Chem.-Biol. Interactions, 13 (1976) 153-163 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

THE KINETICS OF THE ALKALINE HYDROLYSIS OF IN DNA SHOOTER Chester Research Institute, of Cancer Research, Royal Cancer Hospital, Fulham Road, London SW3 6JB (Great Britain) (Received March 17th. 1975) (Revision received September 29th. 197 5 ; (Accepted October 2Oth, 1975)

SUMMARY

The degradation in alkali of normal DNA and DNA alkylated with dimethyl sulphate (DMS), N-methyl-IV-nitrosourea (MNUA) and N-ethyl-W nitrosourea (ENUA) has been investigated using analytical ultracentifugation techniques. For control T7-DNA (m.wt. denatured form 12.5 . lo6 daltons) the rate of degradation at 37” varies from 0.14 breaks/molecule/h in 0.1 M NaOH to 1.2 breaks/molecule/h in 0.4 M NaOH. When DNA is alkylated with reagents known to produce phosphotriesters addition of alkali leads to an initial rapid degradation not observed with control DNA. Ethyl phosphotriesters are hydrolysed at about half the rate of methyl phosphotriesters. Approximately one third of the methyl or ethyl phosphotriesters present hydrolyse to give breaks in the DNA chain.

INTRODUCTION

Although it has been suggested many times that reaction of nucleic acids with alkylating agents could yield phosphotriesters (see review, ref. 1) positive evidence for such products in reactions with DNA under near-physiological conditions has oniy recently been presented by Bannon and Verly [ 21. These authors were able to show that methyl and ethyl phosphotriesters in DNA are stable for 90 min at 100” in buffers at neutral pH and they were able to use this property as 3 basis for the analysis of phosphotriester content at a total alkylation of about 10 mmole alkyl/mole DNA-P. Further evidence for the formation of phosphotriesters has been provided by Lawley [3] who isolated, by column chromatography of enzymatic digests of methAbbreviations: DMS. dimethyl sulphate;ENUA. N-ethyl-N-nitrosourea; MNUA. N-methylN-nitrosourea.

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ylated DNA, products with properties consistent with the structure Ap(me)B where A and B denote deoxyribonucleoside residues and p(me) is a methylated phosphate group. This chromatographic method could be used to determine relative amounts of phosphotriesters and other alkylation products but it required a high degree of alkylation (130 mmoles alkyl/mole DNA-P) for accuracy. h contrast to the stability of the Ghosphotriesters in DNA such products formed in RNA hydrolyse readily leading to loss of the alkyl group or to scission of the RNA chain [4,5] . Shooter et al. [6) have studied the alkylation induced degradation of the RNA molecule of bacteriophage R17 in relation to the extent of alkylation produced by a series of methylating reagents-and to the inactivation of biological activity. Assuming that degradation of the RNA was a measure of phosphotriester formation it was possible to show that the amount of this product formed could be correlated with the formation of 06-alkylguanine, i.e. the formation of both of these products increased as the mechanism of alkylation tended from SN2 type to SNl type. Since this progression from one reaction mechanism to another also paralleled the increasing carcinogenic potency of the alkylating agents the biological effects of phosphotriester formation became of conslderable interest. Using the bacteriophage R17 system, further work with ethylating and isopropylating agents showed that a phosphotriester was not itself a lethal lesion [?I. Analysis of the biological effects of reacting the DNAcontaining bacteriophage T7 with ethyi methanesulphonate led Verly et al. [8] to the conclusion that an ethyl phosphotriester in this system was also not a lethal lesion. Despite these negative inferences regarding the biological effect of phosphotriesters in the bacteriophage systems such lesions in the DNA of a mammalian cell might have deleterious effects. Recent work, for example, has indicated some of the more subtle changes which the ‘presence of phosphotriesters -may produce in the biological functioning of’ nucleic acids [9,10]. There is also some evidence that in E. coli phosphotritsters are eliminated along with 06-methylguanine by the DNA repair systems leaving ‘I-methylguanine as the only alkylation product [ll] . It is, therefore, of some importance to be able to assay for phosphotriesters present in low amount and to be able to d! ;tinguish changes in the structure or stability of the DNA resulting from the presence of such lesions from similar changes due to other causes. In this paper we have used a model system to investigate in some detail the kinetics of the hydrolysis of phosphotriesters in alkaline solution to determine whether such techniques can be usefully applied to tknedetection and estimation of these groups in DNA. The alkali lability of phosphotriesters in DNA has already been noted by Rhaese and Freese [123. Walker and Ewart [13] have already attempted to use alkaline hydrolysis to estimate the frequency of phosphotriesters in .the DNA of L-cells treated with IV-methyl-iV’-nitro-N-nitrosoguanidine. Preparations of DNA from bacteriophage T7 have been alkylated with two reagents which have been shown to produce phosphotriesters [6], MNUA and ENIJA, and with DMS, a com-

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pound which does not produce measurable amounts of phosphotriester [6 3. The kinetics of the degradation in alkali have been studied usmg analytical ultracentrifugation methods to follow changes in molecular weight. The principle behind the method is that addition of alkali should lead to the opening of the imidazole ring of purines and thus block the spontaneous loss of alkyiated purines [ 14,151, leaving the phosphotriesters as the only alkali-labile groups. The basic chemistry of these reactions has recently been reviewed by Brown [16]. Studies on the mechanisms and rates of hydrolysis of various alkyl phosphotriesters have been reported (see e.g. Cox and Ramsay [ 171) and it has been found that the rate of hydrolysis of triesters in alkali is much faster than the rate for phosphodiesters [18]. EXPERIMENTAL

Bacteriophage T’7 was grown and the DNA isolated using the methods described by Lawley et al. [ 151. DNA was dissolved in 0.1 M Tris-HCl buffer pH 8.5. For the determination of the extent of alkylation various amounts of radioactively labelled reagents were added to DNA (2 ml, 600 E.cg/ml) and incubated at 37” for 30 min. At the end of the reaction DNA was precipitated with ethanol, washed eight times with ethanol, dissolved in sodium acetate, reprecipitated, washed twice more and then dissolved by warming in 0.1 M HCl. Radioactivity was determined using a Packard Tri-carb model 3375 and the concentration of DNA was estimated from the absorption of the solution (A &$&,, = 26 = 1 mg DNA/ml). [3H] DMS (15.4 Ci/mmole) was obtained from the Radiochemical Centre (Amersham, Bucks, U.K.) and was diluted to 86.9 @i/mg with unlabelled material. Solutions were made up in ether (10 mg/ml) and samples (20-100 pi/ml) were added to the DNA solution. [‘HI MNUA (11.4 mCi/mmole) was synthesised as described by Lawley and Shah [19]. Samples of an ethanol solution (7 mg/ml) were added to solutions of DNA to give MNUA concentrations from 0.04-0.26 mg/ml. [‘HI ENUA (116 mCi/mmok), synthesised as described by Howse [ 201, was diluted with unlabelled material to 2.1 mCi/mmole and dissolved in ethanol (50 mg/ml). Measured volumes were added to the DNA solutions to give concentrations of ENUA ranging from 0.8-5.4 mg/ml. For the first degradation experiments, known amounts of the alkylating agents were added to DNA (90 p&/ml) in 0.1 M Tris-HCl pH 8.5 and incubated for 30 min at 37”. At the end of this time one fifth volume of M NaOH was added and the incubation at 37” continued. At suitable intervals 0.3 ml of the alkaline solution was taken and added to 0.3 ml 1.8 M NaCl. Boundary velocity sedimentation experiments were then performed using a BeckmanSpinco analytical ultracentrifuge equipped with UV absorption optics. From the photographs of the boundary, integral distribution curves of sedimentation coefficients were calculated using the methods described by Shooter and Butler [21]. In further experiments DNA, after alkylation with MNUA, wx incubated

153

with concentrations of sodium hydroxide from 0.1 to ‘3.4 M. Samples were taken at intervsrls and diluted to 35-40 pg DNA/ml in 0.1 M NaOH, 0.9 M NaCl for the sedimentation experiments. Under the conditions described above the ~dimen~tion c~fficient of undegraded, single-stranded T7-DNA was found to be 31.4 S. For the calculations of molecular weight this value has been taken as S,, the effects of concentration being neglected. The molecular weight of the undegraded single chains of the DNA has been taken as 12.5 - lo6 daltons [22] and the relation between ~imentation coefficient and molecular weight [23] as S = kMa4. Theoretical distribution curves of s~imen~tion coefficients were calculated from this data using the equations given by Lehman and Ormerod 124 J and by Schans et al. [ 251. The shapes of the observed distribution curves paralleled those predicted by theory (Fig. 1). Estimates of the average number of breaks per molecule at low frequencies were obtained by comparison of observed and theoretics curves. At high frequencies of breaks/ molecule calculations were based on measurements of the mean sedimentation coefficient, In Fig. 2 the relationship between mean sedimentation coefficient and chain breaks calculated using the approach of Schans et al.

Sedirr.entotion coeff ictent (* 1013 set)

Fig. 1. A comparison of observed and theoretical distribution curves of sedimentation coefficients. Full lines. theoretical curves calculated using the equations given in refs. 24 and 25: the number beside each curve gives the average number of breaks per molecuIe of 12.5 - lo6 daltons. The increment in concentration or absorption, E, at a sedimen~tion coefficient s is given by E(s) = [ 2 + p( 1 - .R)]pR .

(RJW’ _a e-pR CY

the norm~ised

E’ by

increment

l-e* E’(s) = E(s) -_ zE(sI where p is the average number of breaks per molecule, R is the ratio of molecular weights Ml&, and CYis the exponent in the equation connecting sedimentation coefficient and molecular weight. Dashed lines. examples of observed distribution curves.

154

I

10

I

I

20 Breaks/molecule

30

1 40

DNA

Fig. 2. The relationship between mean sedimentation I r Iefficient and breaks/molecule. Full line, taken from the curves of Fig. 1; dashed line, caiii ulated assuming MUI= 2M,, = M, (SIS,)‘~~ and breaks/molecule

=

[M,-,/M,, - l]

with M, = 12.5 * lo6 daltons and a! = 0.4

[25] is compared with estimates obtained on the assumption that the weight average molecular weight calculated from the mean sedimentation coefficient is equal to twice the number average molecular weight. The figure clearly illustrates the change in sensitivity of the sediment&ion coefficient with increasing numbers of breaks/molecule. RESULTS

In this section the number of breaks formed and the rate of chain breakage have, in all cases, been related to a denatured, unbroken, single chain of T7DNA of molecular weight 12.5 lo6 daltons. The general pattern of the degradation process and the reproducibility of the result-s is illustrated in Fig. 3 using data obtained in an experiment in which T7-DNA alkylated with MNUA (0.08 mgiml) was incubated in 0.17 M NaOH. Comparison of the curves for control and alkylated DNA shows that methylation introduces alkali-labile sites into the DNA which are all hydrolysed in about 10 h. After this initial phase of rapid degradation the rate of chain break formation becomes much slower (0.22 breaks/molecule/h), approximately paralleling the degradation observed in the control DNA (0.15 breaks/molecule/h). Extrapolating the data for the slow degradation phase back to zero time gives an estimate of labile sites introduced on alkylal

155

Treated

DNA

/

Fig. 3. General pattern and reproducibility of the hydrolysis kinetics. T7-DNA in 0.1 M Tris pH 8.5 was treated with 0.08 mg MNUAlml for 30 min at 37’. 0.2 vol. of MNaOH were added and the solution incubated at 37’. Boundary sedimentation experiments were performed and the number of breaks/molecule calculated as described in the text. The open and full circles represent two separate experiments with control and MNUA-treated DNA.

tion. These rapidly formed breaks may be attributed to the hydrolysis of phosphotriester groups and the subsequent, slow degradation to hydrolysis of the phosphodiester groups. To check that the number of breaks introduced depended upon the extent of alkylation and not on the size of the DNA molecules the experiment illustrated in Fig. 3 was repeated using calf thymus DNA that had been alkylated using 0.08 mg/ml MNUA. The untreated DNA had a number average molecular weight, after denaturation, of 2.8 * lo6 daltons, equivalent to a molecule of 12.5 0 lo6 ddtons with 8 random breaks introduced. During the initial rapid degradation in alkali the number of breaks per molecule increased from 8 to 18 i.e. in agreement with the data on T7-DNA, the alkylation, at this concentration of MNUA, introduced 10 alkali-labile sites per molecule of 12.5 - lo6 daltons. In a further experiment T7-DNA was degraded by irradiation with ultrasonics to a weight average molecular weight of 1.5 * 10” daltons and incubated in 0.2 M NaOH. Over the period of incubation 24-72 h the rate of degradation was 0.18 breaks/molecule/h which is close to the rate (0.15) obtained with undegraded T7-DNA. The degradation of control-DNA in alkali is not therefore dependent upon the size of the DNA molecules. Further data on the degradation of T7-DNA alkylated using different concentrations of DMS, MNUA and ENUA are shown in Figs. 4a, b and c. At the highest concentrations of DMS and ENUA duplicate experiments were performed both to check the reproducibility of results and to investigate in more detail the kinetics of the early stages of the alkaline degradation (the dashed lines in Figs. 4a and c). With all three compounds the initial rapid hydrolysis of the DNA was over within 24 h and for the next 3 days of incubation the rate of formation of chainbreaks in the DNA was approximately constant. The number of breaks introduced in the initial phase of the 156

mg EMS/ml

HOW5 at 37”

MNUA/ml 0.2

a 1

Q.16 0.12 0.08 0.04

Hours at 37*

mgENU.-/ml

0.25 0

HOWS at 3?O

Fig. 4. T7 DNA in 0.1 M Tris-HCI pH 8.5 was incubated with alkylating agent for 30 min at 37’. 0.2 vol. of M NaOH were added and the solutions incubated at 37’ for up to 4 days. Estimates of the number of breaks formed/molecule were made as described in the text. The figures to the right of the lines give the amount of each reagent added. (a) DMS, (b) MNUA and (c) ENUA. The dashed lines in Figs. 4a and c illustrate replicate experiments at the highest concentration of reagent used and also show the early stages of the degradation reactions. 157

0.2

0.4

0.6

0.

mg reagent/ml

Fig. 5. The number of alkali-labile sites/molecule introduced by various concentra the three reagents have been calculated by extrapolatirrg the dam SF Piga 41, b a zero time of incubation. q, DMS; 0, MNUA; a, ENUA.

degradation,, estimated for each experiment by e time, have been plotted in Fig. 6. In the experiments with the number of breaks formed increases linearly with tie con reagent. With DMS the curve is non-linear, the number o only rising appreciably after methyl&ion at re 0.4 mg/ml. The estimates of the number of bre pound at a concentration of 1 mg/m extent of alkylation observed at this re sets of data the percentage of alkylatio has been calculated.

TABLE I ESTIMATES OF THE EXTENT OF ALKYLATION AND THE NUMBER OF ALKALILABILE GROUPS INTRODUCED INTO DNA These calculations are related to a single-stranded DNA mol 12.5 * lo6 daltons. The figure in brackets after swh mean irethe estimate, Degradation in alkali: Breakslmoleculelmg reagent/ml Extent of reaction: moles alkyllmole DNA/mg reagent/ml Extent of degradation: breaks/100 elkyl groups bound

158

rn the text. (a). MNUA-treated OH; 0.0.4 M NaOH.

m line of Table 1 show that only a small proportion e formation of chain 1 groups introduced residues this result under the alkaline t hydrolysis in alkali can be otriester groups. In 0.17 AM Won requiring 10 h or more at 37” for cant number of additional chain dative reactions. Further to determine if the rate of hydrolysis ing the concentration of alkali. The control, untreated T’I-DNA and T7-DNA UA were incubated in 0.1,0.2 and 0.4 M concentration of control and alkylated DNA, s the same estimate, within s per molecule attributable to the so show that the rate on of alkali increases. ule determined after incuerror, equal to the number

159

TAIILE II THE EFFECT OF NaOH CONCENTRATION DEGRADATION OF DNA METHYLATED

ON THE RATE AND &WE WlTH N-M~HYL*N-NITRg)SQ)tllRfr.l”

T7-DNA was treated as described in the legend to Fig. 6. Erti

breaks (+ standard error)/molecule at ze_m time were made by r data for incubations from 20-80 h at 37”. ~_--~_p____~_~~---p~p-~~ ~_-.- .-j=_iz_is____-=__i Q* NaOH concentration during incubation (M) 0.1 Breaks/molecule at zero time

9&l

10,

Bate of degradation during 20--110-h period (breaks/molecule/h) methylated DNA control DNA

0.23 0.14

0.32

t

DIXXJSSION

The degradation of alkylated DNA on incubation in alkali can into two phases, an initial, rapid breakdown complete in 5-24 h and a slower, continuocr mately linear with time over at least 5 day exhibits the slow, continuous degradation. and Fig. 6b the formation of breaks in control DNA is slow after some hours does it become linear with time. Th that chain scission is probably the end product of a which lead finally to the hydrolysis of pho ments with alkylated DNA, the rate of d greater than for control DNA and increase tion, Figs. 4a, b and c, Fig. 6a and Table II. Alky introduces groups into DNA which are readily hy sensitises the DNA in the slow degradation phase. In the initial, rapid degradation breaks can be form by hydrolysis of phosphotriesters and experiments with DMS it is probabl observed derive from apurinic sites. The data for this re show that appreciable numbers of incubation at 37” only when the groups/mole DNA (25 mmoles/mole DNA-B). In the MNUA and ENUA where the maximum extents of alkyle not exceed 200 and 90 moles bound/mole DNA fraction of the observed breaks will h The data shown in Fig. 9 can be u of methyl phosphotriesters in DNA. From Table I at the concentration of MNUA used since the single strands of T7-DNA eat 160

met-hyl groups approx. 20% will be timate with the concentrame ronmntration of methyl phosnmt.isn of alkali, 0.167 M, is much uaticsn for the bimolecular

From Fig. 3, the half life ation gives K = 4-6 * 10-O for the alkaline rs in DNA are the half life for the estimated from this alter than that reported 3.73 * 10ms (mole/l)-’ ysts reactions which involve

MNUA have shown that 80% of r ti alkylated bases [27] the (3). The data of Tabie I show that ut one third of the phosphotriesters, ion is consistent with in the previous paray reaction with ENUA have shown mnted for as alkylated bases a detailed study of the remaining

osphate

compared

with

hydrolysis can be used many problems to consider e. More information from uency with which the alkyl would be welcome. The hydrolysis of phosphoret&ion of data

161

has the disadvantage that it can only be used in experiments with rapidly reacting alkylating agents when loss of alkylated purines can be kept at a very low level. ACKNOWLEDGEMENTS

I am indebted to Dr. P.D. Lawley for stimulating discu course of this work. I thank Miss W.L. Hou and Mr. R.K. Me technical assistance. This work was supported by grants to the Chester Beatty Institute of Cancer Research, Royal Cancer Hospital, Research Council and the Cancer Research Campaign. REFERENCES

5 6 7 8 9 10 11 12 13 14 15

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ence

Diibutiorr

properties

of O*-ethylguanine in rat-brain nesis by ethylnitrosourea.

of

DNA: Proc.

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