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Suppression by caffeine and EDTA of exposure of 3′-OH termini in the DNA of γ-irradiated barley seed
279
Mutation Research, 44 (1977) 279--282 © Elsevier/North-Holland Biomedical Press
Short communication SUPPRESSION BY CAFFEINE AND EDTA OF EXPOSURE OF 3'-OH TERMINI IN THE DNA OF 7-IRRADIATED BARLEY SEED *
HIKOYUKI YAMAGUCHI and ATSUSHI TATARA
Laboratory of Radiation Genetics, Faculty of Agriculture, The University of Tokyo, Tokyo 113 (Japan) (Received December 12th, 1976) (Revision received March 7th, 1977) (Accepted March 10th, 1977)
Damage induced by ionizing radiations in plant seeds is enhanced with caffeine, and this has been ascribed to the inhibition of repair [1--4,7,9]. Recently, Yamaguchi et al. [6,7] suggested that lesions of irradiated barley seed are repairable with unscheduled DNA synthesis during G1 phase of early germination, concurrently with fibrous diffusion of chromatin fibres. Since the 3'-OH group is required on DNA templates for polymerization catalysed by the DNA polymerase, it was assumed that the unscheduled repair synthesis takes place at about 7 h after soaking of 30-kR-irradiated seeds, based on the experiment using Escherichia coli DNA polymerase I [8] in vitro. Here, the effects of caffeine and EDTA upon the accessibility of 7-ray-induced 3'-OH termini toward DNA polymerase were studied, and a mechanism of the suppression of DNA repair by caffeine and EDTA in irradiated barley seed is discussed. The materials and methods are given in detail in a previous paper [8]. Dry seeds (moisture c o n t e n t of about 10%) of barley cultivar Fuji 2-Jyo were exposed to 0, 15 or 30 kR of 137Cs gamma rays with 500 R per min. Then they were allowed to grow on the filter paper in Petri dishes for 5 h at 25°C in the dark. Two ml of 1 mM EDTA 2Na or 5 mM caffeine dissolved in 50 mM Tris-HC1 buffer (pH 7.0) was put into each Petri dish. At 5 h after initiation of seed soaking, a part of them was fixed by immersion in the fixative of acetic and alcohol (1 : 3), and the other was transferred into freshly prepared dishes containing 2 ml of distilled water. They continued to grow for a further 10 or 12 h, corresponding to 15 or 17 h from the initiation of seed soaking, followed by fixation. Control samples of seeds were soaked in petri dishes containing 2 ml of distilled water at 25°C and fixed after 5, 15 or 17 h soaking. After embryos excised from these soaked seeds were stained with Feulgen reagent, the first leaf meristims were cut off and squashed on the slide glasses. * Research Mutation
carried o u t u n d e r the F A O / I A E A C o - o r d i n a t e d R e s e a r c h P r o g r a m m e on I m p r o v e m e n t Breeding T e c h n i q u e s ( R e s e a r c h A g r e e m e n t No. 1206).
of
280 The cover glasses were flipped off by the dry ice method, and the preparations were dried naturally at room temperature. The reaction mixture for the DNA polymerase which contained 1 to 2 units of Escherichia coli DNA polymerase I (C.F. Boehringer & Soehne GmbH, Mannheim, Germany), 0.13 mM deoxyribonucleoside triphosphate of cytosine, thymine, adenine and guanine, 8 mM MgC12 and 50 mM potassium phosphate buffer (pH 7.0) was prepared as mentioned in the previous paper [8]. Fifty pl of the solution were dripped upon the meristem cells of the slides. Tritiumlabelled thymidine triphosphate (thymidine methyl-3H-5'-triphosphate ammonium salt, 15.6 Ci/mM, The Radiochemical Centre, England} was used in this experiment. After the reaction had continued for 2 h at 37°C in the dark, the reaction mixture was rinsed away with 5% trichloroacetic acid for the slides. They were air dried and then subjected to autoradiography. The nuclei over which silver grains had been spread were scored as the 3Hincorporated nuclei. Yamaguchi et al. [7] reported that in interphase nuclei of the first leaf meristem, fibrous chromatin dispersed before the onset of chromosomal DNA replication when barley seeds were soaked in distilled water at 25°C. Thus, the effect of caffeine and EDTA on the chromatin diffusion was also studied in the present experiment. The percentage of 3H-labelled nuclei against radiation dose is illustrated in Fig. 1. Tritium-labelled nuclei of no-post-treated controls increased to about 45% in 30-kR seeds, while those of EDTA or caffeine post-treatment increased only to 10 to 11%, even though irradiated with the same dose; that is, an increment of 3H-labelled nuclei after gamma-ray irradiation was greatly inhibited with soaking in the presence of EDTA or caffeine. Previously, Yamaguchi et al. [7] showed that an increase of 3H-labelled nuclei in vivo, after gamma-ray irradiation, was suppressed only to one-tenth with EDTA post-treatment. Fur-
5ol
50
0 4C
40
3C
•_~ 3 o c
~- 2 0
i
/
E
E
o ~
,~
Dose of gamma ray (kR)
~o
% Diffuse nuclei (%)
Fig. 1. E f f e c t o f E D T A o r c a f f e i n e p o s t - t r e a t m e n t o n t h e t r i t i u m i n c o r p o r a t i o n i n t o the first l e a f meristern cells o f 7-irradiated barley seed. N o n - p o s t - t r e a t e d (o); E D T A - p o s t - t r e a t e d (e); c a f f e i n e p o s t - t r e a t e d (~). Fig. 2. I n t e r r e l a t i o n b e t w e e n 3 H - l a b e U e d n u c l e i and d i f f u s e c h r o m a t i n n u c l e i in irradiated or non-irrad i a t e d seed. N o n - p o s t - t r e a t e d (o); E D T A p o s t - t r e a t e d ( e ) ; c a f f e i n e p o s t - t r e a t e d (~).
281 thermore, the diffuse chromatin nuclei induced with gamma radiation also decreased notably with EDTA post-treatment. Fig. 2 shows an interrelation between tritium-incorporated nuclei and diffuse nuclei. In no-post-treated seeds, both labelled and diffuse nuclei were much more with 30-kR irradiation, but no increment of these nuclei was observed with EDTA post-treatment. When the irradiated seeds were allowed to soak in the presence of caffeine, both diffuse and labelled nuclei increased slightly. From these results, it was postulated that EDTA inhibited an exposure of 3'-OH groups of D N A strand breaks as a result of the suppression of the unwinding of chromatin fibres. This may be why EDTA inhibits the repair synthesis in irradiated barley seeds. On the other hand, it seems that caffeine inhibits the repair of radiation damage by combining with the D N A strand breaks rather than by suppressing the conformational change of chromatin fibres. T'so and Lu [5] have reported that caffeine binds to single-stranded and denatured DNA. References 1 A h n s t r 6 m , G. a n d A . T . N a t a r a j a n , R e p a i r o f g a m m a - r a y a n d n e u t r o n - i n d u c e d lesions in g e r m i n a t i n g b a r l e y s e e d s , I n t e r n . J. R a d i a t i o n Biol., 1 9 ( 1 9 7 1 ) 4 3 3 - - 4 4 3 . 2 Ahnstr~Sm, G., R e p a i r p r o c e s s e s in g e r m i n a t i n g seeds: c a f f e i n e e n h a n c e m e n t o f d a m a g e i n d u c e d by gamma-radiation and aikylating chemicals, Mutation Res., 26 (1974) 99--103. 3 S o o m r o , A.M. a n d H. Y a m a g u c h i , C a f f e i n e e n h a n c e m e n t o f the d a m a g e i n d u c e d b y g a m m a r a y s a n d a i k y l a t i n g a g e n t s in rice seeds, J a p a n . J. B r e e d i n g 2 7 ( 1 9 7 7 ) 1 3 - - 1 8 . 4 S w i e t l i n s k a , Z. a n d J. Z u k , E f f e c t o f c a f f e i n e o n c h r o m o s o m e d a m a g e i n d u c e d b y c h e m i c a l m u t a g e n s a n d i o n i z i n g r a d i a t i o n in Vicia faba and Secale cereale, M u t a t i o n Res., 2 6 ( 1 9 7 4 ) 89---97. 5 T'so, P . O . P . a n d P. L u , I n t e r a c t i o n o f nucleic a c i d . I. P h y s i c a l b i n d i n g o f t h y m i n e , a d e n i n e , s t e r o i d , a n d a r o m a t i c h y d r o c a r b o n s a n d n u c l e i c acids, P r o c . N a t l . A c a d . Sci. (U.S.), 51 ( 1 9 6 4 ) 1 7 - - 2 4 . 6 Y a m a g u c b i , H., T. N a i t o a n d A. T a t a r a , T h e i n h i b i t i n g e f f e c t o f E D T A o n the repair o f g a m m a - r a y i n d u c e d l e s i o n s in g e r m i n a t i n g barley s e e d s , Japan. J. B r e e d i n g , 2 4 ( 1 9 7 4 ) 1 8 8 - - 1 9 3 . 7 Y a m a g u c h i , H., A. T a t a r a a n d T. N a i t o , U n s c h e d u l e d D N A s y n t h e s i s i n d u c e d in barley s e e d s b y g a m m a rays a n d 4 - n i t r o q u i n o l i n e 1 - o x i d e , J a p a n , J. G e n e t i c s , 5 0 ( 1 9 7 5 ) 3 0 7 - - 3 1 8 . 8 Y a m a g u c h i , H., A. T a t a r a a n d T. N a i t o , A c c e s s i b i l i t y o f 7 - r a y i n d u c e d p r i m e r t o w a r d D N A p o l y m e r a s e I o f Escherichia coli d u r i n g s o a k i n g o f barley s e e d , E n v i r o n m . a n d E x p t l . B o t a n y , 16 ( 1 9 7 6 ) 1 4 1 - - 1 4 4 . 9 Y a m a m o t o , K. a n d H . Y a m a g u c h i , I n h i b i t i o n b y c a f f e i n e o f the repair o f ? - r a y - i n d u c e d c h r o m o s o m e breaks in b a r l e y , M u t a t i o n Res. 8 ( 1 9 6 9 ) 4 2 8 - - 4 3 0 .
Mutation Research, 44 (1977) 279--282 © Elsevier/North-Holland Biomedical Press
Short communication SUPPRESSION BY CAFFEINE AND EDTA OF EXPOSURE OF 3'-OH TERMINI IN THE DNA OF 7-IRRADIATED BARLEY SEED *
HIKOYUKI YAMAGUCHI and ATSUSHI TATARA
Laboratory of Radiation Genetics, Faculty of Agriculture, The University of Tokyo, Tokyo 113 (Japan) (Received December 12th, 1976) (Revision received March 7th, 1977) (Accepted March 10th, 1977)
Damage induced by ionizing radiations in plant seeds is enhanced with caffeine, and this has been ascribed to the inhibition of repair [1--4,7,9]. Recently, Yamaguchi et al. [6,7] suggested that lesions of irradiated barley seed are repairable with unscheduled DNA synthesis during G1 phase of early germination, concurrently with fibrous diffusion of chromatin fibres. Since the 3'-OH group is required on DNA templates for polymerization catalysed by the DNA polymerase, it was assumed that the unscheduled repair synthesis takes place at about 7 h after soaking of 30-kR-irradiated seeds, based on the experiment using Escherichia coli DNA polymerase I [8] in vitro. Here, the effects of caffeine and EDTA upon the accessibility of 7-ray-induced 3'-OH termini toward DNA polymerase were studied, and a mechanism of the suppression of DNA repair by caffeine and EDTA in irradiated barley seed is discussed. The materials and methods are given in detail in a previous paper [8]. Dry seeds (moisture c o n t e n t of about 10%) of barley cultivar Fuji 2-Jyo were exposed to 0, 15 or 30 kR of 137Cs gamma rays with 500 R per min. Then they were allowed to grow on the filter paper in Petri dishes for 5 h at 25°C in the dark. Two ml of 1 mM EDTA 2Na or 5 mM caffeine dissolved in 50 mM Tris-HC1 buffer (pH 7.0) was put into each Petri dish. At 5 h after initiation of seed soaking, a part of them was fixed by immersion in the fixative of acetic and alcohol (1 : 3), and the other was transferred into freshly prepared dishes containing 2 ml of distilled water. They continued to grow for a further 10 or 12 h, corresponding to 15 or 17 h from the initiation of seed soaking, followed by fixation. Control samples of seeds were soaked in petri dishes containing 2 ml of distilled water at 25°C and fixed after 5, 15 or 17 h soaking. After embryos excised from these soaked seeds were stained with Feulgen reagent, the first leaf meristims were cut off and squashed on the slide glasses. * Research Mutation
carried o u t u n d e r the F A O / I A E A C o - o r d i n a t e d R e s e a r c h P r o g r a m m e on I m p r o v e m e n t Breeding T e c h n i q u e s ( R e s e a r c h A g r e e m e n t No. 1206).
of
280 The cover glasses were flipped off by the dry ice method, and the preparations were dried naturally at room temperature. The reaction mixture for the DNA polymerase which contained 1 to 2 units of Escherichia coli DNA polymerase I (C.F. Boehringer & Soehne GmbH, Mannheim, Germany), 0.13 mM deoxyribonucleoside triphosphate of cytosine, thymine, adenine and guanine, 8 mM MgC12 and 50 mM potassium phosphate buffer (pH 7.0) was prepared as mentioned in the previous paper [8]. Fifty pl of the solution were dripped upon the meristem cells of the slides. Tritiumlabelled thymidine triphosphate (thymidine methyl-3H-5'-triphosphate ammonium salt, 15.6 Ci/mM, The Radiochemical Centre, England} was used in this experiment. After the reaction had continued for 2 h at 37°C in the dark, the reaction mixture was rinsed away with 5% trichloroacetic acid for the slides. They were air dried and then subjected to autoradiography. The nuclei over which silver grains had been spread were scored as the 3Hincorporated nuclei. Yamaguchi et al. [7] reported that in interphase nuclei of the first leaf meristem, fibrous chromatin dispersed before the onset of chromosomal DNA replication when barley seeds were soaked in distilled water at 25°C. Thus, the effect of caffeine and EDTA on the chromatin diffusion was also studied in the present experiment. The percentage of 3H-labelled nuclei against radiation dose is illustrated in Fig. 1. Tritium-labelled nuclei of no-post-treated controls increased to about 45% in 30-kR seeds, while those of EDTA or caffeine post-treatment increased only to 10 to 11%, even though irradiated with the same dose; that is, an increment of 3H-labelled nuclei after gamma-ray irradiation was greatly inhibited with soaking in the presence of EDTA or caffeine. Previously, Yamaguchi et al. [7] showed that an increase of 3H-labelled nuclei in vivo, after gamma-ray irradiation, was suppressed only to one-tenth with EDTA post-treatment. Fur-
5ol
50
0 4C
40
3C
•_~ 3 o c
~- 2 0
i
/
E
E
o ~
,~
Dose of gamma ray (kR)
~o
% Diffuse nuclei (%)
Fig. 1. E f f e c t o f E D T A o r c a f f e i n e p o s t - t r e a t m e n t o n t h e t r i t i u m i n c o r p o r a t i o n i n t o the first l e a f meristern cells o f 7-irradiated barley seed. N o n - p o s t - t r e a t e d (o); E D T A - p o s t - t r e a t e d (e); c a f f e i n e p o s t - t r e a t e d (~). Fig. 2. I n t e r r e l a t i o n b e t w e e n 3 H - l a b e U e d n u c l e i and d i f f u s e c h r o m a t i n n u c l e i in irradiated or non-irrad i a t e d seed. N o n - p o s t - t r e a t e d (o); E D T A p o s t - t r e a t e d ( e ) ; c a f f e i n e p o s t - t r e a t e d (~).
281 thermore, the diffuse chromatin nuclei induced with gamma radiation also decreased notably with EDTA post-treatment. Fig. 2 shows an interrelation between tritium-incorporated nuclei and diffuse nuclei. In no-post-treated seeds, both labelled and diffuse nuclei were much more with 30-kR irradiation, but no increment of these nuclei was observed with EDTA post-treatment. When the irradiated seeds were allowed to soak in the presence of caffeine, both diffuse and labelled nuclei increased slightly. From these results, it was postulated that EDTA inhibited an exposure of 3'-OH groups of D N A strand breaks as a result of the suppression of the unwinding of chromatin fibres. This may be why EDTA inhibits the repair synthesis in irradiated barley seeds. On the other hand, it seems that caffeine inhibits the repair of radiation damage by combining with the D N A strand breaks rather than by suppressing the conformational change of chromatin fibres. T'so and Lu [5] have reported that caffeine binds to single-stranded and denatured DNA. References 1 A h n s t r 6 m , G. a n d A . T . N a t a r a j a n , R e p a i r o f g a m m a - r a y a n d n e u t r o n - i n d u c e d lesions in g e r m i n a t i n g b a r l e y s e e d s , I n t e r n . J. R a d i a t i o n Biol., 1 9 ( 1 9 7 1 ) 4 3 3 - - 4 4 3 . 2 Ahnstr~Sm, G., R e p a i r p r o c e s s e s in g e r m i n a t i n g seeds: c a f f e i n e e n h a n c e m e n t o f d a m a g e i n d u c e d by gamma-radiation and aikylating chemicals, Mutation Res., 26 (1974) 99--103. 3 S o o m r o , A.M. a n d H. Y a m a g u c h i , C a f f e i n e e n h a n c e m e n t o f the d a m a g e i n d u c e d b y g a m m a r a y s a n d a i k y l a t i n g a g e n t s in rice seeds, J a p a n . J. B r e e d i n g 2 7 ( 1 9 7 7 ) 1 3 - - 1 8 . 4 S w i e t l i n s k a , Z. a n d J. Z u k , E f f e c t o f c a f f e i n e o n c h r o m o s o m e d a m a g e i n d u c e d b y c h e m i c a l m u t a g e n s a n d i o n i z i n g r a d i a t i o n in Vicia faba and Secale cereale, M u t a t i o n Res., 2 6 ( 1 9 7 4 ) 89---97. 5 T'so, P . O . P . a n d P. L u , I n t e r a c t i o n o f nucleic a c i d . I. P h y s i c a l b i n d i n g o f t h y m i n e , a d e n i n e , s t e r o i d , a n d a r o m a t i c h y d r o c a r b o n s a n d n u c l e i c acids, P r o c . N a t l . A c a d . Sci. (U.S.), 51 ( 1 9 6 4 ) 1 7 - - 2 4 . 6 Y a m a g u c b i , H., T. N a i t o a n d A. T a t a r a , T h e i n h i b i t i n g e f f e c t o f E D T A o n the repair o f g a m m a - r a y i n d u c e d l e s i o n s in g e r m i n a t i n g barley s e e d s , Japan. J. B r e e d i n g , 2 4 ( 1 9 7 4 ) 1 8 8 - - 1 9 3 . 7 Y a m a g u c h i , H., A. T a t a r a a n d T. N a i t o , U n s c h e d u l e d D N A s y n t h e s i s i n d u c e d in barley s e e d s b y g a m m a rays a n d 4 - n i t r o q u i n o l i n e 1 - o x i d e , J a p a n , J. G e n e t i c s , 5 0 ( 1 9 7 5 ) 3 0 7 - - 3 1 8 . 8 Y a m a g u c h i , H., A. T a t a r a a n d T. N a i t o , A c c e s s i b i l i t y o f 7 - r a y i n d u c e d p r i m e r t o w a r d D N A p o l y m e r a s e I o f Escherichia coli d u r i n g s o a k i n g o f barley s e e d , E n v i r o n m . a n d E x p t l . B o t a n y , 16 ( 1 9 7 6 ) 1 4 1 - - 1 4 4 . 9 Y a m a m o t o , K. a n d H . Y a m a g u c h i , I n h i b i t i o n b y c a f f e i n e o f the repair o f ? - r a y - i n d u c e d c h r o m o s o m e breaks in b a r l e y , M u t a t i o n Res. 8 ( 1 9 6 9 ) 4 2 8 - - 4 3 0 .