o r d i n a t e m e a s u r e s t h e 3 H / I 4C ratio as a p e r c e n t a g e o f t h a t in u n l r r a diated controls.
185
Photoreactivating light did not significantly affect cells which had not been ultraviolet-irradiated. In the eight experiments which provided the results for Fig. 3, the amount of thymidine incorporation in unirradiated cells which were incubated in the light was (95 ± 4}% of the incorporation into cells incubated in the dark. Discussion In an attempt to obtain information on the role that pyrimidine dimers play in interrupting DNA synthesis in animal cells, we have studied the characteristics of DNA synthesis in ultraviolet-irradiated chick e m b r y o fibroblasts, making use of the active photoreactivation system present in these cells [20]. Since the characteristics of postreplication repair (Fig. 1) and the ultravioletinduced inhibition of DNA synthesis (Fig. 3) were similar in chick and mammalian systems, it is probable though not certain, that the conclusions reached are applicable to mammalian systems. Our findings that chick cells have a postreplication repair mechanism implies that chick cells can cope with pyrimidine dimers in the dark and are not totally dependent on the light-mediated process, as suggested b y Paterson et al. [20]. During a 2 h incubation under growth conditions in the presence of visible light, most of the pyrimidine dimers disappeared from the DNA as a consequence of photoreactivation (Table I). The number of gaps in daughter-strand DNA molecules was also considerably less in the photoreactivated cells than in the non-photoreactivated cells (Fig. 2). The average number of gaps present in a stretch of DNA at any one time is given by 1 / M n - - 1 / M o (1), where Mn and Mo are the number-average molecular weights of the DNA in test cells and unirradiated cells respectively. Accurate estimation of the number of daughter-strand gaps produced per unit length of DNA is very difficult to obtain for the following reasons: (1) For a random distribution of uniformly-labelled molecules it is possible to measure Mn accurately by a graphical method [31]. In our pulse-labelling experiments, many of the molecules will not have been uniformly labelled, and also the molecular weight distributions were often non-random. Summation methods for determining Mn are subject to large errors caused by small amounts of spurious slowly sedimenting material, and these methods also are only valid for uniformly-labelled molecules. (2) Eqn 1 is valid only if the same length of DNA (i.e., the same number of nucleotides per replication fork) is labelled in unirradiated and test cells. Although attempts have been made to label the cells so that the same amount of DNA is labelled in both sets of cells (a) it is difficult to obtain this situation exactly; and also (b) the amount of radioactivity in cells treated in different ways may be dependent on other factors apart from the length of DNA synthesized at each replication fork (see below). (3) If a gap is sealed before the next gap is formed, or if gaps are sealed during the pulse-label period, the frequency of gaps which have been produced (rather than gaps open at any one time), will be underestimated. From calculations of average molecular weights in experiments like the one shown in Fig. 2, we have estimated that approx. 70% of the gaps present in
186 cells ultraviolet-irradiated with an exposure of 12.5 J • m -2 were not present if the cells were photoreactivated after ultraviolet-irradiation. In view of the uncertainties listed above, this must be regarded as a rather approximate estimate. We may conclude, therefore, that a large number, but not all, of the gaps are opposite pyrimidine dimers, or to be more rigorous, that the dimers are at least responsible for the production of a large proportion of the gaps in the daughter strands. Very recently, Buhl et al. [21] using a marsupial cell line have also reached the conclusion that pyrimidine dimers were responsible for most of the daughter-strand gaps. As in mammalian cells (see for example refs 30,32), the rate of DNA synthesis in chick cells (Fig. 3) was inhibited by ultraviolet-irradiation. Photoreactivation overcame this inhibition to some extent, indicating that the inhibition of DNA synthesis is at least partly caused by the pyrimidine dimers. However, under conditions of photoreactivation in which most of the dimers were lost from the DNA (see Table I) an appreciable inhibition of DNA synthesis still remained. This suggests that this inhibition was not simply or solely the result of a short delay in the rate of progress of the replication fork at each dimer, but that it was partly caused by some other means. Possible mechanisms include (1) some of the dimers irreversibly blocking the progress of the replication fork, (2) the replication fork being delayed by non-dimer lesions, (3) some of the dimers causing blocks in initiation of DNA synthesis which might be irreversible. Further experimentation is needed to distinguish between these and other possibilities.
Acknowledgements We are grateful to Dr C.F. Arlett for instructing us in culture techniques for chick embryo fibroblasts and to Drs C.F. Arlett and B.A. Bridges for helpful criticisms of the manuscript. References 1 2 3 4
5 6 7 8 9 10 11 12 13 14 15 16
L e h m a n n . A . R . ( 1 9 7 4 ) Life S c i e n c e s 1 5 , 2 0 0 5 - - 2 0 1 6 R u p p , W.D. a n d H o w a r d - F l a n d e r s , P. ( 1 9 6 8 ) J. Mol. Biol. 3 1 , 2 9 1 - - 3 0 4 Cleaver. J . E . a n d T h o m a s . G . H . ( 1 9 6 9 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 3 6 , 2 0 3 - - 2 0 8 R u p p , W . D . , Z i p s e r , E., y o n Essen, C., R e n o , D., P r o s n i t z , L. a n d H o w a r d - F i a n d e r s , P., ( 1 9 7 0 ) In T i m e a n d D o s e R e l a t i o n s h i p in R a d i a t i o n B i o l o g y as A p p l i e d t o R a d i o t h e r a p y , p p . 1 - 1 3 , B r o o k h a v e n Natl. Lab. Publication BNL 50203 (c-57) M e y n . R . E . a n d H u m p h r e y , R . M . ( 1 9 7 1 ) B i o p h y s . J . 11, 2 9 5 - - 3 0 1 L e h m a n n , A . R . ( 1 9 7 2 ) J . Mol. Biol. 6 6 , 3 1 9 - - 3 3 7 F u j i w a r a , Y. ( 1 9 7 2 ) E x p . Cell Res. 7 5 . 4 8 3 - - 4 8 9 B u h l , S.N., S t i l l m a n , R . M . , S e t l o w , R . B . a n d R e g a n , J . D . ( 1 9 7 2 ) B i o p h y s . J. 12, 1 1 8 3 - - 1 1 9 1 H o w a r d - F l a n d e r s , P.0 R u p p , W . D . , Wilkins, B.M. a n d Cole, R.S. ( 1 9 6 8 ) C o l d S p r i n g H a r b o r S y m p . Q u a n t . Biol. 3 3 , 1 9 5 - - 2 0 7 S m i t h . K . C . a n d M e u n , D . H . C . ( 1 9 7 0 ) J. Mol. Biol. 510 4 5 9 - - 4 7 2 Bridges, B.A. a n d S e d g w i c k , S.G. ( 1 9 7 4 ) J . B a c t . 1 1 7 , 1 0 7 7 - - 1 0 8 1 C o o k , J . S . ( 1 9 7 0 ) In P h o t o p h y s i o l o g y ( A . C . Giese, e d . ) , Vol. 5, p p . 1 9 1 - - 2 3 3 , A c a d e m i c Press, N e w York H a r m , W., R u p e r t , C.S. a n d H a r m , H. ( 1 9 7 1 ) In P h o t o p h y s i o l o g y ( A . C . Giese, ed.)0 Vol. 6, p p . 2 7 9 - - 3 2 4 , A c a d e m i c Press, N e w Y o r k Cleaver, J . E . ( 1 9 6 6 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 24, 5 6 9 - - 5 7 6 P a t e r s o n , M.C., L o h m a n , P . H . M . , W e s t e r v e l d , A. a n d S l u y t e r , M.L. ( 1 9 7 4 ) B i o p h y s . J . 14, 8 3 5 - - 8 4 5 S u t h e r l a n d , B.M., R i c e , M. a n d W a g n e r , E . K . ( 1 9 7 5 ) P r o c . Natl. A c a d . Sei. U.S. 7 2 , 1 0 3 - - I 07
187
17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Zipser, E.V. ( 1 9 7 3 ) Ph.D. thesis, Yale Univ. C o o k , J.S. a n d M c G r a t h , J . R . ( 1 9 6 7 ) Proc. Natl. A c a d . Sci. U.S. 58, 1 3 5 9 - - 1 3 6 5 P f e f f e r k o r n , E.R. a n d C o a d y , H.M. ( 1 9 6 8 ) J . Virol. 2. 4 7 4 - - 4 7 9 P a t e r s o n , M.C., L o h m a n , P.H.M., de Weerd-Kastelein, E.A. a n d Westerveld, A. ( 1 9 7 4 ) Biophys. J. 14, 454--466 Buhl, S.N., Setlow, R.B. a n d Regan, J . D . ( 1 9 7 4 ) Biophys. J . 14, 7 9 1 - - 8 0 3 M a r m u r , J. ( 1 9 6 1 ) J . Mol. Biol. 3, 2 0 8 - - 2 1 8 Carrier, W.L. a n d Setlow, R.B. ( 1 9 7 1 ) In M e t h o d s in E n z y m o l o g y (L. G r o s s m a n a n d K. Moldave, eds), Vol. 21, pp. 2 3 0 - - 2 3 7 , A c a d e m i c Press, New York G o l d m a n n , K. a n d F r i e d b e r g , E.C. ( 1 9 7 3 ) Anal. Biochem. 53, 1 2 4 - - 1 3 1 Wilkins, R.J. ( 1 9 7 3 ) Int. J. R a d i a t . Biol. 2 4 , 6 0 9 - - 6 1 3 L e h m a n n , A.R. and Kirk-Bell, S. ( 1 9 7 2 ) Eur. J. Biochem. 3 1 , 4 3 8 - - 4 4 5 L e h m a n n , A.R. a n d O m e r o d , M.G. ( 1 9 6 9 ) N a t u r e 221, 1 0 5 3 - - 1 0 5 6 L e h m a n n . A.R. a n d Kirk-Bell, S. ( 1 9 7 4 ) Mutat. Res. 26, 7 3 - - 8 2 L e h m a n n , A.R., Kirk-Bell, S., Arlett, C.F., P a t e r s o n , M.C., L o h m a n , P.H.M., de Weerd-Kastelein, E.A. a n d B o o t s m a , D. ( 1 9 7 5 ) Proc. Natl. Acad. Sci. U.S. 72, 2 1 9 - - 2 2 3 R a u t h , A.M. ( 1 9 7 0 ) In Cttrrent Topics in R a d i a t i o n Research (M. Ebert a n d A. H o w a r d , eds), Vol. 6, pp. 195---248, N o r t h Holland, A m s t e r d a m Dean, C J . , O m e r o d , M.G., Serianni, R.W. a n d A l e x a n d e r , P. ( 1 9 6 9 ) N a t u r e 222, 1 0 4 2 - - 1 0 4 4 Cleaver, J . E . ( 1 9 6 7 ) R a d i a t . Res. 30, 7 9 5 - - 8 1 0
Biochimica et Biophysica Acta, 402 (1975) 179--187 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98372
POSTREPLICATION R E P A I R OF DNA IN CHICK CELLS: STUDIES USING PHOTOREACTIVATION
A.R. LEHMANN and S. STEVENS
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BNI 9QG (U.K.) (Received February 26th, 1975)
Summary During replication of DNA after ultraviolet irradiation, gaps are left in the newly-synthesized DNA strands in both bacterial and animal cells and these gaps are subsequently sealed by a process known as postreplication repair. In order to test whether it is the ultraviolet-induced pyrimidine dimers which are responsible for the production of these daughter-strand gaps in animal cells, we have used chick e m b r y o fibroblasts. In these cells the pyrimidine dimers are photoreactivable, i.e. they can be split b y an enzymatic process dependent on visible or near ultraviolet light. Our results indicate that chick cells possess a postreplication repair system similar to that in mammalian cells; gaps are produced in the newly-synthesized strands and then filled in. If the ultravioletirradiated cells are first photoreactivated to remove most of the dimers, the number of daughter-strand gaps produced is much less than without photoreactivation. This suggests that the dimers are indeed responsible for the formation of many of the gaps in the newly-synthesized DNA. Ultraviolet light also inhibits the overall rate of DNA synthesis. This inhibition is, however, only partly overcome by photoreactivation.
Introduction
Postreplication repair of DNA in damaged cells refers to the process whereby lesions remaining in the DNA of the cells are circumvented during or after replication (reviewed in ref. 1). As with bacteria [2], experiments using alkaline sucrose gradients have shown that gaps are left in the daughter strands of DNA synthesized in ultraviolet-irradiated cells derived from a variety of species of mammals, and these gaps are sealed on further incubation [3--8]. Experiments with Escherichia coli have provided good indirect evidence suggesting that the gaps in the daughter strands are opposite the pyrimidine dimers in the parental strands [9--11]. These experiments made use of the process of
180 photoreactivation (the enzymic splitting of the pyrimidine dimers in situ in the presence of visible or near ultraviolet light (reviewed in refs 12 and 13}). Previous attempts to demonstrate photoreactivation in vivo in placental mammals have been unsuccessful [12,14,15]; only very recently has a successful demonstration been reported [16]. Thus, the only evidence to suggest that daughter-strand gaps might be opposite the pyrimidine dimers in ultravioletirradiated animal cells, is a very approximate correspondence in some cell lines between the average interdimer distance in the parental strands, and the average size of the newly-synthesized pieces of DNA [6,8,17]. For technical reasons, neither of these measurements can be made accurately so that this comparison is a rather crude approximation. In order to investigate this problem in a more satisfactory way we have studied postreplication repair in chick e m b r y o fibroblasts. Cook and McGrath [18] showed that extracts from chick embryos contained photoreactivating enzyme, and Pfefferkorn and Coady [19] and more recently Paterson et al. [20] showed that in the presence of visible light, pyrimidine dimers (measured directly or as sites susceptible to an ultravioletspecific endonuclease) were rapidly lost from the DNA of ultraviolet-irradiated chick embryo cells during incubation at 37°C. In contrast only a very small fraction of the dimers were removed during prolonged incubation periods in the dark. This suggests that these cells can efficiently remove dimers from their DNA by photoreactivation but only to a very limited extent b y excision-repair. Since these cells do have an efficient photoreactivation system, we have been able to carry o u t experiments similar to those with E. coli [10]. Our results have provided information on the role of the pyrimidine dimer in interrupting DNA synthesis in these cells after ultraviolet irradiation. While this manuscript was in preparation, a report appeared of similar work by Buhl et al. [21] using a marsupial (Potorous tridactylis) cell line. Materials and methods
Cell culture 12
181 the petri dishes put into sealed containers. These were placed on a sheet of perspex, 6 mm thick, which lay on two Osram 20 W fluorescent tubes, in a warm r o o m (37°C) and incubated for 2 h. Control cells were treated identically b u t incubated in the dark.
Measurement of pyrimidine dimers 0.5 • 106--1.0 • 106 cells were seeded on 9 cm petri dishes and incubated for 16--24 h with 5 pCi/ml [ 3H]thymidine, 10 -~ M unlabelled thymidine. The cells were ultraviolet-irradiated and photoreactivated if required; (a) Direct measurement. The cells were trypsinized and DNA was extracted by a modified Marmur [22] procedure. The percentage of radioactivity in pyrimidine dimers was determined as described b y Carrier and Setlow [ 2 3 ] , with the modification using thin-layer chromatography (Polygram - CEL300 Camlab, Cambridge) [24]. (b) Measurement of ultraviolet-endonuclease-susceptible sites. The method used was essentially the same as described b y Wilkins [ 2 5 ] . The cells were scraped off the plates into 1 ml EDTA in buffered saline (0.2 g/l), spun down and resuspended in 0.15 ml 10% sucrose in 0.1 M Tris • HC1 (pH 7.6) for 10 rain at 4°C. 1.4 ml H2 O were added and the cells left for a further 10 rain. To 80 gl were added 10 pl of buffer (0.5 M Tris • HC1/0.1 M mercaptoethanol/ 0.01 M EDTA, 60 pg/ml DNA, pH 8.0). The cells were vortexed for 10 s and then 20 pl of purified ultraviolet-endonuclease from Micrococcus luteus (generously supplied by Dr R.A. Oosterbaan, Medical Biological Laboratory, TNO, Rijswijk, Netherlands) were added. After 20 rain the cells were chilled, exposed to 2 krad 7-irradiation (see below), lysed on top of alkaline sucrose gradients and the DNA centrifuged and analysed as described previously [26].
Post-replication repair 2 • 105 Cells were incubated for 16--24 h in 5 cm petri dishes, ultravioletirradiated and photoreactivated if required. All subsequent procedures t o o k place in the dark. The newly-synthesized DNA was labelled with 17 pCi/ml [ all] thymidine (20--25 Ci/mmol) for various periods of time, such that the amount of DNA labelled in cultures treated differently was approximately the same (This avoids labelling artefacts [6,27] ). In pulse-chase experiments, the radioactive medium was removed and the cells washed, and then incubated in fresh medium containing 10 -s M thymidine, 10 -s M deoxycytidine for various periods. In some experiments caffeine was present at 0.3 mg/ml during the pulse and chase periods. The cells were then scraped off the dishes with a piece of silicone rubber into 0.3 ml buffered saline containing 0.2 g/l sodium EDTA and exposed to 2 krad of 7-irradiation to overcome entanglement problems during subsequent centrifugation [6]. Approx. 2 • 104 cells were lysed on t o p of alkaline sucrose gradients, which were centrifuged and analysed, as described previously [26].
Thymidine incorporation experiments 2 • l 0 s cells were incubated for 16--24 h with 0.05 p Ci/ml [~4C] thymidine (62 Ci/mol). The medium was removed, the cells irradiated and photoreactivated as required. The medium was then replaced with fresh medium contain-
182 ing 5 #Ci/ml [ 3H] thymidine {20--25 Ci/mmol) and incubated for 1 - 5 h. The medium was removed, the cells washed and scraped off the plates into 0.3 ml EDTA solution (see above). Duplicate 0.1-ml samples were taken onto Whatman 3MM paper squares, which had been previously soaked in sodium dodecyl sulphate and dried. Two drops of sodium dodecyl sulphate were added to complete cell lysis, and the papers were subsequently washed 3 times in 5% trichloracetic acid, twice in ethanol, dried and counted in an Intertechnique SL40 liquid scintillation counter. The 3H/~4C ratio gave a measure of the a m o u n t of 3H incorporated per cell. In some experiments, before pulse-labelling, fluorodeoxyuridine was added to a concentration of 10 '~ M for 15 min at 37°C in the dark. The medium was then replaced with fresh medium containing 10 -6 M fluorodeoxyuridine, 2.5 pM thymidine, 10 pCi/ml [~H] thymidine and the cells incubated for 1.5 h. Results
1. Post replication repair in chick cells Fig. 1 shows sucrose gradient profiles of newly-synthesized DNA from unirradiated and ultraviolet-irradiated chick cells. In unirradiated cells, DNA synthesized in a short pulse-label was heterogeneous in size {Fig. la} and on subsequent incubation for 1.5 h it was converted to high molecular weight (Fig. lb). This was presumably the result of the normal processes of chain elongation and ligation of adjacent replicating units. After an ultraviolet fluence of 12.5 J • m -2 the newly-synthesized DNA was much smaller than in unirradiated cells 8 6 /., 2 " ] "~ o
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183 (Fig. lc) indicating the presence of daughter-strand gaps. After a further incubation for 3 h and 6 h these gaps were sealed and high molecular weight DNA was produced (Figs l d , e). With a higher ultraviolet fluence (Fig. l f ) newly synthesized DNA was even smaller and it then took some 6 h until high molecular weight DNA was produced (Figs lg, h). These characteristics of postreplication repair in chick cells are similar to those of cells from various mammalian species (e.g. see refs 6,8). Caffeine inhibits postreplication repair in many rodent cell lines [28] but not in normal human cell lines [29]. Under comparable conditions it did not inhibit this process in the chick cells (results not shown}.
2. Photoreactivation The results in Table I show that during a 2 h incubation of the chick cells at 37 °C in medium in the presence of visible light, pyrimidine dimers were lost from the DNA. After an ultraviolet fluence of 12.5 J • m -2 almost all the dimers disappeared whereas after 25.0 J • m -2 about 30% remained. Similar results were obtained whether the dimers were measured directly or by the indirect, but more sensitive technique of measuring the number of sites in the DNA (presumed to be pyrimidine dimers) which are susceptible to the ultravioletspecific endonuclease from M. luteus [25] (Table I). These findings are in accordance with the results of Paterson et al. [20]. 3. Effect o f photoreactivation on size of newly-synthesized DNA As shown in Fig. 1 the size of DNA synthesized in ultraviolet-irradiated cells was smaller than that in unirradiated cells. Very similar profiles were obtained if the cells were incubated for 2 h in the dark after irradiation prior to pulse-labelling (Fig. 2). In contrast, if the cells were incubated for 2 h in the light, and then pulse-labeled, the size of newly-synthesized DNA was bigger than that from the dark-incubated cells, and approached that of the unirradiated cells (Fig. 2). Thus after photoreactivation the newly-synthesized DNA contained fewer gaps than the DNA from cells which had been ultraviolet-irradiated but not photoreactivated. The profiles from unirradiated cells which were subsequently incubated in the dark or in the light were not significantly different. TABLE I P H O T O R E A C T I V A T I O N (PR) O F D I M E R S Ceils l a b e l l e d o v e r n i g h t w e r e u l t r a v i o l e t - i r r a d i a t e d a n d i n c u b a t e d for 2 h in t h e d a r k o r light, a n d t h e p y r i m i d i n e d i m e r s w e r e m e a s u r e d e i t h e r d i r e c t l y [ 2 3 ] ( c o l u m n s 3, 4, 5) or as u l t r a v i o l e t - e n d o n u c l e a s e s u s c e p t i b l e sites { 2 5 ] ( c o l u m n s 6, 7). Ultraviolet exposure (J • m -2)
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184 4. Inhibition o f D N A synthesis by ultraviolet light and the effect o f photoreactivation As in mammalian systems [30] ultraviolet-light inhibited DNA synthesis in the chick cells, as measured b y incorporation of [3 HI thymidine into DNA. The kinetics of this inhibition are shown in Fig. 3 (lower curve). Photoreactivation prior to measuring the rate of thymidine incorporation decreased this inhibition. However, after fluences of 12.5 J • m -2 and 25.0 J • m -2, 93% and 70% of the pyrimidine dimers were removed from the DNA by photoreactivation (Table I) but the inhibition of thymidine incorporation was relieved by photoreactivation to a much smaller extent (Fig. 3). In order to check whether the thymidine incorporation data of Fig. 3 were a true measure of DNA synthesis rather than the result of changes in pool sizes, nucleoside transport rates etc., these experiments were repeated in the presence of 1 pM fluorodeoxyuridine and 3 pM thymidine. Under these conditions endogenous synthesis of thymidine monophosphate is inhibited, so that exogenous thymidine is the sole source of thymidine nucleotides. Essentially the same results were obtained under these conditions as those shown in Fig. 3. UV E x p o s ~ e ~Jm-~) I00 r
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Fig. 2. E f f e c t of p h o t o r e a c t i v a t i o n o n size o f n e w l y - s y n t h e s i z e d D N A . Cells w e r e u n i r r a d i a t e d , i n c u b a t e d for 2 h in t h e d a r k ( ) a n d pulse-labelled for 25 rain, o r u l t r a v i o l e t - i r r a d i a t e d ( 1 2 . 5 J . m -2) a n d i n c u b a t e d for 2 h in t h e d a r k ( . . . . . . ) o r light ( . . . . . . ) a n d pulse-labelled f o r 60 or 40 rain r e s p e c t i v e l y w i t h [ 3HI t h y m i d i n e . C e n t r i f u g a t i o n in alkaline s u c r o s e g r a d i e n t s a t 3 8 0 0 0 r e v . / m i n f o r 70 rain. Sedim e n t a t i o n f r o m r i g h t to left. Fig. 3. I n h i b i t i o n o f D N A S y n t h e s i s b y u l t r a v i o l e t light. Cells labelled o v e r n i g h t w i t h [ ! 4 C ] t h y m i d i n e were u l t r a v i o l e t - i r r a d i a t e d , i n c u b a t e d f o r 2 h in t h e d a r k (o o) o r light (o o) a n d p u i s e - l a b e n e d for 1.5 h with [ 3HI t h y m i d i n e . T h e cells w e r e s c r a p e d o f f t h e p l a t e s a n d t h e a m o u n t o f acid-insoluble 3H a n d 14C r a d i o a c t i v i t y d e t e r m i n e d . T h e o r d i n a t e m e a s u r e s t h e 3 H / I 4C ratio as a p e r c e n t a g e o f t h a t in u n l r r a diated controls.
185
Photoreactivating light did not significantly affect cells which had not been ultraviolet-irradiated. In the eight experiments which provided the results for Fig. 3, the amount of thymidine incorporation in unirradiated cells which were incubated in the light was (95 ± 4}% of the incorporation into cells incubated in the dark. Discussion In an attempt to obtain information on the role that pyrimidine dimers play in interrupting DNA synthesis in animal cells, we have studied the characteristics of DNA synthesis in ultraviolet-irradiated chick e m b r y o fibroblasts, making use of the active photoreactivation system present in these cells [20]. Since the characteristics of postreplication repair (Fig. 1) and the ultravioletinduced inhibition of DNA synthesis (Fig. 3) were similar in chick and mammalian systems, it is probable though not certain, that the conclusions reached are applicable to mammalian systems. Our findings that chick cells have a postreplication repair mechanism implies that chick cells can cope with pyrimidine dimers in the dark and are not totally dependent on the light-mediated process, as suggested b y Paterson et al. [20]. During a 2 h incubation under growth conditions in the presence of visible light, most of the pyrimidine dimers disappeared from the DNA as a consequence of photoreactivation (Table I). The number of gaps in daughter-strand DNA molecules was also considerably less in the photoreactivated cells than in the non-photoreactivated cells (Fig. 2). The average number of gaps present in a stretch of DNA at any one time is given by 1 / M n - - 1 / M o (1), where Mn and Mo are the number-average molecular weights of the DNA in test cells and unirradiated cells respectively. Accurate estimation of the number of daughter-strand gaps produced per unit length of DNA is very difficult to obtain for the following reasons: (1) For a random distribution of uniformly-labelled molecules it is possible to measure Mn accurately by a graphical method [31]. In our pulse-labelling experiments, many of the molecules will not have been uniformly labelled, and also the molecular weight distributions were often non-random. Summation methods for determining Mn are subject to large errors caused by small amounts of spurious slowly sedimenting material, and these methods also are only valid for uniformly-labelled molecules. (2) Eqn 1 is valid only if the same length of DNA (i.e., the same number of nucleotides per replication fork) is labelled in unirradiated and test cells. Although attempts have been made to label the cells so that the same amount of DNA is labelled in both sets of cells (a) it is difficult to obtain this situation exactly; and also (b) the amount of radioactivity in cells treated in different ways may be dependent on other factors apart from the length of DNA synthesized at each replication fork (see below). (3) If a gap is sealed before the next gap is formed, or if gaps are sealed during the pulse-label period, the frequency of gaps which have been produced (rather than gaps open at any one time), will be underestimated. From calculations of average molecular weights in experiments like the one shown in Fig. 2, we have estimated that approx. 70% of the gaps present in
186 cells ultraviolet-irradiated with an exposure of 12.5 J • m -2 were not present if the cells were photoreactivated after ultraviolet-irradiation. In view of the uncertainties listed above, this must be regarded as a rather approximate estimate. We may conclude, therefore, that a large number, but not all, of the gaps are opposite pyrimidine dimers, or to be more rigorous, that the dimers are at least responsible for the production of a large proportion of the gaps in the daughter strands. Very recently, Buhl et al. [21] using a marsupial cell line have also reached the conclusion that pyrimidine dimers were responsible for most of the daughter-strand gaps. As in mammalian cells (see for example refs 30,32), the rate of DNA synthesis in chick cells (Fig. 3) was inhibited by ultraviolet-irradiation. Photoreactivation overcame this inhibition to some extent, indicating that the inhibition of DNA synthesis is at least partly caused by the pyrimidine dimers. However, under conditions of photoreactivation in which most of the dimers were lost from the DNA (see Table I) an appreciable inhibition of DNA synthesis still remained. This suggests that this inhibition was not simply or solely the result of a short delay in the rate of progress of the replication fork at each dimer, but that it was partly caused by some other means. Possible mechanisms include (1) some of the dimers irreversibly blocking the progress of the replication fork, (2) the replication fork being delayed by non-dimer lesions, (3) some of the dimers causing blocks in initiation of DNA synthesis which might be irreversible. Further experimentation is needed to distinguish between these and other possibilities.
Acknowledgements We are grateful to Dr C.F. Arlett for instructing us in culture techniques for chick embryo fibroblasts and to Drs C.F. Arlett and B.A. Bridges for helpful criticisms of the manuscript. References 1 2 3 4
5 6 7 8 9 10 11 12 13 14 15 16
L e h m a n n . A . R . ( 1 9 7 4 ) Life S c i e n c e s 1 5 , 2 0 0 5 - - 2 0 1 6 R u p p , W.D. a n d H o w a r d - F l a n d e r s , P. ( 1 9 6 8 ) J. Mol. Biol. 3 1 , 2 9 1 - - 3 0 4 Cleaver. J . E . a n d T h o m a s . G . H . ( 1 9 6 9 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 3 6 , 2 0 3 - - 2 0 8 R u p p , W . D . , Z i p s e r , E., y o n Essen, C., R e n o , D., P r o s n i t z , L. a n d H o w a r d - F i a n d e r s , P., ( 1 9 7 0 ) In T i m e a n d D o s e R e l a t i o n s h i p in R a d i a t i o n B i o l o g y as A p p l i e d t o R a d i o t h e r a p y , p p . 1 - 1 3 , B r o o k h a v e n Natl. Lab. Publication BNL 50203 (c-57) M e y n . R . E . a n d H u m p h r e y , R . M . ( 1 9 7 1 ) B i o p h y s . J . 11, 2 9 5 - - 3 0 1 L e h m a n n , A . R . ( 1 9 7 2 ) J . Mol. Biol. 6 6 , 3 1 9 - - 3 3 7 F u j i w a r a , Y. ( 1 9 7 2 ) E x p . Cell Res. 7 5 . 4 8 3 - - 4 8 9 B u h l , S.N., S t i l l m a n , R . M . , S e t l o w , R . B . a n d R e g a n , J . D . ( 1 9 7 2 ) B i o p h y s . J. 12, 1 1 8 3 - - 1 1 9 1 H o w a r d - F l a n d e r s , P.0 R u p p , W . D . , Wilkins, B.M. a n d Cole, R.S. ( 1 9 6 8 ) C o l d S p r i n g H a r b o r S y m p . Q u a n t . Biol. 3 3 , 1 9 5 - - 2 0 7 S m i t h . K . C . a n d M e u n , D . H . C . ( 1 9 7 0 ) J. Mol. Biol. 510 4 5 9 - - 4 7 2 Bridges, B.A. a n d S e d g w i c k , S.G. ( 1 9 7 4 ) J . B a c t . 1 1 7 , 1 0 7 7 - - 1 0 8 1 C o o k , J . S . ( 1 9 7 0 ) In P h o t o p h y s i o l o g y ( A . C . Giese, e d . ) , Vol. 5, p p . 1 9 1 - - 2 3 3 , A c a d e m i c Press, N e w York H a r m , W., R u p e r t , C.S. a n d H a r m , H. ( 1 9 7 1 ) In P h o t o p h y s i o l o g y ( A . C . Giese, ed.)0 Vol. 6, p p . 2 7 9 - - 3 2 4 , A c a d e m i c Press, N e w Y o r k Cleaver, J . E . ( 1 9 6 6 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 24, 5 6 9 - - 5 7 6 P a t e r s o n , M.C., L o h m a n , P . H . M . , W e s t e r v e l d , A. a n d S l u y t e r , M.L. ( 1 9 7 4 ) B i o p h y s . J . 14, 8 3 5 - - 8 4 5 S u t h e r l a n d , B.M., R i c e , M. a n d W a g n e r , E . K . ( 1 9 7 5 ) P r o c . Natl. A c a d . Sei. U.S. 7 2 , 1 0 3 - - I 07
187
17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Zipser, E.V. ( 1 9 7 3 ) Ph.D. thesis, Yale Univ. C o o k , J.S. a n d M c G r a t h , J . R . ( 1 9 6 7 ) Proc. Natl. A c a d . Sci. U.S. 58, 1 3 5 9 - - 1 3 6 5 P f e f f e r k o r n , E.R. a n d C o a d y , H.M. ( 1 9 6 8 ) J . Virol. 2. 4 7 4 - - 4 7 9 P a t e r s o n , M.C., L o h m a n , P.H.M., de Weerd-Kastelein, E.A. a n d Westerveld, A. ( 1 9 7 4 ) Biophys. J. 14, 454--466 Buhl, S.N., Setlow, R.B. a n d Regan, J . D . ( 1 9 7 4 ) Biophys. J . 14, 7 9 1 - - 8 0 3 M a r m u r , J. ( 1 9 6 1 ) J . Mol. Biol. 3, 2 0 8 - - 2 1 8 Carrier, W.L. a n d Setlow, R.B. ( 1 9 7 1 ) In M e t h o d s in E n z y m o l o g y (L. G r o s s m a n a n d K. Moldave, eds), Vol. 21, pp. 2 3 0 - - 2 3 7 , A c a d e m i c Press, New York G o l d m a n n , K. a n d F r i e d b e r g , E.C. ( 1 9 7 3 ) Anal. Biochem. 53, 1 2 4 - - 1 3 1 Wilkins, R.J. ( 1 9 7 3 ) Int. J. R a d i a t . Biol. 2 4 , 6 0 9 - - 6 1 3 L e h m a n n , A.R. and Kirk-Bell, S. ( 1 9 7 2 ) Eur. J. Biochem. 3 1 , 4 3 8 - - 4 4 5 L e h m a n n , A.R. a n d O m e r o d , M.G. ( 1 9 6 9 ) N a t u r e 221, 1 0 5 3 - - 1 0 5 6 L e h m a n n . A.R. a n d Kirk-Bell, S. ( 1 9 7 4 ) Mutat. Res. 26, 7 3 - - 8 2 L e h m a n n , A.R., Kirk-Bell, S., Arlett, C.F., P a t e r s o n , M.C., L o h m a n , P.H.M., de Weerd-Kastelein, E.A. a n d B o o t s m a , D. ( 1 9 7 5 ) Proc. Natl. Acad. Sci. U.S. 72, 2 1 9 - - 2 2 3 R a u t h , A.M. ( 1 9 7 0 ) In Cttrrent Topics in R a d i a t i o n Research (M. Ebert a n d A. H o w a r d , eds), Vol. 6, pp. 195---248, N o r t h Holland, A m s t e r d a m Dean, C J . , O m e r o d , M.G., Serianni, R.W. a n d A l e x a n d e r , P. ( 1 9 6 9 ) N a t u r e 222, 1 0 4 2 - - 1 0 4 4 Cleaver, J . E . ( 1 9 6 7 ) R a d i a t . Res. 30, 7 9 5 - - 8 1 0