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On the existence of intrastrand crosslinks in DNA alkylated with sulfur mustard
657
SHORT COMMUNICATIONS
BBA 93572
On the existence of introstrond crosslinks in D N A olkyloted with sulfur mustord It is well known that bifunctional alkylating agents such as nitrogen and sulfur mustard are more toxic to eucaryotic and procaryotic cells than their monofunctional analogs 1,2. The basis of this enhanced toxicity was thought to be the result of bifunctional agents crosslinking the opposite strands of a DNA duplex via the alkylation of two guanines positioned nearly opposite each other in the duplex a. It was, for a time, considered that essentially all diguaninyl alkylation products were derived from such interstrand crosslinks e,~. More recently, indirect evidence has been reported which suggests that intrastrand crosslinks (occurring between adjacent guanines on a given DNA strand) are three or four times more common following sulfur mustard treatment than crosslinks of the interstrand typO. Furthermore, these intrastrand crosslinks appeared to represent a highly significant factor in the production of cellular toxicity. To provide, in this paper, evidence of a more direct nature for the existence of such crosslinks we have selected some unusual DNA fractions for alkylation with sulfur mustard. These DNA's are from m a m m a l i a n sources and represent the so-called mouse and guinea pig satellites 5-s. Both the isolated single strands 5,e and the duplexes 9,6 were subjected to alkylation with sulfur mustard as described in Fig. I and acid hydrolysates were prepared for chromatographic separation of alkylation products. A typical chromatographic
4oI L
i'°[ ~)rigin
20
40
60
80
Froctions
Fig. I. P a p e r c h r o m a t o g r a p h i c s e p a r a t i o n of the alkylation p r o d u c t s of an acid h y d r o l y s a t e of D N A previously alkylated w i t h 35S-labeled m u s t a r d gas (63o mC/mmole; Nuclear of Chicago). Total m o u s e D N A (2o/,g/ml) was t r e a t e d for 3° rain in i ml of aqueous I mM s o d i u m acetate (pH 7.2) at 3°° w i t h o.o 4 mM 8~S-labeled m u s t a r d gas; e x t e n t of reaction, approx. 3 mmole]mole D N A nucleotide. The reaction m i x t u r e w a s diluted 2-fold with a o ° solution containing o.o8 M NaC1, o.o2 M Tris-HC1 and I mM E D T A (pH 8.0) and dialyzed against the same solution for 2 h in the cold. Acid hydrolysis of alkylated D N A and descending p a p e r c h r o m a t o g r a p h y of the h y d r o lysate in a 2-propanol-conc. H C l - w a t e r (17:4.1:3. 9, b y vol.) solvent s y s t e m was carried o u t as described b y LAWLEY et al. 4. R a d i o a c t i v i t y m e a s u r e m e n t s of laterally sectioned strips were obtained f r o m a P a c k a r d Tri-Carb, Model 544. F r a c t i o n I corresponds to the diguaninyl derivative [di(guanin-7-yl-ethyl) sulfide] and F r a c t i o n I [ to the m o n o g u a n i n y l derivative [7-(2'-hydroxyethylthioethyl)guanine~. Biochim. Biophys. Acta, 224 (197o) 657-659
658
SHORT COMMUNICATIONS
separation of an acid-hydrolyzed, alkylated DNA is shown in Fig. I in which total mouse DNA was employed. The first peak labelled (I) represents the diguaninyl product [di-(guaninyl-7-yl-ethyl ) sulfidel while (II) corresponds to the monoguaninyl [7- (2'-hydroxyethylthioethyl)guanineJ. The special advantage afforded by the satellite DNA's relates to their interstrand compositional bias and to the fact that their individual complementary strands can be isolated 5,~. As shown by the data of Table I, isolated single strands which, according to earlier studies, do not interact but remain single-stranded ~,8, contain both mono- and diguaninyl products following alkylation. The relatively high proportion of diguaninyl derivative associated with alkylated single-strands argues in favor of intrastrand crosslinks since the hydrolysis of the unreacted arm of the mustard would probably preclude most intermolecular reactions. It should, however, be recognized that on account of structural differences, duplexed DNA would not necessarily behave in a manner similar to single strands (i.e., develop intrastrand crosslinks). For this reason, the e-satellite duplex of the guinea pig was examined, following alkylation, for the presence of diguaninyl derivatives. This is relevant since of the two strands of the e-satellite, the H~ contains 36 % guanine while its complement, the Le, contains only 2 % guanine *. TABLE 1 PERCENTAGE
OF D I G U A N I N Y L
PRODUCTS FOUND IN
DNA's
OF D I F F E R I N G
BASE COMPOSITION
DNA Sample"
Mole °/o guanine in DNA
% Diguaninyl to total alkylated guanine
Total mouse, native Total mouse, denatured Mouse satellite, H-strand Mouse satellite, L-strand Guinea pig, He-strand Guinea pig, s-satellite
21 2I 14 22 36 t9
I5.9 12.9 18. 5 28. 7 3o-o 26.0
" DNA was extracted as previously reported 9. This imbalance in the distribution of guanine means that only a small proportion of the alkylated guanines on the H e strand would have an opportunity of undergoing a second reaction with a guanine on the opposite strand. Hence, if diguaninyl products arise solely from interstrand crosslinks, their propoltion among total alkylated guanines would be very low (approx. 5 %). On the other hand, if diguaninyl products are also derived from intrastrand crosslinks this proportion might be substantially higher. Since the proportion is 26 % (Table I), we conclude that intrastrand crosslinking of adjacent guanines does indeed occur upon the reaction of sulfur mustard with DNA. National Institute o] Environmental Health Sciences, National Institutes o] Health, Public Health Service Department o] Health, Education and Wel]are, Research Triangle Park, N.C. 277o 9 (U.S.A.) Bioehim. t?iophys, dcta, 224 (I97o) 657-659
W. G. FLAMM N. J. BERNHEIM
L. FISHBEIN
SHORT COMMUNICATIONS I 2 3 4 5 6 7 8 9
659
13. BROOKES AND P. D. LAWLEY, Biochem. J., 80 (1961) 496. E. FREESE, in J. H. TAYLOR, Molecular Genetics, Academic 1Dress, N e w York, 1963, p. 207. 13. D. LAWLEY AND 13. BROOKES,J. Mol. Biol., 25 (1967) 143. 13. D. LAWLEY, J. H. LETHBRIDGE, P. A. EDWARDS AND K. V. SHOOTER, J. Mol. Biol., 39 (1969) 181. W. G. FLAMM,M~. 2tV[CCALLUMAND 13. ~V~.B. WALKER, Proc. Natl. Acad. Sci. U.S., 57 (1967) 1729. G. CORNED, E. GINELLI, C. SOAVE AND Cr. BERNARDI, Biochemistry, 7 (1968) 4373. Vv'. G. I~'LAMM, 13. 1Vf. B. WALKER AND M. 1V[CCALLUM, J . Mol. Biol., 4 ° (1969) 423 . W. (3-. FLAMM, P. M. B. WALKER AND 1V[. IV[CCALLUM, J. Mol. Biol., 4 z (i969) 441. W. O. FLAMM, H. E. BOND AND H. E. BURR, Biochim. Biophys. Acta, 129 (1966) 652.
Received September 28th, 197o Biochim. Biophys. Acta, 224 (197 o) 657-659
SHORT COMMUNICATIONS
BBA 93572
On the existence of introstrond crosslinks in D N A olkyloted with sulfur mustord It is well known that bifunctional alkylating agents such as nitrogen and sulfur mustard are more toxic to eucaryotic and procaryotic cells than their monofunctional analogs 1,2. The basis of this enhanced toxicity was thought to be the result of bifunctional agents crosslinking the opposite strands of a DNA duplex via the alkylation of two guanines positioned nearly opposite each other in the duplex a. It was, for a time, considered that essentially all diguaninyl alkylation products were derived from such interstrand crosslinks e,~. More recently, indirect evidence has been reported which suggests that intrastrand crosslinks (occurring between adjacent guanines on a given DNA strand) are three or four times more common following sulfur mustard treatment than crosslinks of the interstrand typO. Furthermore, these intrastrand crosslinks appeared to represent a highly significant factor in the production of cellular toxicity. To provide, in this paper, evidence of a more direct nature for the existence of such crosslinks we have selected some unusual DNA fractions for alkylation with sulfur mustard. These DNA's are from m a m m a l i a n sources and represent the so-called mouse and guinea pig satellites 5-s. Both the isolated single strands 5,e and the duplexes 9,6 were subjected to alkylation with sulfur mustard as described in Fig. I and acid hydrolysates were prepared for chromatographic separation of alkylation products. A typical chromatographic
4oI L
i'°[ ~)rigin
20
40
60
80
Froctions
Fig. I. P a p e r c h r o m a t o g r a p h i c s e p a r a t i o n of the alkylation p r o d u c t s of an acid h y d r o l y s a t e of D N A previously alkylated w i t h 35S-labeled m u s t a r d gas (63o mC/mmole; Nuclear of Chicago). Total m o u s e D N A (2o/,g/ml) was t r e a t e d for 3° rain in i ml of aqueous I mM s o d i u m acetate (pH 7.2) at 3°° w i t h o.o 4 mM 8~S-labeled m u s t a r d gas; e x t e n t of reaction, approx. 3 mmole]mole D N A nucleotide. The reaction m i x t u r e w a s diluted 2-fold with a o ° solution containing o.o8 M NaC1, o.o2 M Tris-HC1 and I mM E D T A (pH 8.0) and dialyzed against the same solution for 2 h in the cold. Acid hydrolysis of alkylated D N A and descending p a p e r c h r o m a t o g r a p h y of the h y d r o lysate in a 2-propanol-conc. H C l - w a t e r (17:4.1:3. 9, b y vol.) solvent s y s t e m was carried o u t as described b y LAWLEY et al. 4. R a d i o a c t i v i t y m e a s u r e m e n t s of laterally sectioned strips were obtained f r o m a P a c k a r d Tri-Carb, Model 544. F r a c t i o n I corresponds to the diguaninyl derivative [di(guanin-7-yl-ethyl) sulfide] and F r a c t i o n I [ to the m o n o g u a n i n y l derivative [7-(2'-hydroxyethylthioethyl)guanine~. Biochim. Biophys. Acta, 224 (197o) 657-659
658
SHORT COMMUNICATIONS
separation of an acid-hydrolyzed, alkylated DNA is shown in Fig. I in which total mouse DNA was employed. The first peak labelled (I) represents the diguaninyl product [di-(guaninyl-7-yl-ethyl ) sulfidel while (II) corresponds to the monoguaninyl [7- (2'-hydroxyethylthioethyl)guanineJ. The special advantage afforded by the satellite DNA's relates to their interstrand compositional bias and to the fact that their individual complementary strands can be isolated 5,~. As shown by the data of Table I, isolated single strands which, according to earlier studies, do not interact but remain single-stranded ~,8, contain both mono- and diguaninyl products following alkylation. The relatively high proportion of diguaninyl derivative associated with alkylated single-strands argues in favor of intrastrand crosslinks since the hydrolysis of the unreacted arm of the mustard would probably preclude most intermolecular reactions. It should, however, be recognized that on account of structural differences, duplexed DNA would not necessarily behave in a manner similar to single strands (i.e., develop intrastrand crosslinks). For this reason, the e-satellite duplex of the guinea pig was examined, following alkylation, for the presence of diguaninyl derivatives. This is relevant since of the two strands of the e-satellite, the H~ contains 36 % guanine while its complement, the Le, contains only 2 % guanine *. TABLE 1 PERCENTAGE
OF D I G U A N I N Y L
PRODUCTS FOUND IN
DNA's
OF D I F F E R I N G
BASE COMPOSITION
DNA Sample"
Mole °/o guanine in DNA
% Diguaninyl to total alkylated guanine
Total mouse, native Total mouse, denatured Mouse satellite, H-strand Mouse satellite, L-strand Guinea pig, He-strand Guinea pig, s-satellite
21 2I 14 22 36 t9
I5.9 12.9 18. 5 28. 7 3o-o 26.0
" DNA was extracted as previously reported 9. This imbalance in the distribution of guanine means that only a small proportion of the alkylated guanines on the H e strand would have an opportunity of undergoing a second reaction with a guanine on the opposite strand. Hence, if diguaninyl products arise solely from interstrand crosslinks, their propoltion among total alkylated guanines would be very low (approx. 5 %). On the other hand, if diguaninyl products are also derived from intrastrand crosslinks this proportion might be substantially higher. Since the proportion is 26 % (Table I), we conclude that intrastrand crosslinking of adjacent guanines does indeed occur upon the reaction of sulfur mustard with DNA. National Institute o] Environmental Health Sciences, National Institutes o] Health, Public Health Service Department o] Health, Education and Wel]are, Research Triangle Park, N.C. 277o 9 (U.S.A.) Bioehim. t?iophys, dcta, 224 (I97o) 657-659
W. G. FLAMM N. J. BERNHEIM
L. FISHBEIN
SHORT COMMUNICATIONS I 2 3 4 5 6 7 8 9
659
13. BROOKES AND P. D. LAWLEY, Biochem. J., 80 (1961) 496. E. FREESE, in J. H. TAYLOR, Molecular Genetics, Academic 1Dress, N e w York, 1963, p. 207. 13. D. LAWLEY AND 13. BROOKES,J. Mol. Biol., 25 (1967) 143. 13. D. LAWLEY, J. H. LETHBRIDGE, P. A. EDWARDS AND K. V. SHOOTER, J. Mol. Biol., 39 (1969) 181. W. G. FLAMM,M~. 2tV[CCALLUMAND 13. ~V~.B. WALKER, Proc. Natl. Acad. Sci. U.S., 57 (1967) 1729. G. CORNED, E. GINELLI, C. SOAVE AND Cr. BERNARDI, Biochemistry, 7 (1968) 4373. Vv'. G. I~'LAMM, 13. 1Vf. B. WALKER AND M. 1V[CCALLUM, J . Mol. Biol., 4 ° (1969) 423 . W. (3-. FLAMM, P. M. B. WALKER AND 1V[. IV[CCALLUM, J. Mol. Biol., 4 z (i969) 441. W. O. FLAMM, H. E. BOND AND H. E. BURR, Biochim. Biophys. Acta, 129 (1966) 652.
Received September 28th, 197o Biochim. Biophys. Acta, 224 (197 o) 657-659