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The influence of spermidine (polyamines) on DNA depurination in vitro
Biochimica et Biophysica Acta, 698 (1982) 100-101
100
Elsevier Biomedical Press
BBA Report BBA 90010
THE INFLUENCE OF SPERMIDINE (POLYAMINES) ON DNA DEPURINATION IN VITRO O L E G A. A N D R E E V * and O L E G K. K A B O E V **
Leningrad Nuclear Physics Institute, U.S.S.R. Academy of Sciences, Gatchina, Leningrad district 188350 (U.S.S.R.) (Received March 24th, 1982)
Key words: DNA depurination," Polyamine," Spermidine
The influence of the spermidine, spermine and putrescine on the DNA depurination rate was studied. These polyamines protect DNA against depurination. The rate of Col E1 DNA depurination at pH 4.3 was decreased over 10-fold by addition of 10 mM polyamines.
Greer and Zamenhof [l], and Lindahl and Nyberg [2] provided the evidence that free purines were released from DNA after heating. From these data it may be proposed that DNA from mammalian cells loosed about 10000 purines every 20 h after heating at 37°C [3]. However, the DNA in cells interacts with naturally occurring polyamines [4,5]. How they to influence DNA depurination has not as yet been determined. This report describes protection of DNA against depurination by spermidine, spermine and putrescine. 14C-labelled Col E1 DNA (100 #g/ml, 10000 cpm//~g, 80% superhelical forms) was prepared as described previously [6] and stored in 5 mM TrisHC1, pH 8.0/0.5 mM EDTA/0.1 M NaC1/1 mM NaN 3 (Buffer A). The mixture for DNA depurination (0.1 ml) contained 8/~g Col E1 D N A / 4 mM Tris-HC1/0.4 mM EDTA/80 mM NaC1/50 mM sodium acetate buffer/0-10 mM polyamines. The final pH value was 4.3 (measured at room temperature). After heating, the partly depurinated DNA was desalted by Sepharose 4B gel-filtration (the column was equilibrated with Buffer A). To de* Present address: Institute of Cytology, Academy of Sciences of the U.S.S.R., Leningrad, U.S.S.R. **To whom correspondence should be addressed. 0167-4781/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
termine the number of apurinic sites per Col EI molecule two methods were used. The first was based on the measurement of apurinic-endonuclease-sensitive sites according to Bekker et al.
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5PERHIDINE j m/u[ Fig. 1. Correlation between spermidine concentration and the decrease of the D N A depurination rate. The time of incubation was 10 min at 60°C, p H 4.3. The n u m b e r of AP-sites was determined as described in the text.
101 TABLE I THE EFFECT OF DECREASING DNA DEPURINATION BY POLYAM1NES AP-endo, apurinic/apyrimidinicDNA-endonuclease. The Col E1 DNA was depurinated by heating for 10 min at 54°C, pH 4.3, with 0.15 M NaC1. Compounds added
None 6 mM MgCI2 6 mM putrescine.2 HCI 6 mM spermidine.3 HC1 6 mM spermine. 4 HC1 12 mM spermine. 4 HC1
Nicks/per Col E1 molecule (S.D. --+0.04) Plus AP-endo
Minus AP-endo
AP-sites
1.24 1.02 0.68 0.56 0.49 0.42
0.22 0.24 0.29 0.30 0.32 0.34
1.02 0.78 0.39 0.26 0.17 0.08
[6] ( i n c u b a t i o n of Col E1 D N A with excess A P - e n d o n u c l e a s e was p e r f o r m e d for 15 m i n at 60°C). A P - e n d o n u c l e a s e was p u r i f i e d f r o m Bacillus stearothermophilus n e a r to h o m o g e n e i t y b y p h o s phocellulose, D N A - a g a r o s e a n d h y d r o x y a p a t i t e c h r o m a t o g r a p h y a n d was free f r o m unspecific end o n u c l e a s e c o n t a m i n a t i o n . T h e r e was n o effect of p o l y a m i n e s (10 m M ) or MgC12 (10 m M ) on the e n z y m e activity. I n the s e c o n d m e t h o d the nicks in the d e p u r i n a t e d Col E1 D N A were i n t r o d u c e d b y t r e a t m e n t of D N A with 0.1 M p o t a s s i u m phosp h a t e buffer, p H 12.2, for 4 h at 37°C a c c o r d i n g to K u n l e i n et al. [7]. T h e n u m b e r o f the a p u r i n i c sites was c a l c u l a t e d b y a s s u m i n g a Poisson d i s t r i b u t i o n o f nicks p e r molecule. T h e results f r o m e x p e r i m e n t s are p r e s e n t e d in Fig. 1 a n d T a b l e I. W i t h increasing a m o u n t s of spermidine, the r a t e o f Col E1 D N A d e p u r i n a t i o n was d e c r e a s e d (Fig. 1). C o m p a r i s o n s of the p r o t e c tive effect of spermidine, spermine, p u t r e s c i n e a n d MgC12 showed that there were no c o n s i d e r a b l e differences b e t w e e n p o l y a m i n e s ( T a b l e I). A l i n e a r A r r h e n i u s p l o t gives an a c t i v a t i o n energy for C o l E1 d e p u r i n a t i o n at p H 4.3 a b o u t 2 5 - 2 6 k c a l / m o l with o r w i t h o u t 5 m M s p e r m i d i n e ( d a t a n o t shown). 15 m M p u r i n e nucleoside h a d n o effect b u t 10 m M A T P e v o k e d 3-fold r e d u c t i o n of the p r o t e c t i v e a c t i o n of 5 m M spermidine.
P r o b a b l y , the m e c h a n i s m of influence on the D N A d e p u r i n a t i o n is c o n n e c t e d with a screen o f the N - g l y c o s i d e b o n d s (i.e., the o x y g e n of d e o x y r i b o s e [8] b y p o l y a m i n e s u n d e r p r o t o n a t i o n conditions). T o sum up, the D N A d e p u r i n a t i o n rate for the D N A - p o l y a m i n e c o m p l e x e s should b e decreased. W e t h a n k L. N o s k i n a n d G. B a g y a n for helpful d i s c u s s i o n a n d M. A b a z e v a for t y p i n g the m a n u script.
References 1 Greer, S. and Zamenhof, S. (1962) J. Mol. Biol. 4, 123-126 2 Lindahl, T. and Nyberg, B. (1972) Biochemistry 19, 36103618 3 Lindahl, T. (1977) Cellular senescence and somatic cell genetics, DNA repair processes (Warren, W. et al., eds.), pp. 225-240, New-York 4 Barton, D., Forsen, S. and Reimarson, P. (1981) Nucleic Acid Res. 9, 1219-1228 5 Tabor, C. and Tabor, H. (1976) Annu. Rev. Biochem. 45, 285-306 6 Bekker, M., Kaboev, O., Akhmedov, A. and Luchkina, L. (1980) J. Bacteriol. 142 322-324 7 Kunlein, U. Penhoet, U., Lea, N. and Linn, s. (1979) Nutr. Res. 64, 167-182 8 Kochetcov, H., Budovskiy, E. Sverdlov, E., Semukova, N., Turchinsky, M. and Shibaev, B. (1970) Organic chemistry of nucleic acid, pp. 485-508, Chemistry, Moscow
100
Elsevier Biomedical Press
BBA Report BBA 90010
THE INFLUENCE OF SPERMIDINE (POLYAMINES) ON DNA DEPURINATION IN VITRO O L E G A. A N D R E E V * and O L E G K. K A B O E V **
Leningrad Nuclear Physics Institute, U.S.S.R. Academy of Sciences, Gatchina, Leningrad district 188350 (U.S.S.R.) (Received March 24th, 1982)
Key words: DNA depurination," Polyamine," Spermidine
The influence of the spermidine, spermine and putrescine on the DNA depurination rate was studied. These polyamines protect DNA against depurination. The rate of Col E1 DNA depurination at pH 4.3 was decreased over 10-fold by addition of 10 mM polyamines.
Greer and Zamenhof [l], and Lindahl and Nyberg [2] provided the evidence that free purines were released from DNA after heating. From these data it may be proposed that DNA from mammalian cells loosed about 10000 purines every 20 h after heating at 37°C [3]. However, the DNA in cells interacts with naturally occurring polyamines [4,5]. How they to influence DNA depurination has not as yet been determined. This report describes protection of DNA against depurination by spermidine, spermine and putrescine. 14C-labelled Col E1 DNA (100 #g/ml, 10000 cpm//~g, 80% superhelical forms) was prepared as described previously [6] and stored in 5 mM TrisHC1, pH 8.0/0.5 mM EDTA/0.1 M NaC1/1 mM NaN 3 (Buffer A). The mixture for DNA depurination (0.1 ml) contained 8/~g Col E1 D N A / 4 mM Tris-HC1/0.4 mM EDTA/80 mM NaC1/50 mM sodium acetate buffer/0-10 mM polyamines. The final pH value was 4.3 (measured at room temperature). After heating, the partly depurinated DNA was desalted by Sepharose 4B gel-filtration (the column was equilibrated with Buffer A). To de* Present address: Institute of Cytology, Academy of Sciences of the U.S.S.R., Leningrad, U.S.S.R. **To whom correspondence should be addressed. 0167-4781/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
termine the number of apurinic sites per Col EI molecule two methods were used. The first was based on the measurement of apurinic-endonuclease-sensitive sites according to Bekker et al.
£/
/.t
/.3
0.9
o.£ I
i
J
I
t
o
2
4
6
8
5PERHIDINE j m/u[ Fig. 1. Correlation between spermidine concentration and the decrease of the D N A depurination rate. The time of incubation was 10 min at 60°C, p H 4.3. The n u m b e r of AP-sites was determined as described in the text.
101 TABLE I THE EFFECT OF DECREASING DNA DEPURINATION BY POLYAM1NES AP-endo, apurinic/apyrimidinicDNA-endonuclease. The Col E1 DNA was depurinated by heating for 10 min at 54°C, pH 4.3, with 0.15 M NaC1. Compounds added
None 6 mM MgCI2 6 mM putrescine.2 HCI 6 mM spermidine.3 HC1 6 mM spermine. 4 HC1 12 mM spermine. 4 HC1
Nicks/per Col E1 molecule (S.D. --+0.04) Plus AP-endo
Minus AP-endo
AP-sites
1.24 1.02 0.68 0.56 0.49 0.42
0.22 0.24 0.29 0.30 0.32 0.34
1.02 0.78 0.39 0.26 0.17 0.08
[6] ( i n c u b a t i o n of Col E1 D N A with excess A P - e n d o n u c l e a s e was p e r f o r m e d for 15 m i n at 60°C). A P - e n d o n u c l e a s e was p u r i f i e d f r o m Bacillus stearothermophilus n e a r to h o m o g e n e i t y b y p h o s phocellulose, D N A - a g a r o s e a n d h y d r o x y a p a t i t e c h r o m a t o g r a p h y a n d was free f r o m unspecific end o n u c l e a s e c o n t a m i n a t i o n . T h e r e was n o effect of p o l y a m i n e s (10 m M ) or MgC12 (10 m M ) on the e n z y m e activity. I n the s e c o n d m e t h o d the nicks in the d e p u r i n a t e d Col E1 D N A were i n t r o d u c e d b y t r e a t m e n t of D N A with 0.1 M p o t a s s i u m phosp h a t e buffer, p H 12.2, for 4 h at 37°C a c c o r d i n g to K u n l e i n et al. [7]. T h e n u m b e r o f the a p u r i n i c sites was c a l c u l a t e d b y a s s u m i n g a Poisson d i s t r i b u t i o n o f nicks p e r molecule. T h e results f r o m e x p e r i m e n t s are p r e s e n t e d in Fig. 1 a n d T a b l e I. W i t h increasing a m o u n t s of spermidine, the r a t e o f Col E1 D N A d e p u r i n a t i o n was d e c r e a s e d (Fig. 1). C o m p a r i s o n s of the p r o t e c tive effect of spermidine, spermine, p u t r e s c i n e a n d MgC12 showed that there were no c o n s i d e r a b l e differences b e t w e e n p o l y a m i n e s ( T a b l e I). A l i n e a r A r r h e n i u s p l o t gives an a c t i v a t i o n energy for C o l E1 d e p u r i n a t i o n at p H 4.3 a b o u t 2 5 - 2 6 k c a l / m o l with o r w i t h o u t 5 m M s p e r m i d i n e ( d a t a n o t shown). 15 m M p u r i n e nucleoside h a d n o effect b u t 10 m M A T P e v o k e d 3-fold r e d u c t i o n of the p r o t e c t i v e a c t i o n of 5 m M spermidine.
P r o b a b l y , the m e c h a n i s m of influence on the D N A d e p u r i n a t i o n is c o n n e c t e d with a screen o f the N - g l y c o s i d e b o n d s (i.e., the o x y g e n of d e o x y r i b o s e [8] b y p o l y a m i n e s u n d e r p r o t o n a t i o n conditions). T o sum up, the D N A d e p u r i n a t i o n rate for the D N A - p o l y a m i n e c o m p l e x e s should b e decreased. W e t h a n k L. N o s k i n a n d G. B a g y a n for helpful d i s c u s s i o n a n d M. A b a z e v a for t y p i n g the m a n u script.
References 1 Greer, S. and Zamenhof, S. (1962) J. Mol. Biol. 4, 123-126 2 Lindahl, T. and Nyberg, B. (1972) Biochemistry 19, 36103618 3 Lindahl, T. (1977) Cellular senescence and somatic cell genetics, DNA repair processes (Warren, W. et al., eds.), pp. 225-240, New-York 4 Barton, D., Forsen, S. and Reimarson, P. (1981) Nucleic Acid Res. 9, 1219-1228 5 Tabor, C. and Tabor, H. (1976) Annu. Rev. Biochem. 45, 285-306 6 Bekker, M., Kaboev, O., Akhmedov, A. and Luchkina, L. (1980) J. Bacteriol. 142 322-324 7 Kunlein, U. Penhoet, U., Lea, N. and Linn, s. (1979) Nutr. Res. 64, 167-182 8 Kochetcov, H., Budovskiy, E. Sverdlov, E., Semukova, N., Turchinsky, M. and Shibaev, B. (1970) Organic chemistry of nucleic acid, pp. 485-508, Chemistry, Moscow