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The rate of hydrolysis of methyl phosphotriesters in DNA under conditions used in alkaline sucrose gradients
155
Biochimica et Biophysica Acta, 521 (1978) 155--159 © Elsevier/North-Holland Biomedical Press
BBA 99282
THE RATE OF H Y D R O L Y S I S OF M E T H Y L P H O S P H O T R I E S T E R S IN DNA U N D E R CONDITIONS USED IN A L K A L I N E SUCROSE G R A D I E N T S
K.V. SHOOTER and R.K. MERRIFIELD
Carcinogenesis Division, Institute of Cancer Research, Pollards Wood Research Station, Nightingales Lane, Chalfont St. Giles, Bucks. HP8 4SP (U.K.) (Received February 23rd, 1978)
Summary Methyl phosphotriesters have been introduced into DNA, in vitro, b y reaction with N-methyl-N-nitrosourea and the rate of degradation in alkali has been followed b y measurements of the mean sedimentation coefficient using an analytical ultracentrifuge. In 0.3 M NaOH/0.7 M NaC1, a solution c o m m o n l y used in alkaline sucrose gradient experiments, hydrolysis of the methyl phosphotriesters present is complete after 15 h at 20°C, or after 2--3 h at 37°C. In addition to breaks formed b y the latter reaction there was a continuous background degradation of the DNA giving rise to 6.3 and 63 breaks/106 nucleotides per h at 20 ° and 37°C, respectively. The problem of obtaining quantitative data on phosphotriester concentrations from results of alkaline sucrose gradient experiments has been discussed.
Introduction
There is n o w considerable evidence that the process of initiation in chemical carcinogenesis m a y result from the reaction of the chemical or one of its metabolites with cellular DNA. Detailed studies have shown that most of the lesions induced in DNA b y these reactions are subject to repair b y enzymatic systems in cells or are lost b y hydrolysis [1]. The kinetics of the processes b y which lesions in DNA are eliminated can be followed using the technique of sedimentation on alkaline sucrose gradients, a m e t h o d which provides information on the size of the single strands o f the DNA. Repair processes lead at first to a progressive reduction in the length of single DNA strands as cuts or breaks are made near lesions and this is followed by a return towards normal strand lengths as rejoining o f the broken strands occurs [2,3]. Elimination of lesions b y hydrolysis (e.g. o f 7-alkylguanine) follows the same apparent pattern since apurinic sites left by loss of the altered bases are converted to single strand
156
breaks either by specific nucleases within cells as part of the repair process or, if such sites are present, on addition of alkali. In general, such studies indicate that cellular repair of lesions in DNA is a fairly rapid process. Other work on the persistence of alkylated bases in DNA has confirmed these observations and has shown that the loss of alkylated bases by hydrolysis is also fairly rapid, e.g. elimination of 7-alkylguanine occurs with a half-life of about 2--3 days [1]. From the sucrose gradient work there is, however, evidence that, after treatment with some carcinogens, cellular DNA contains alkali-labile sites which persist for long periods [2,4]. It was suggested by den Engelse and Philippus [4] that these persistent lesions in the DNA could be alkyl phosphotriesters. The alkaline hydrolysis of methyl and ethyl phosphotriesters has been studied in some detail and a method for estimating their concentration in DNA in the presence of single-strand breaks and apurinic sites has been developed [5,6]. Using this method of assay the persistence of methyl and ethyl phosphotriesters in DNA in vivo and their accumulation following repeated injections of small doses of appropriate alkylating agents has been investigated and it was concluded that these lesions were not excised by the enzymatic repair system of cells and, in the absence of gross tissue injury, the phosphotriesters were stable for several weeks at least [7,8]. It seems highly probable, therefore, that the persistent alkali-labile sites detected in alkaline sucrose gradient experiments could be alkyl phosphotriesters [4]. The question remains, do the measurements of the frequency of chain breaks in alkaline sucrose gradients give a quantitative measure of the concentration of phosphotriesters in DNA? Results of experiments described below suggest that only a small proportion of the phosphotriesters present hydrolyse under the conditions normally used for lysing cells or nuclei and freeing the DNA but that hydrolysis of these lesions is completed during the centrifugation period.
Experimental Bacteriophage T7 was grown and the DNA isolated by the phenol method as previously described [5]. Methyl phosphotriesters were introduced into the DNA (400/~g/ml in 0.1 M Tris • HC1 pH 8.5) by adding 0.08 mg/ml. N-methylN-nitrosourea and incubating at 37°C for 30 min. At the end of this time the DNA solution was diluted to 120 #g/ml in 0.3 M NaOH/0.7 M NaC1, a medium commonly used in alkaline sucrose gradients and in the preliminary lysis stage [e.g. 9--12]. The rate of hydrolysis in such solutions was determined at 20 ° and 37°C. After various incubation times samples were taken and diluted to 40 #g/ml D N A in 0.1 M NaOH/0.9 M NaC1 and the mean sedimentation rate of the D N A determined by the boundary velocity method using a Beckman Model E ultracentrifuge fitted with ultraviolet absorption optics. Varian@s and standard deviations of the estimates of sedimentation coefficients were calculated from the scatter of .observed data in the log (radial distance) ~ time plot. The average number of breaks present in each single strand of phage T7 D N A (Mr 12.5 • 106, 38000 nucleotides) was calculated from the equation No. of breaks, B = 2(8o/S)2"s _ 1
157
where S is the mean sedimentation coefficient of treated, incubated DNA and So the sedimentation coefficient of the unbroken DNA single strands. In the present experiments, under the conditions and concentration used S0 = 32.6 (S.D. = 0.2). The variance o f the estimate of the number o f breaks V(B) was calculated from the variances of the sedimentation coefficients V(So) and V(S) using the equation V(B) = (5S -2"5 X S0~'s)2V(S0) + (58 -3.s X S~'s)2V(S) In both of the experiments the mean sedimentation coefficient o f the DNA was measured immediately after adding alkali to the reaction mixture to provide a zero time point from which subsequent degradation could be followed. At the beginning of the experiment in which DNA was incubated at 20°C the average number o f breaks per single strand was 3.1 (S.D. 0.08). By the time the experiments at 37°C was performed the initial number of breaks per strand, effectively the number in the untreated, control DNA, had increased to 6.4 (S.D. 0.15). Results The time course o f the degradation o f the alkylated DNA over 3--4 days is shown in Fig. l a and the early stages o f this degradation are shown in Fig. lb. The breaks observed at zero time, i.e. immediately after adding the alkali, will include the single-strand breaks present in the DNA of the original preparation plus breaks formed at any apurinic sites which may have formed during the initial reaction with the nitrosourea. On addition of alkali the imidazole ring of the purines is opened and this reaction blocks any further loss of alkylated b 4C
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F i g . 1. T h e d e g r a d a t i o n of methylated DNA on incubation i n 0 . 3 M N a O H , 0 . 7 M N a t l . a. s h o w s t h e d e g r a d a t i o n o b s e r v e d o v e r 3 - - 4 d a y S , a n d b. s h o w s in m o r e d e t a i l t h e e a r l y s t a g e s d u r i n g w h i c h h y d r o l y s i s of methyl phosphotxiestere is o c c u r r i n g . T h e b a r s o n t h e d a t a o f b s h o w t h e e s t i m a t e o f t h e n u m b e r o f b r e a k s + t h e s t a n d a r d d e v i a t i o n , e , i n c u b a t e d i n a l k a l i at 2 0 ° C ; o, a t 3 7 ° C .
158
purines. During the early stages of the incubation in alkali the majority of breaks formed result from the hydrolysis of the methyl phosphotriester present (a more detailed discussion of this point is contained in refs. 5, 6). At later times, and presumably during the early stage of the incubation as well, degradation of the DNA continues, the rate of formation of breaks in the DNA chain being proportional to the time of incubation. Extrapolation from the data obtained after the longer periods of incubation back to zero time provides an estimate of the number of breaks due to hydrolysis of the phosphotriesters plus the breaks observed immediately on addition of the alkali. The extrapolated data give intercepts at 18.1 (S.D. 1.5) breaks after the incubation at 37°C and 16.3 (S.D. 0.4) for the 20°C incubation. Deducting from these values the corresponding estimates of the numbers of breaks present at the beginning of the incubation 6.4 (S.D. 0.15) and 3.1 (S.D. 0.08) respectively gives the number of breaks per single strand of phage T7 DNA due to phosphotriesters determined at the t w o incubation temperatures as 11.7 (S.D. 1.7) and 13.2 (S.D. 0.5). Inspection of the curves of Fig. l b shows that the time required for the hydrolysis of all the phosphotriesters present is about 15 h at 20°C and 2--3 hours at 37 ° C. However, at these end points, the continuous degradation of the DNA has introduced an extra 3--4 breaks per molecule at 20 ° and a b o u t 6 breaks per molecule at 37°C. Previous experiments have demonstrated that control DNA is progressively degraded in alkali when incubated at 37 ° b u t only after a lag period of 12--24 h [5]. Alkylation of the DNA was found to increase the rate of degradation and also to eliminate the lag period (Fig. 4 a in ref. 5). In the present experiments the observed rates of the background degradation are, at 20 ° and 37°C, respectively, 0.24 and 2.4 breaks/single-strand T7 DNA per h, or 6.3 and 63 breaks/106 nucleotides per h. It may be concluded from these observations that, if cells are lysed in medium containing 0.3 M NaOH, only a b o u t one fifth of the alkylphosphotriesters in the DNA will be hydrolysed in 1 h at 20°C, b u t all such lesions present would be hydrolysed by the end of a 16 h centrifugation at this temperature. Incubation for 1 h at 37°C would introduce breaks equivalent to the number which would be produced b y complete hydrolysis of phosphotriesters present and this procedure would probably lead to a best estimate of the concentration of these lesions. Factors such as the rate of background degradation of the DNA, as discussed above, would have to be considered in assessing the accuracy of the estimate. It must also be noted that the nature of the alkyl group in the phosphotriester may affect b o t h the rate of hydrolysis in alkali and the fraction of such lesions which, on hydrolysis, lead to chain breaks rather than to the simple loss of the alkyl group.
Acknowledgements We thank Miss W.L. House for technical assistance. This work was supported b y grants to the Institute of Cancer Research from the Medical Research Council and the Cancer Research Campaign.
References 1 Lawley P.D. (1976) in Screening T e s t s in Chemical Carcinogenesis (Montesano, R.0 Bat~sch, H. and Tomatis, L,, eds.), pp. 181--208, I.A.R.C. publication No. 12, L y o n s
159 2 Damjanov, I., Cox, R., Sarma, D.S.R. and F~rber, E. (1973) Cancer Res. 33, 2122--2128 3 Cleaver, J.E. (1974) in Repair processes for p h o t o c h e m i c a l damage in m a m m a l i a n cells (Lett, J.T., Adler, M. and Zelle, M. eds.) pp. 1--75, Academic Press New Y o r k 4 Den Engelse, L. and Phillippus, E.J. (1977) Chem.-Biol. I n t e r a c t i o n s 19, 111--124 5 Shooter, K.V. (1976) Chem.-Biol. I n t e r a c t i o n s 13, 151--163 6 Shooter, K.V. and Merrifield, R.K. (1976) Chem.-Biol. In t e ra c t i ons 13, 223--236 7 Shooter, K.V. and Slade, T.A. (1977) Chem.-Biol. I n t e r a c t i o n s 19, 353--361 8 Shooter, K.V., Slade, T.A. and O'Connor, P.J. (1977) Chem.-Biol. Int e ra c t i ons 19, 363--367 9 Coyle, M.B. and Strauss, B.S. ( 1 9 6 9 / 7 0 ) Chem.-Biol. I n t e r a c t i o n s 1, 89--98 10 Walker, I.G. and Ewart, D.F. (1973) Mutation Res. 1 9 331--341 11 Parodi, S., Mulivor, R.A., Martin, J.T., Nicolini, C., Sarma, D.S.R. and Farber, E. (1975) Biochim. Biophys. Acta 407, 174--190 12 Crathorn, A.R. and Schackleton, J. (1976) Chem.-Biol. I n t e r a c t i o n s 15, 117--130
Biochimica et Biophysica Acta, 521 (1978) 155--159 © Elsevier/North-Holland Biomedical Press
BBA 99282
THE RATE OF H Y D R O L Y S I S OF M E T H Y L P H O S P H O T R I E S T E R S IN DNA U N D E R CONDITIONS USED IN A L K A L I N E SUCROSE G R A D I E N T S
K.V. SHOOTER and R.K. MERRIFIELD
Carcinogenesis Division, Institute of Cancer Research, Pollards Wood Research Station, Nightingales Lane, Chalfont St. Giles, Bucks. HP8 4SP (U.K.) (Received February 23rd, 1978)
Summary Methyl phosphotriesters have been introduced into DNA, in vitro, b y reaction with N-methyl-N-nitrosourea and the rate of degradation in alkali has been followed b y measurements of the mean sedimentation coefficient using an analytical ultracentrifuge. In 0.3 M NaOH/0.7 M NaC1, a solution c o m m o n l y used in alkaline sucrose gradient experiments, hydrolysis of the methyl phosphotriesters present is complete after 15 h at 20°C, or after 2--3 h at 37°C. In addition to breaks formed b y the latter reaction there was a continuous background degradation of the DNA giving rise to 6.3 and 63 breaks/106 nucleotides per h at 20 ° and 37°C, respectively. The problem of obtaining quantitative data on phosphotriester concentrations from results of alkaline sucrose gradient experiments has been discussed.
Introduction
There is n o w considerable evidence that the process of initiation in chemical carcinogenesis m a y result from the reaction of the chemical or one of its metabolites with cellular DNA. Detailed studies have shown that most of the lesions induced in DNA b y these reactions are subject to repair b y enzymatic systems in cells or are lost b y hydrolysis [1]. The kinetics of the processes b y which lesions in DNA are eliminated can be followed using the technique of sedimentation on alkaline sucrose gradients, a m e t h o d which provides information on the size of the single strands o f the DNA. Repair processes lead at first to a progressive reduction in the length of single DNA strands as cuts or breaks are made near lesions and this is followed by a return towards normal strand lengths as rejoining o f the broken strands occurs [2,3]. Elimination of lesions b y hydrolysis (e.g. o f 7-alkylguanine) follows the same apparent pattern since apurinic sites left by loss of the altered bases are converted to single strand
156
breaks either by specific nucleases within cells as part of the repair process or, if such sites are present, on addition of alkali. In general, such studies indicate that cellular repair of lesions in DNA is a fairly rapid process. Other work on the persistence of alkylated bases in DNA has confirmed these observations and has shown that the loss of alkylated bases by hydrolysis is also fairly rapid, e.g. elimination of 7-alkylguanine occurs with a half-life of about 2--3 days [1]. From the sucrose gradient work there is, however, evidence that, after treatment with some carcinogens, cellular DNA contains alkali-labile sites which persist for long periods [2,4]. It was suggested by den Engelse and Philippus [4] that these persistent lesions in the DNA could be alkyl phosphotriesters. The alkaline hydrolysis of methyl and ethyl phosphotriesters has been studied in some detail and a method for estimating their concentration in DNA in the presence of single-strand breaks and apurinic sites has been developed [5,6]. Using this method of assay the persistence of methyl and ethyl phosphotriesters in DNA in vivo and their accumulation following repeated injections of small doses of appropriate alkylating agents has been investigated and it was concluded that these lesions were not excised by the enzymatic repair system of cells and, in the absence of gross tissue injury, the phosphotriesters were stable for several weeks at least [7,8]. It seems highly probable, therefore, that the persistent alkali-labile sites detected in alkaline sucrose gradient experiments could be alkyl phosphotriesters [4]. The question remains, do the measurements of the frequency of chain breaks in alkaline sucrose gradients give a quantitative measure of the concentration of phosphotriesters in DNA? Results of experiments described below suggest that only a small proportion of the phosphotriesters present hydrolyse under the conditions normally used for lysing cells or nuclei and freeing the DNA but that hydrolysis of these lesions is completed during the centrifugation period.
Experimental Bacteriophage T7 was grown and the DNA isolated by the phenol method as previously described [5]. Methyl phosphotriesters were introduced into the DNA (400/~g/ml in 0.1 M Tris • HC1 pH 8.5) by adding 0.08 mg/ml. N-methylN-nitrosourea and incubating at 37°C for 30 min. At the end of this time the DNA solution was diluted to 120 #g/ml in 0.3 M NaOH/0.7 M NaC1, a medium commonly used in alkaline sucrose gradients and in the preliminary lysis stage [e.g. 9--12]. The rate of hydrolysis in such solutions was determined at 20 ° and 37°C. After various incubation times samples were taken and diluted to 40 #g/ml D N A in 0.1 M NaOH/0.9 M NaC1 and the mean sedimentation rate of the D N A determined by the boundary velocity method using a Beckman Model E ultracentrifuge fitted with ultraviolet absorption optics. Varian@s and standard deviations of the estimates of sedimentation coefficients were calculated from the scatter of .observed data in the log (radial distance) ~ time plot. The average number of breaks present in each single strand of phage T7 D N A (Mr 12.5 • 106, 38000 nucleotides) was calculated from the equation No. of breaks, B = 2(8o/S)2"s _ 1
157
where S is the mean sedimentation coefficient of treated, incubated DNA and So the sedimentation coefficient of the unbroken DNA single strands. In the present experiments, under the conditions and concentration used S0 = 32.6 (S.D. = 0.2). The variance o f the estimate of the number o f breaks V(B) was calculated from the variances of the sedimentation coefficients V(So) and V(S) using the equation V(B) = (5S -2"5 X S0~'s)2V(S0) + (58 -3.s X S~'s)2V(S) In both of the experiments the mean sedimentation coefficient o f the DNA was measured immediately after adding alkali to the reaction mixture to provide a zero time point from which subsequent degradation could be followed. At the beginning of the experiment in which DNA was incubated at 20°C the average number o f breaks per single strand was 3.1 (S.D. 0.08). By the time the experiments at 37°C was performed the initial number of breaks per strand, effectively the number in the untreated, control DNA, had increased to 6.4 (S.D. 0.15). Results The time course o f the degradation o f the alkylated DNA over 3--4 days is shown in Fig. l a and the early stages o f this degradation are shown in Fig. lb. The breaks observed at zero time, i.e. immediately after adding the alkali, will include the single-strand breaks present in the DNA of the original preparation plus breaks formed at any apurinic sites which may have formed during the initial reaction with the nitrosourea. On addition of alkali the imidazole ring of the purines is opened and this reaction blocks any further loss of alkylated b 4C
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F i g . 1. T h e d e g r a d a t i o n of methylated DNA on incubation i n 0 . 3 M N a O H , 0 . 7 M N a t l . a. s h o w s t h e d e g r a d a t i o n o b s e r v e d o v e r 3 - - 4 d a y S , a n d b. s h o w s in m o r e d e t a i l t h e e a r l y s t a g e s d u r i n g w h i c h h y d r o l y s i s of methyl phosphotxiestere is o c c u r r i n g . T h e b a r s o n t h e d a t a o f b s h o w t h e e s t i m a t e o f t h e n u m b e r o f b r e a k s + t h e s t a n d a r d d e v i a t i o n , e , i n c u b a t e d i n a l k a l i at 2 0 ° C ; o, a t 3 7 ° C .
158
purines. During the early stages of the incubation in alkali the majority of breaks formed result from the hydrolysis of the methyl phosphotriester present (a more detailed discussion of this point is contained in refs. 5, 6). At later times, and presumably during the early stage of the incubation as well, degradation of the DNA continues, the rate of formation of breaks in the DNA chain being proportional to the time of incubation. Extrapolation from the data obtained after the longer periods of incubation back to zero time provides an estimate of the number of breaks due to hydrolysis of the phosphotriesters plus the breaks observed immediately on addition of the alkali. The extrapolated data give intercepts at 18.1 (S.D. 1.5) breaks after the incubation at 37°C and 16.3 (S.D. 0.4) for the 20°C incubation. Deducting from these values the corresponding estimates of the numbers of breaks present at the beginning of the incubation 6.4 (S.D. 0.15) and 3.1 (S.D. 0.08) respectively gives the number of breaks per single strand of phage T7 DNA due to phosphotriesters determined at the t w o incubation temperatures as 11.7 (S.D. 1.7) and 13.2 (S.D. 0.5). Inspection of the curves of Fig. l b shows that the time required for the hydrolysis of all the phosphotriesters present is about 15 h at 20°C and 2--3 hours at 37 ° C. However, at these end points, the continuous degradation of the DNA has introduced an extra 3--4 breaks per molecule at 20 ° and a b o u t 6 breaks per molecule at 37°C. Previous experiments have demonstrated that control DNA is progressively degraded in alkali when incubated at 37 ° b u t only after a lag period of 12--24 h [5]. Alkylation of the DNA was found to increase the rate of degradation and also to eliminate the lag period (Fig. 4 a in ref. 5). In the present experiments the observed rates of the background degradation are, at 20 ° and 37°C, respectively, 0.24 and 2.4 breaks/single-strand T7 DNA per h, or 6.3 and 63 breaks/106 nucleotides per h. It may be concluded from these observations that, if cells are lysed in medium containing 0.3 M NaOH, only a b o u t one fifth of the alkylphosphotriesters in the DNA will be hydrolysed in 1 h at 20°C, b u t all such lesions present would be hydrolysed by the end of a 16 h centrifugation at this temperature. Incubation for 1 h at 37°C would introduce breaks equivalent to the number which would be produced b y complete hydrolysis of phosphotriesters present and this procedure would probably lead to a best estimate of the concentration of these lesions. Factors such as the rate of background degradation of the DNA, as discussed above, would have to be considered in assessing the accuracy of the estimate. It must also be noted that the nature of the alkyl group in the phosphotriester may affect b o t h the rate of hydrolysis in alkali and the fraction of such lesions which, on hydrolysis, lead to chain breaks rather than to the simple loss of the alkyl group.
Acknowledgements We thank Miss W.L. House for technical assistance. This work was supported b y grants to the Institute of Cancer Research from the Medical Research Council and the Cancer Research Campaign.
References 1 Lawley P.D. (1976) in Screening T e s t s in Chemical Carcinogenesis (Montesano, R.0 Bat~sch, H. and Tomatis, L,, eds.), pp. 181--208, I.A.R.C. publication No. 12, L y o n s
159 2 Damjanov, I., Cox, R., Sarma, D.S.R. and F~rber, E. (1973) Cancer Res. 33, 2122--2128 3 Cleaver, J.E. (1974) in Repair processes for p h o t o c h e m i c a l damage in m a m m a l i a n cells (Lett, J.T., Adler, M. and Zelle, M. eds.) pp. 1--75, Academic Press New Y o r k 4 Den Engelse, L. and Phillippus, E.J. (1977) Chem.-Biol. I n t e r a c t i o n s 19, 111--124 5 Shooter, K.V. (1976) Chem.-Biol. I n t e r a c t i o n s 13, 151--163 6 Shooter, K.V. and Merrifield, R.K. (1976) Chem.-Biol. In t e ra c t i ons 13, 223--236 7 Shooter, K.V. and Slade, T.A. (1977) Chem.-Biol. I n t e r a c t i o n s 19, 353--361 8 Shooter, K.V., Slade, T.A. and O'Connor, P.J. (1977) Chem.-Biol. Int e ra c t i ons 19, 363--367 9 Coyle, M.B. and Strauss, B.S. ( 1 9 6 9 / 7 0 ) Chem.-Biol. I n t e r a c t i o n s 1, 89--98 10 Walker, I.G. and Ewart, D.F. (1973) Mutation Res. 1 9 331--341 11 Parodi, S., Mulivor, R.A., Martin, J.T., Nicolini, C., Sarma, D.S.R. and Farber, E. (1975) Biochim. Biophys. Acta 407, 174--190 12 Crathorn, A.R. and Schackleton, J. (1976) Chem.-Biol. I n t e r a c t i o n s 15, 117--130