The nature of single-strand breaks in DNA following treatment of L-cells with methylating agents

Mutation Research, 19 (1973) 331-341 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

331

T H E N A T U R E OF S I N G L E - S T R A N D B R E A K S IN DNA F O L L O W I N G T R E A T M E N T OF L-CELLS W I T H M E T H Y L A T I N G AGENTS

I. G. WALKER* AND D. F. EWART Department of Biochemistry, University o[ Western Ontario, London, Ontario (Canada N6A 3K7) (Received February 27th, I973)

SUMMARY DNA from untreated L-cells had a weight average molecular weight (Mw) of 5.7 4- 0.58. lO 8 daltons as measured b y sedimentation in an alkaline sucrose gradient. This value was reduced b y one half after the cells were treated for I h with 8/zg/ml of N-methyl-N-ultrosourea (MNUA), 34/~g/ml of methyl methanesulfonate (MMS) or o.16/,g/ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). T h a t dose of MNUA produced 52 methylations per 5.7"1o8 daltons DNA. 2o% of these were not purine derivatives and were assumed to contairx some phosphotriesters. T h a t dose of MMS (above) produced 290 methylations per 5-7" lO8 daltons DNA and about 14% of these were not purine derivatives. The rates of loss of methylated purines from DNA were 2.3% per hour for 7-methylguanine (7-MEG), 7-4% per hour for 3-methyladenine (3-MeA) and no detectable loss of O6-methylguanine (O6-MeG) over a 12 h period. Since phosphotriesters are alkali-labile the single-strand breaks probably arose from this structure and did not form within the cell. This conclusion is supported b y the following considerations. MNUA was more effective than MMS at reducing the molecular weight of DNA, as measured in alkaline medium. The greater SN1 character of MNUA would cause a greater formation of phosphotriesters than would MMS.

INTRODUCTION Methylating agentsT,*8, *' ionizing radiation (ref. 20, and refeiences therein) and ultraviolet light (refs. 6, I9, and references therein) induce repair replication and the unscheduled synthesis of DNA in normal human cells and in established lines of human and rodent cells. Cells from individuals with the rare autosomal recessive disease xeroderma pigmentosum also respond in this way to the first two agentsS, ' * To whom requests for reprints should be sent. Abbreviations: 3-MeA, 3-methyladenine; 7-MEG, 7-methylguanine; Oe-MeG, O6-methylguanine; MMS, methyl methanesnlfonate; Mn, number average molecular weight; MNNG, N-methyl-N" nitro-N-nitrosoguanidine; MNUA, N-methyl-N-nitrosourea; Mw, weight average molecular weight; SDS, sodium dodecyl sulfate.

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I. G. W A L K E R , D. F. E W A R T

but the response to ultraviolet light is nmch reduced4, 5. It is believed that xeroderma pigmentosum cells lack an endonuclease which in the unaffected cell hydrolyses the DNA strand near the ultraviolet light-induced alteration thereby starting off the repair replication process~, ~4. Ionizing radiation causes breaks in the DNA strand as a primary event and hence no endonuclease action is required to initiate repair replication. In analogy with ionizing radiation it was suggested that methylating agents also produce breaks non-enzymatically and, indeed, the production of single-strand breaks in the DNA of mammalian cellsSO° and bacteria 18 following treatment with methylating agents has been reported. The extent of this breakage was assessed by examining the sedimentation of the DNA in an alkaline sucrose gradient. Recently, the alkaline sucrose gradient method, particularly as applied to the examination of mammalian cell DNA, has been reinvestigated~O6, 21. Conditions were identified which allow mammalian DNA to sediment reproducibly with an Mw of about 5" lO8 daltons. In view of these developments it seemed appropriate to reexamine the ability of methylating agents to induce single-strand breaks in the DNA of mammalian cells. MATERIALS AND METHODS

Cells and radioactive compounds Strain L-mouse cells were grown routinely at 37 ° in suspension culture in thymidine-free alpha-MEM medium (Flow Laboratories, Rockville, Md., U.S.A.) containing 7 % horse serum, penicillin and streptomycin. The doubling time of these cells was about 18 h. All radioactive compounds were obtained from New England Nuclear (Canada) Ltd. E14C~MMS, 56 mCi/mmole, was in ether solution. For an experiment, a portion of the ether solution was evaporated under a stream of air and a methanol solution of unlabelled MMS was added to reduce the specific activity IO times. E3HIMNUA, 49 mCi/mmole, was also diluted IO times with unlabelled MNUA in methanol. [SHlThymidine and E14C1thymidine had specific activities of 20 Ci/mmole and 50 mCi/mmole respectively. Alkaline sucrose gradient sedimentation analysis A suspension culture of cells was grown for 24 h in medium containing I/~Ci/ml of [3Hlthymidine and then alkylating agent dissolved in methanol was added. After I h the cells were centrifuged, resuspended in ice-cold phosphate-buffered saline and kept cold until being placed on the lysis layer of the gradient. Linear 5 - 2 0 % sucrose gradients (4.7 ml in 1/2 × 2" cellulose nitrate tubes) were made on an Isco Model 57 ° gradient former. The gradient solution contained 0.3 M NaOH, o . o i % SDS and o.ooi M EDTA. On top of the gradient, 0.3 ml of lysing solution (0.5 M NaOH, 0.2% SDS, and o.oi M EDTA) was carefully layered just prior to lysis. An Agla syringe affixed to a microscope body was used to dispense carefully i . lO 4 cells in 0.02 ml into the lysing layer. The cells were allowed to lyse for 12 h at room temperature after which tubes were centrifuged at 20 ° using a SW 5o.1 rotor in a Beckman L2-65B preparative ultracentrifuge at 15ooo rev./min for 27 ° rain. After centrifugation, 26 o.2-ml fractions were collected from each gradient using an Isco Model 640 density gradient fractionator. The DNA in each fraction was precipitated with ice-cold 5% trichloracetic acid and collected on a glass fibre filter. The filters were washed twice with cold 5 % TCA, then once with 75 % ethanol, dried and put into

EFFECT OF METHYLATING AGENTS ON L-CELLS

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scintillation vials. To each vial was added 4 ml of scintillation fluid and radioactivity was measured. From the radioactivity data the Mw of the DNA was calculated using a P D P - I o computer and a program written in Fortran IV. The centrifugal constant necessary for the calculation was obtained b y using bacteriophage T 4 labelled with [3H]thymidine. Details of the calculations and an analysis of the factors involved in measuring molecular weights of DNA are provided in the paper by PALClC AND SKARSGARD ~1.

Methylation of cellular DNA (i) With [x*C]MMS. To a 5- or Io-ml suspension containing 8 .lO 7 cells was added 0.5 mg of [~4C]MMS dissolved in 0.05 ml of methanol. A sample was withdrawn to determine specific activity. After I h the cells were centrifuged and DNA was extraced. (ii) With [3H]MNUA. Cells were grown overnight in medium containing 2.5" lO-4 #Ci/ml of [x4C]thymidine. The cells were centlifuged, suspended in 50 nfl of fresh medium (8.IO e cells/ml) and 5.0 mg of [3H]MNUA dissolved in 0. 5 ml of methanol was added. After I h a Io-ml sample was centrifuged and stored frozen for eventual isolation of DNA. The remainder of the suspension was diluted to 800 ml and incubated. After 3, 6, 9 and 12 h 2oo-ml samples were taken for DNA isolation.

Reaction of M N U A with DNA in vitro To 2 ml of DNA solution (2 mg/ml of salmon sperm DNA dissolved in 14 m M sodium chloride-I. 5 m M sodium citrate) was added 0.02 ml of methanol containing 27 #g [14C]MNUA (1.88 mCi/mmole), a gift from Dr. J. V. Frei, Department of Pathology. After I h at room temperature the solution was made 0.25 M with sodium acetate in a volume of io ml. The DNA was precipitated by adding 2 vol. of ethanol. After another dissolution-precipitation cycle the DNA was hydrolyzed and chromatographed.

Isolation, hydrolysis, chromatography of DNA The method used to isolate DNA has been described~L To about o.5 mg of dry DNA was added 0.2 ml of water. Next day after adding 0.2 ml of 0.2 N HC1 the DNA was hydrolyzed in a closed tube at IOO° for 30 min. Portions were taken for radioactivity and A260 measurements from which the extent of methylation was calculated. A o.i-ml sample was chromatographed on W h a t m a n No. 3MM paper b y downward flow using as solvent a freshly prepared solution containing t-butanol:methyl ethylketone: concentrated ammonium hydroxide: water, in the volume ratio 40 : 3o : 20 : IO. A double run was employed. In the first, the solvent was allowed to migrate 25 cm from the origin. In the second, the solvent ran to about 4 ° cm. In between runs the paper was dried thoroughly. This method, developed b y Dr. S. Maitra, Department of Pathology, was designed to give compact spots and to separate the puline bases from their methyl delivatives. In the experiment with [14C]MMS the chromatograms were cut into i-cm strips and placed in scintillation solution for radioactivity measurement. In the experiment with [aH]MNUA the I-cm strips were placed in counting vials containing 1. 5 ml of 0.05 N HC1 and allowed to extract for 24 h. Then Io ml of liquid scintillation solution (Aquasol, New England Nuclear (Canada) Ltd.) were added.

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Measurement of radioactivity Radioactivity was measured in a Nuclear Chicago instrument which allowed aH and "C to be counted in separate channels. Appropriate correction was made for the "C in the 3H channel and absolute counting rates were determined using the internal standard method.

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335

EFFECT OF METHYLATING AGENTS ON L-CELLS RESULTS

Single-strand molecular weight of DN A after methylation Alkaline sedimentation patterns of DNA from control L-cells and cells that were treated with various concentrations of MNUA are shown in Fig. I while Fig. 2 shows similar patterns for DNA from L-cells treated with various concentlations of MMS. The methylating agent MNNG gave similar Iesults but at much lower concentrations. From 9 sedimentation analyses the Mw 4- S.D. of DNA from untreated cells was found to be 5-7 i 0.58. lO 8 daltons. This value compares favourably with the value of PALCIC AND SKARSGARD.1 who found Mw for L-cell DNA to be 4.9" lO9 daltons and with that of LETT et al. TM who found it to be 5.2" lO 8 daltons for the DNA of CHO, L5178Y and H e L a cells. For each experiment the numbel of single-strand breaks in the DNA from treated cells was calculated from the equation: Mn of control DNA Number of breaks per weight of control DNA ------I Mn of treated DNA Mn was considered to equal 0.5 Mw since CI~ARLESBY8 has shown this to be true if the distribution of molecules is random and PALCIC AND SKARSGARD11 have demonstrated randomness of distribution in DNA sedimentation patterns from control and X-irradiated L-cells. Since the molecular weight of control DNA varied slightly between experiments the number of breaks per weight of DNA was normalized by proportion to give the value, number of breaks per 5.7" IO8 daltons. Whelt these values were plotted as a function of concentration of methylating agent a reasonably linear relation was obtained up to about 2 breaks per 5.7" lO8 daltons of DNA (Fig. 3). As the dose of methylating agent was increased further the number of breaks per unit of DNA fell off (Fig. 4).

Extent of methylation of DNA by M M S and M N U A The extent to which DNA was methylated following treatment of L-cells with MMS was determined using 1*C-labelled agent. After doses of 50 and IOO #g/ml for I h there were 202 and 523 methyls per IOe nucleotides, respectively, so that on average o

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D. F. EWART

the extent of methylation was 4.68 methyls per Io 8 nucleotides per/~g of MMS/ml. The previously determined value for MNUA was 3.50 methyls per lO 6 nucleotides per #g of MNUA/ml ~7.

Relation between methylation of DNA and single strand break formation In order to measure the extent to which DNA was methylated by MMS or MNUA cell suspensions were concentrated and treated with the radioactive agents. When single-strand breaks were being examined it was more convenient not to concentrate the cells. Thus the two sets of data cannot be combined to give an exact relation between methylation of DNA and strand breakage. However, it is proper to combine these data to obtain a value that is proportional to the relationship. The concentrations of MMS and MNUA that produced one single-strand break per 5.7' lO8 daltons of DNA were 34 and 8/zg/ml, respectively (Figs. 3 and 4)- These concentrations of MMS and MNUA would yield 290 and 52 meth3ds per 5.7'1o 8 daltons of DNA. Thus a given extent of methylation from MNUA treatment was about 5 times as effective at producing single strand breaks as an equivalent extent of methylation from MMS treatment.

Nature of the methylation products and their rate of removal DNA from cells that were treated with ['CJMMS was hydrolyzed in o.I N HC1 at IOO° for 30 rain and the products were chromatographed on paper. The chromatogram (Fig. 5) shows the expected 1~ products 7-MEG (72%), 3-MeA (9.5%) along with unidentified products (14%) running between the origin and 7-MEG. This is the area in which, apnrinic DNA and pyrimidine aligonucleotides are found. Located at 36 cm from the origin is a very tiny peak (0.25%) which from its location is thought to be OS-MeG. A similar study of DNA methylation was made using [3HIMNUA but in this case the cells had been grown previously in ['C]thymidine. In addition, some of these treated cells were incubated in order to measure the rate of disappearance of methylation products. A chromatogram of the hydrolysed DNA from cells immediately after

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EFFECT OF METHYLATING AGENTS ON L-CELLS

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treatment with MNUA is shown in Fig. 6. The identifiable alkylation products are 7-MEG (64%), 3-MeA (5.4%) and O6-MeG (3.5 %). There are also unidentified products (19.7%) that migrate more slowly than 7-MEG and some (5.I ~/o)near the solvent front. The location of apurinic DNA and pyrimidine oligonucleotides is marked by the 14C-label. When the ratio, total 8H:total ~4C was calculated for each time interval following treatment with MNUA in order to measule the overall rate of loss of methylation products the value increased with time. It turned out that this was an artifact due to incorporation of the hydrolysis products of I3HIMNUA into the methyl group of thymine and positions 2 and 8 of the purine ring. In Fig. 7, the chromatogram of I0

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Fig. 6. C h r o m a t o g r a m of h y d r o l y s e d D N A o b t a i n e d f r o m L-cells a f t e r t r e a t m e n t for ; h ~ i t h IOO ,ug/ml of [SH]MNUA. T h e cells h a d been p r e v i o u s l y g r o w n in m e d i u m c o n t a i n i n g [14C~ thymidine. • e , aH label ; © . . . . ©, 1~C label.

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Fig. 7. C h r o m a t o g r a m of h y d r o l y s e d D N A o b t a i n e d f r o m L-cells x2 h a f t e r t r e a t m e n t w i t h [aH1M N U A . L-cells p r e v i o u s l y g r o w n in m e d i u m c o n t a i n i n g [14C]thyrnidine were t r e a t e d for x h w i t h xoo p g / m l of t h e M N U A a n d were t h e n d i l u t e d (details in text) a n d i n c u b a t e d for ~2 h. e O, aH label; © . . . . ©, 14C label.

338

I. G. WALKER, D. F. EWART

the hydrolysed DNA obtained 12 h after treatment shows that 3H activity migrating between the origin and 7-MEG (thymine containing compounds) and at the location of adenine is much higher, relative to total "C, than in the previous chromatogram. Guanine shows very little uptake of ~H as expected because the hydrogen associated with the "one carbon" is largely lost during the biosynthetic steps. Since 7-MEG, 3-MeA and Oe-MeG occurred as discrete spots on the chromatograms, it was possible to measure the rate at which they were removed from DNA during incubation of the cells following treatment. The data for 7-MEG and 3-MeA are plotted in Fig. 8. For 7-MEG the rate of loss was 2.3% per hour; for 3-MeA the rate was 7.4% per hour. In contrast, the amount of OS-MeG did not change significantly over the i2-h period. The 3H/"C values for O6-MeG at o, 3, 6, 9 and 12 h were 0.0242, 0.0295, o.o312, o.o199 and 0.0295 respectively. The 3H-labelled products found between the origin and 7-MEG at zero time must contain methylation products and not just thymine derivatives because the ~H profile does not follow the 14C profile the way it does at 12 h when incorporation of ~H into thymine was extensive. In addition the reaction between MNUA and DNA in vitro also produced methylation products that migrated between the origin and 7-MEG (Fig. 9).

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Fig. 8. The rate of loss of 7-MEG and 3-MeA from the D N A of L-cells following t r e a t m e n t with MNUA. Fig. 9. C h r o m a t o g r a m o b t a i n e d after h y d r o l y s i n g D N A which h a d been reacted with M N U A in vitro. DISCUSSION

There are two reports that provide compelling evidence for the notion that ethyl methanesulfonate can esterify the phosphate of DNA and form a phosphotriesterl, 22. The ability of ethyl methanesulfonate to react in this way is explained by its SN1 character 15. MNUA is also considered to possess considerable SN1 chalacter in contrast to MMS which reacts mainly by an SN, mechanism ~5. MNUA and MMS produced unidentified methylation products which chromatographed in the same area as apurinic DNA. (Figs. 5 and 6). It seems reasonable to assume that some of

EFFECT OF METHYLATING AGENTS ON L-CELLS

339

these methylation products were in the form of phosphotriesters. If this is so then the single-strand breakage observed probably arose at the phosphotriester sites after the DNA was subjected to the highly alkahne conditions necessary for the sedimentation analysis. Hydrolysis of 2 out of 3 of the bonds would lead to cleavage of the nucleotide chain. Alkaline hydrolysis of 7-MEG would occur also but would not be expected to lead to strand breaks2,11. The contention that the strand breakage arose from alkaline hydrolysis of phosphotriester groups is supported by the following considerations. The methylation of DNA by MNUA was more effective than the methylation by MMS at yielding single-strand breaks. The greater SN1 character of MNUA would cause a greater formation of phosphotriesters than would MMS. It can be argued that the single-strand breaks arose from alkaline hydrolysis at apurinic sites which in turn arose by a spontaneous or enzyme-mediated loss of methylated purines in vivo. Thus, MNUA has a half-life of about 15 min so that at the end of I h treatment and allowing for manipulation time there has been virtually i h for depurination to occur. After a dose of 8/~g MNUA per ml there was i single-strand break and about 33 7-MEG per 5.7" lO8 daltons DNA. The contribution of 3-MeA to depurination would be very small in a I-h period since it comprised only 5.4% of the methylation products. At a depurination rate of 2.3°1/o per hour for 7-MEG almost i apurinic site could have formed. However, when MMS produced similar amounts of 7-MEG and 3-MeA the strand breaking efficiency was only one fifth. Thus the singlestrand breaks observed at the end of a I-h treatment with methylating agents do not seem to have their basis in the loss of alkylated purines. The rate of loss of methylation products given in this paper differs from those reported ealher 27. The previous values were in error because account was not taken of the incorporation of label from hydrolysed MNUA into the purine skeleton and the methyl of thymine. The presently reported rate for the loss of 7-MEG from the DNA of L-cells (2.3% per hour) is now close to the overall rate of loss of methylation products from the DNA of HeLa cells (3.3% per hour) reported by ROBERTS et al. ~s. It is also apparent that L-cells like HeLa cells ~7 exhibit an even more rapid loss of 3-MeA (7.4% per hour for L-cells). In this respect mammalian cells are similar to E. coli B/r 14. However, E. coli B/r also excised O6-MeG rapidly but there was no evidence of its loss from the DNA of L-cells in this study or from the DNA of HeLa c e U s 27.

The effectiveness of low doses of MNNG at producing single-strand breaks in DNA of 1.-cells is noteworthy and reflects the efficiency of methylation by this agent. Equal extents of strand breakage were obtained when L-cells were treated with 0.16/~g/ml MNNG, 8/~g/ml MNUA and 34 #g/ml MMS. MNNG is unlike the other methylating agents in two respects; it is unreactive in aqueous solution at neutral pH but is made highly reactive by thiol containing compounds~3,17 and perhaps by the guanidine group of arginine ~6. Since there is no free thiol in the culture medium ~5 MNNG does not react appreciably until it has penetrated the cell. Two groups of investigators, have reported that after mammalian cells were treated with MMS the DNA suffered single-strand breaks as indicated by its sedimentation behaviour in an alkaline sucrose gradient. In the study by COYLE AND STRAUSS8 the conditions for lysing the cells and sedimenting the DNA caused the DNA from untreated cells to sediment to the bottom of the tube. This DNA was estimated to have an S value of 491 and undoubtedly consisted of some kind of

34 °

I. G. WALKER, D. F. EWART

complex. After the cells were treated with 2.io-SM MMS for I h the DNA had an S value of 81. If the cells were allowed to incubate for 5 h in fresh medium after drug treatment the S value rose to 376 indicating that some kind of repair process had occurred. The nature of this repair remains unclear because of the unknown nature of the sedimenting species of DNA. A number of studiesg,l~, ~I and the present one indicate that the size of the single-stranded DNA unit obtained with minimal shear and sedimentation conditions that avoid tangling of DNA strands is near I65S. In the study b y F o x AND FOX 1° MMS treatment led to the formation of more slowly sedimenting DNA but again the precise nature of the effect is not clear. No increase in S value occurred when the cells were incubated after drug treatment. These authors also made the interesting observation that the sedimentation rate of newly formed DNA was decreased by low doses of MMS and that post treatment incubation restored this DNA to almost its original size. In the present study the repairability of the single-strand breaks has not been tested. If the breaks are the result of the alkaline hydrolysis of phosphotriesters then it is unlikely that a repair process will be observed unless the phosphotriester group is removed and the gap repaired. On the other hand, studies with xeroderma pigmentosum cells suggested that methylating agents behaved like X-rays and induced breaks in DNA strands within the celP. Since X-ray-induced breaks are healed (ref. 9 for example) then methylating agentinduced breaks could be expected to be healed. These questions are being investigated. ACKNOWLEDGEMENTS

Technical assistance was provided by Mrs. JANE PoczI. We are grateful to Dr. G. F. WHITMORE, Toronto for providing the computer program; to Mr. WALTER KOCHA for adapting the program to our computer; to Dr. B. PALCIC for helpful discussions; to Dr. K. EBISUZAKI for help in preparing the labelled T2 bacteriophage. This work was supported b y a grant and the award of a Postdoctoral Fellowship to Dr. D. F. EWART from the Medical Research Council of Canada. REFERENCES

I BANNON, P., AND W. VERLY, Alkylation of phosphates and stability of phosphate triesters in DNA, European J. Biochem., 31 (1972) IO3-III. 2 BROOKES, P., AND P. D. LAWLEY, The alkylation of guanosine and guanosinic acid, J. Chem. Soc., (I96I) 3923-3928. 3 CHARLESBY, A., Molecular-weight changes in the degradation of long-chain polymers, Proc. Royal Soc. A, 224 (1954) 12o-128. 4 CLEAVER, J. E., Defective repair replication of DNA in xeroderma pigmentosum, Nature, 2~8 (1968) 652-656. 5 CLEAVER, J. E., Xeroderma pigmentosum : A human disease in which an initial stage of DNA repairs is defective, Proc. Natl. Acad. Sci. (U.S.), 63 11969) 428-435. 6 CLEAVER, J. E., Repair replication in Chinese hamster cells after damage from ultraviolet light, Photochem. Photobiol., 12 (197 o) 17-28. 7 CLEAVER, J. E., Repair of alkylation damage in ultraviolet sensitive (xeroderma pigmentosum) human cells, Mutation Res., 12 (i97 I) 453-462. 8 COYLE, MARIE B., AND B. S. STRAUSS,Characteristics of DNA synthesized by methyl methanesulfonate-treated HEp-2 cells, Chem.-Biol. Interactions, I (1969-7 o) 89-98. 9 ELKIND, M. i . , AND CHIN-MEI CHANG-LIlY, Repair of a DNA complex from X-irradiated Chinese hamster cells, Int. J. Radiation Res., 22 (1972) 313-324 . io Fox, B. W., AND M. Fox, Sensitivity of the newly synthesized and template DNA oi lymphoma cells to damage by methyl methanesulphonate, and the natule of the associated "repair" process, Mutation Res., 8 (1969) 629-638.

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