The response of mammalian cells to alkylating agents

BIOCHIMICA ET BIOPHYSICA ACTA

179

BBA 96131

T H E R E S P O N S E OF MAMMALIAN CELLS TO A L K Y L A T I N G AGENTS II. ON T H E MECHANISM OF T H E REMOVAL OF SULFUR-MUSTARDI N D U C E D CROSS-LINKS*

B. D. R E I D ' " AND I. G. W A L K E R * "

Cancer Research Laboratory and Department of Biochemistry, University of Western Ontario, London (Canada) (Received S e p t e m b e r I6th, 1968)

SUMMARY

I. The ability of L-cells to remove alkylation products produced by sulfur mustard di-(2-chloroethyl)-sulfide from their DNA has been studied. 2. The loss of total alkylation products as well as the loss of the diguaninyl mustard product, di-(guanin-7-yl)-ethyl sulfide, were followed as a function of time using asS-labelled mustard. Both processes were exponential and occurred with halflives of 18 h. 3. Parallel measurements of the amount of renaturable cross-linked DNA present were made using methylated albumin kieselguhr column chromatography or ultracentrifugation in CsC1 gradients to separate renaturable, i.e., bihelical, DNA from non-renaturable, i.e., denatured, DNA. These studies showed that (a) the loss of renaturable DNA occurred with a half-life of 2 h; (b) the loss of renaturable DNA occurred more rapidly than did the loss of either the total or the diguaninyl alkylation products; (c) this loss of renaturable DNA occurred in the absence of preferential excision of the diguaninyl alkylation product. 4. The following mechanism is proposed to account for these findings. It appears that L-cells are able to excise both monoguaninyl (7-hydroxyethylthioethylguanine) and diguaninyl alkylation products from their DNA and further, that the latter product is removed by a two-step process that involves first, the 'unhooking' of one arm of the cross-link and second, excision at some later time of the remaining product.

INTRODUCTION

Sulfur mustard and the nitrogen mustard HN2 react with DNA in vitro and in vivo to form alkyl derivatives of the bases. Because these mustards possess two * P a r t I of this series b y I. G, WALKER AND C. J. THATCHER a p p e a r e d in Radiation Res., 34 (1968) IiO. ** P r e s e n t address: D e p a r t m e n t of Biochemistry, U n i v e r s i t y of Massachusetts, A m h e r s t , Mass., U.S.A. "** P r e s e n t address: Chester B e a t t y I n s t i t u t e , Pollards W o o d Division, Nightingales Lane, Chalfont St. Giles, Bucks, G r e a t Britain.

Biochim. Biophys. Acta, 179 (1969) 179-188

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B . D . REID, I. G. WALKER

reactive chloroethyl groups it is possible for some of the alkylation products to be in the form of cross-links between the strands of the double helix I. Evidence for this concept was first obtained by BROOKES AND LAWLEV2. These authors identified diguaninyl derivatives obtained from DNA which had been alkylated by sulfur or nitrogen mustard. The mustard moiety joined a pair of guanines through their N- 7 positions. A lower portion of this material was formed when aqueous solutions of RNA or denatured DNA were alkylated, suggesting that the majority if not all of the diguaninyl compound arose by interstrand and not intrastrand linking of guanines. Further support for this concept was provided by the same authors when they treated DNA in 9 ° % methanol with sulfur mustard and no diguaninyl product was formed 3. In this instance the DNA was entirely in the single-stranded form, suggesting that the diguaninyl compound arose only from guanines that resided on neighbouring strands. Physical evidence for the cross-linking of two strands in the DNA molecule has come from studies of the reversible denaturation of mustard-treated DNA 4-6. Mustard treatment allowed the DNA to reform a double helix after denaturation under conditions where nontreated DNA remained single stranded. From the biological point of view we have been interested in the effect produced on organisms when their DNA was alkylated by mustards. The question being asked is, how is the organism that survives mustard treatment able to cope with this kind of insult to its hereditary apparatus ? KOHN, STEIGBIGELAND SPEARS7 showed that the resistant strain of fscherichia coli B/r, but not the sensitive strain, Bs-x, could remove mustard-induced cross-links from its DNA. LAWLEY AND BROOKES 8 showed that E. coli B/r could remove both mono-and diguaninyl alkylation products from its DNA, the diguaninyl product being removed preferentially. VENITT9, using low doses of sulfur mustard has confirmed the findings of LAWLEV A~'D BROOKESs and was able to correlate the loss of diguaninyl product with the loss of cross-links. There is apparently only one report of a similar study with mammalian cells. CRATHORN AND ROBERTS 1° demonstrated that H e L a cells could excise mono- and diguaninyl products from their DNA following sulfur-mustard treatment with neither product being removed preferentially. In the present study, the process of excision of sulfur-mustard-alkylation products from the DNA of L-cells has been followed by chemical and physical methods. Our results indicate that the mustard-induced cross-link is removed by a two-step process. In the first step which is quite rapid, one arm of the cross-link is broken to leave a DNA molecule which will not renature. However, chemical analysis still reveals the presence of covalently bound diguaninyl compound which is also excised eventually.

MATERIALS AND METHODS Materials The following special chemicals were obtained commercially: [asS]sulfur mustard, 0.25 to I.O C/mmole, (Radioehemical Centre, Amersham, England); 5-[125I] iodo-2'-deoxyuridine, 5, 6 or 92 C/mmole, (Nuclear Chicago, Des Plaines, Ill.; and Schwartz BioResearch, Inc., Orangeburg, N.Y., respectively); 5-iodo-2'-deoxyuridine (Calbiochem, Los Angeles, Calif.); CsC1, optical grade (Gallard-Schlessinger, Carle Place, N.Y.); unlabelled sulfur mustard was obtained from the Defence Research Biochim. Biophys. Acta, 179 (1969) 179-188

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181

Establishment, Suffield, Alberta. The fluor system used for liquid-scintillation counting of radioactive samples contained 4.0 g of 2,5-diphenyloxazole and 5° mg of 1, 4bis-(5-phenyloxazole-2)-benzene per 1 of toluene.

Methods Cell cultures. Spinner-flask cultures of a sub-strain of L-cells11 were maintained at 37 ° in medium CMRL-Io66, minus thymidine and coenzymes and supplemented with 5 % bovine serum, ioo/~g/ml of streptomycin and 60 #g/ml of penicillin. Population densities were determined with the Coulter electronic counter and were maintained by dilution between 0. 5. lO5 and 5.O-lO 5 per ml. Treatment o/cells with sul/ur mustard. Approx. 800 ml of cells at a population density of 4" lO5 per ml were centrifuged in sterile 25o-ml centrifuge tubes at 16o × g for IO rain. The cell pellet was gently resuspended in a final vol. of 20 ml of fresh medium at 37 °. The cell suspension was then treated with i/~g/ml of sulfur mustard (I mg/ml in methanol). The cells were kept in suspension by means of a magnetic stirrer. Samples of 5 ml were taken IO rain and I h after treatment, and the remaining suspension was diluted to a vol. of IOO or 200 ml with fresh medium. Samples of 5° and ioo ml, respectively, were then taken 6 and 24 h after treatment. The samples were washed twice with phosphate-buffered saline and the cell pellet either frozen or used immediately for DNA isolation. Cells that had been prelabelled with E125Iliododeoxyuridine were not concentrated before being treated with unlabelled mustard. Otherwise the washing and storing were identical. Isolation and measurement o/DNA. DNA was isolated according to the method of DIAMOND, DEFENDI AND BROOKE812 and if stored, was frozen in O.Ol5 M NAC1o.oo15 M sodium citrate solution. DNA concentrations were determined colorimetrically by the method of BURTON13 using calf-thymus DNA as a standard. Measurement o/mono- and diguaninyl alkylation products. The method of LAWLEV AND BROOKESs was followed. Samples of DNA from cells treated with [35Slsulfur mustard were reprecipitated with alcohol, wound on a glass rod and transferred to a tube for hydrolysis in I M HC1 at IOO° for 15 rain. The hydrolysate was chromatographed on Whatman No. I paper using methanol-water-conc. HC1 (7 : 2 : i, by vol.) as solvent. The chromatogram was cut into sections, and these were placed in toluene-fluor for liquid-scintillation counting. Diguaninyl mustard, di-(guanin-7-yl)-ethylsulfide, appears near the origin. Monoguaninyl mustard, 7-hydroxyethylthioethylguanine, has an RF of 0. 4. Measurement o/radioactivity. All radioactive samples were counted in Nuclear Chicago equipment. Samples of DNA isolated from cells treated with E35Slsulfur mustard were digested and prepared for liquid-scintillation counting by the method of JEFFA¥, OLUBAJO AND JEWEL14. a2P-labelled DNA samples were counted as aqueous solutions in the liquid-scintillation counter at the settings for 8H, making use of Cerenkov radiation 1~. 125I-labelled DNA samples were counted in a well-type v-ray counter. Labelling o/ cells with ElZ5I]iododeoxvuridine or 82Pi. Ex25I]Iododeoxyuridine: cells were grown in the presence of 0.79 #g/ml of unlabelled iododeoxyuridine and o.I/~C/ml of ~a25I]iododeoxyuridine (specific activity 92 C/mmole) for approx. 16 h. Biochim. Biophys. Acta, 179 (1969) 179-188

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B . D . BREID, I. G. WALKER

32Pt: carrier free 32P1 was added to the culture medium which contained I mM phosphate to give a concentration of I/zC/ml and the cells allowed to grow in its presence for 2-3 generations. Alkaline denaturation o/DNA. To 5 o - I o o # g of DNA in I ml of o.o15 M NAC1o.ool 5 M sodium citrate solution were added 2.0 ml of 0.08 M NaOH. The solution was incubated for io min at 37 ° and then neutralized to pH 6. 7 with 3.0 ml of O.Ol7 M citric acid. Methylated albumin kieselguhr chromatography o~ DNA. Methylated albumin and the three layers for the column were prepared according to the method of MANDELL AND HERSHEY~6 as modified by SHEININ17. The finished column (I cm x 18 cm) was washed with 0.6 M NaCI in 0.o5 M phosphate buffer (pH 6.7). Samples containing 5O-lOO/,g of DNA (5°. lO3-3oo • lO3 counts/rain) were loaded on the column in 6.0 ml of 0.6 M NaC1 and eluted in 3-ml fractions with 30 ml of 0. 7 M NaC1 and 30 ml of 0.8 M NaC1, both in 0.05 M phosphate buffer at pH 6.7. The flow rate was 0.5 ml/min. Elution of DNA was monitored by measuring the radioactivity in each fraction. Density-gradient equilibrium centri]ugation. Solid CsC1 was added to the samples for ultracentrifugation and the refractive index adjusted to 1.4OLO. Samples were centrifuged in I2-mm analytical cells fitted with Kel-F centrepieces, in the Beckman Model E ultracentrifuge at 44 77 ° rev./min at 20 ° for 18 h. The samples were photographed on Kodak commercial film using an ultraviolet-light source and chlorine-bromine filters. The films were traced on a Kipp and Zonen microdensitometer. All samples contained a reference DNA, with a buoyant density of 1.7313. The reference DNA was isolated from a Bacillus subtilis copper mutant and was a gift from Dr. P. C. FITZJAMES. Sedimentation-velocity analyses. Molecular weight estimations were obtained from sedimentation-velocity data using the Beckman Model E ultracentrifuge and 12 mm Kel-F centre pieces. Samples were centrifuged in o.15 M NaCl-o.oI 5 M sodium citrate solution at 24 630 rev./min at 20 °. Photographs and tracings were made as outlined above. The value I/S~o,wwas plotted against concentration and extrapolated to zero concentration to give s°20,w. The molecular weight was obtained from Eqn. 3' of EIGNER AND DOTY is, using the s°20,w value obtained as above.

RESULTS

The loss o/alkylation products/rom DNA DNA was isolated from asynchronous cultures of L-cells at various times after treatment with F35S]sulfur mustard and analysed for the extent of alkylation and for the proportion of the guanine alkylation products. The results are shown in Table I. It can be seen in the second column that the extent of alkylation at I h was greater than that at IO rain. This was unexpected since the process of alkylation of DNA with sulfur mustard in ~,,itro in an aqueous medium is thought to have a half-life of approx. 2 min 19 and the half-hydrolysis time of sulfur mustard in medium CMRLlO66 is 3 rain2°. Over the next 23 h, alkylation products were lost from the DNA in a logarithmic manner, with a half-life of approx. 18 h. Proof that the radioactivity associated with the DNA was due to covalently bound sulfur mustard and not to alkylation products physically trapped in the DNA Biochim. Biophys. Acla, 179 (I969) 179-188

183

MECHANISM OF REMOVAL OF SULFUR-MUSTARD-INDUCED CROSS-LINKS

B

A +M

-iI..i ENZYME: I -M

T

B L

C

D

"L

I ENZYME B x -M

I

x

I

ALKALI

)'~ !

2-M

C NEUTRALIZE

j

"

I

1.73

Buoyant density

CHEMICAL ANALYSIS

G-M G-M-G

G-M G-M-G

; G-M

G-M-G DECREASED AMOUNT

Fig. i. Microdensitometer tracings of the ultraviolet a b s o r p t i o n p h o t o g r a p h s of alkaline-denatured and neutralized D N A after 18 h of d e n s i t y - g r a d i e n t ultracentrifugation. Conditions are as described in MATERIALS AND METHODS. The tracings are of D N A isolated from L-cells (A) I h, (B) 2.5 h, (C) 4.5 h a n d (D) 6 h after t r e a t m e n t with I/~g/ml of sulfur m u s t a r d . The peaks are from r i g h t to left, using tracing A, m a r k e r D N A (p 1.7313), d e n a t u r e d DNA, r e n a t u r e d D N A and satellite DNA. Fig. 2. A schematic r e p r e s e n t a t i o n of the reaction of sulfur m u s t a r d (M) w i t h t h e D N A of Lcells a n d the m a n n e r in which its alkylation p r o d u c t s are r e m o v e d (top line); the results expected after alkaline d e n a t u r a t i o n followed b y neutralization (centre portion); and the alkylation products to be found on p a p e r c h r o m a t o g r a p h y of the D N A isolated at each step ( b o t t o m line). (A) N a t i v e u n a l k y l a t e d D N A undergoes s t r a n d s e p a r a t i o n in alkali. W h e n the solution is neutralized the s t r a n d s r e m a i n separated. This D N A will n o t elute f r o m m e t h y l a t e d a l b u m i n kieselguhr c o l u m n and b a n d s in the ' d e n a t u r e d ' position in a CsC1 gradient. Since the D N A has n o t been t r e a t e d w i t h sulfur m u s t a r d at this point, no alkylation p r o d u c t s will be found. (B) Once the D N A has reacted w i t h the sulfur m u s t a r d the s t r a n d s will no longer s e p a r a t e w h e n exposed to alkali, so t h a t w h e n the solution is neutralized, the D N A renatures, and can be recovered f r o m a m e t h y l a t ed a l b u m i n kieselguhr column. I t will also b a n d in the ' n a t i v e ' position in a CsC1 gradient. Chemical analysis s h o w s the presence of b o t h the m o n o g u a n i n y l (G-M) and diguaninyl ( G - M - G ) alkylation products. (C) One a r m of the cross-link has become detached from one of the D N A strands, so t h a t the addition of alkali to this material causes s t r a n d separation similar to t h a t seen w i t h u n a l k y l a t e d DNA. T h u s w h e n this solution of D N A is neutralized, the D N A will not renature, therefore will n o t elute from a m e t h y l a t e d a l b u m i n kieselguhr column and will b a n d in the ' d e n a t u r e d ' position in the ultracentrifuge. However, chemical analysis of this material will still show the presence of b o t h the mono- and diguaninyl alkylation products. (D) F u r t h e r excision of the r e m a i n i n g a r m of the cross-link, or of the m o n o g u a n i n y l p r o d u c t , will n o t change the results obtained w i t h either the m e t h y l a t e d a l b u m i n kieselguhr c o l u m n or the ultracentrifuge from those o b t a i n e d at stage (C), b u t will be seen as a loss of total alkylations.

double helix was o b t a i n e d from the following e x p e r i m e n t . L-cells were t r e a t e d with I ~ g / m l of E35S]sulfur m u s t a r d a n d t h e D N A isolated I a n d 6 h after t r e a t m e n t . The D N A at a c o n c e n t r a t i o n of IOO/,g/ml in 15. lO -4 M N a C l - I 5 . I O - 5 M sodium c i t r a t e Biochim. Biophys. Acta, 179 (1969) 179-188

I84

B . D . REID, I. G. WALKER

TABLE I C H E M I C A L A N D P H Y S I C A L M E A S U R E M E N T S ON AFTER TREATMENT WITH SULFUR MUSTARD

Time a/ter Mustard treatment with (moles/io 5 mustard (h) nucleotides)

0.2 I.O 2"5 4.5 6.0 24.0

DN;~_

ISOLATED

Monoguaninyl mustard

I.I4=ko.2o* ( 9 ) 1.591o.26"*(15) 1'43 (3) 1.31 (3) 1.274-o.33

(15)

o-65~o-I4

(9)

FROM L-CELLS

AT VARIOUS TIMES

Diguaninyl mustard

Total DNA recovered as cross-linked, renaturable DNA by melhy!ated albumin kieselguhr chromatography (%)

(molar ratio)

El~5IjIododeoxyuridine-DN,4 [32PTDNA

2-2° k-°.46 3.I5±O.4O 2.2 4 2.67 2.35±0.46 2"85±0-78

I3.6-ko.2 I7.2~-3.o 7-9 4.0 2.8

(4) (6) (I) (I) (6) (5)

(3) (5) (I) (I) (2)

I9.7

(I)

2. 7 (I) 2.0 (1)

" Mean v a l u e s ± S . D . are given along w i t h t h e n u m b e r of trials in p a r e n t h e s e s . ** T h e I-h value for L-cells is s t a t i s t i c a l l y s i g n i f i c a n t l y different from t h e o.2-h value ( P < o.o I ).

solution was denatured with alkali and renatured as described above and then coprecipitated from solution with bovine serum albumin by adding I M perchloric acid tG a final concentration of o.33 M. The DNA was collected on a nlillipore filter, the filter placed in a counting vial containing 5 ml of a toluene-fluor system and the sample counted. A sample which was not denatured was also counted in this manner. The purpose of denaturing the DNA was to allow complete strand separation so that physically trapped molecules would be released. The counts obtained with or without the denaturation step, were identical, indicating that the radioactivity associated with the DNA was due to covalently bound sulfur mustard. Paper chromatographic studies of acid-hydrolysed DNA (third column, Table I), showed no preferential excision of either the monoguaninyl or the diguaninyl products, that is, the molar ratio of these compounds remained relatively constant. The identity of the diguaninyl product rests on its chromatographic behaviour. Because of its insolubility it has not been possible to obtain a paper chromatographic solvent that would increase its RF value. It seemed possible that some of the radioactivity in the area on the chromatogram assigned to the diguaninyl product could have been due to alkylated apurinic acid 21 which also would remain near the origin. The following experiment was performed to test this possibility. L-cells were treated with I/~g/ml of EasS]sulfur mustard and the DNA isolated i h after treatment as described above. The DNA was hydrolysed in 0.05 ml of I M HCI at IOO° for 15 rain. The hydrolysate was diluted to 2 ml with water and a portion was dialysed against 2 vol. of 0.025 M HC1 for 8 h. Analysis of the solutions inside and outside of the dialysis sac revealed that less then 4 % of the radioactivity was nondialysable. Therefore, the radioactive area near the origin of the chromatograms was not alkvlated apurinic acid.

The loss o/ cross-links /rom DNA Studies using a methylated albumin kieselguhr column For most of these studies L-cells were grown in medium containing r125I~Biochim. Biophys. Acta, 179 (1969) 179-188

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185

iododeoxyuridine as described above in order to provide labelled DNA. Similar results were obtained with cells which had been grown in medium containing 32p1. In nine trials, 96.9 °/o4-9.1 °/o (S.D.) of native DNA applied to methylated albumin kieselguhr columns was recovered in the 0. 7 M plus 0.8 M NaC1 fractions. In four trials with denatured DNA, 0.9 % ± o . I % (S.D.) was recovered in these fractions. DNA from cells that had been alkylated with I ~g/ml of mustard behaved like native DNA. Thus, the method was deemed capable of distinguishing between native and denatured DNA, and alkylation per se had no effect on the elution properties of undenatured DNA. The amount of renaturable DNA following treatment of L-cells with I pg/ml of sulfur mustard was examined next. DNA was isolated from E125Iliododeoxyuridine or 32pl-labelled L-cells at various times after treatment, denatured with alkali, neutralized and applied to a methylated albumin kieselguhr column. The results are given in the last two columns of Table I. The following points should be noted. i. The amount of renaturable DNA i h after sulfur mustard treatment was greater than that IO min after treatment. This effect paralleled the increased total alkylation of DNA seen I h after treatment compared with that io rain after treatment (Table I, column 2). 2. Between I and 6 h after treatment there was a rapid exponential reduction in the amount of renaturable DNA. The process had a half-life of about 2 h. 3. The renaturable DNA present 24 h after treatment is presumed to represent the spontaneously renaturable DNA that is almost always seen even with unalkylated DNA. 4. The rapid loss of renaturable DNA, which occurred between I and 6 h after treatment, is to be compared with the rather slow loss of total alkylation products during that time. In addition there was no appreciable increase in the ratio monoguaninyl to diguaninyl mustard (Table I, column 3), which would have indicated a preferential loss of the diguaninyl compound.

Studies using the ultracentri]uge It appeared anomalous that renaturable DNA was lost at a much greater rate than were the total alkylation products and that this loss should take place when there was no accompanying preferential excision of the diguaninyl product. It seemed worthwhile therefore to assess renaturability by another method, namely CsC1 density-gradient ultracentrifugation. Previous workers had shown that cross-linked DNA which had been exposed to alkali and then neutralized would renature and band in the 'native' position 4,6,7,22. Accordingly cells were treated with i #g/ml of sulfur mustard and the DNA isolated at various times thereafter. The DNA, at a concentration of approx. 75 #g/ml was denatured and neutralized in the usual manner, I ml removed, diluted with an equal volume of water, 2.565 g of CsC1 and 20/zl of reference DNA(approx. 0. 5/~g of DNA per ml) added, the refractive index adiusted to 1.4OLO, and the sample centrifuged 18 h at 20 ° at 44 77 ° rev./min, the final concentration of DNA being approx. 6/~g/ml. The results shown in Fig. I, for DNA isolated from top to bottom are 1, 2.5, 4.5 and 6 h after the cells had been treated with mustard. This pattern was obtained a number of times. The reason for the greater proportion of native to denatured DNA seen I h after treatment, as compared to the value obtained from the methylated albumin kieselguhr column is unknown. Nevertheless, the dramatic reduction in the amount Biochim. Biophys. Acta, 179 (1969) 179-188

186

B.D.

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

of renaturable DNA as a function of time after treatment is apparent. DNA isolated from L-cells 24 h after mustard treatment gave the same pattern as the 6-h sample. The small peak at the left is the mouse satellite DNA (ref. 23).

Sedimentation-velocity studies o/unalkylated and alkylated DNA Since the proportion of cross-linked renaturable DNA would decline if extensive shearing of the DNA molecule had occurred 2~it was necessary to obtain a measure of the molecular weight of all the DNA samples examined. These studies yielded a value of 2. lO7 for the molecular weight of DNA isolated from untreated cells, from cells IO min after treatment and from cells 24 h after treatment. Therefore, the loss of alkylation products did not appear to effect the molecular weight of the DNA isolated.

DISCUSSION

Chemical analysis of DNA isolated from L-cells at various times after treatment with sulfur mustard showed that the rate of loss of alkylation products was logarithmic with a half-life of about 18 h and that there was no preferential excision of the diguaninyl alkylation product. On the other hand, physical measurements revealed that the rate of loss of functional cross-links was also logarithmic but the half-life for the process was about 2 h. The problem then was to rationalize these apparently conflicting results since the presence of diguaninyl compound has been taken as an indicator of a cross-link. First, it could be argued that the radioactivity at the origin of the chromatograms was not entirely due to diguaninyl compound but to a mustard derivative of apurinic acid which also would exhibit a low chromatographic mobility. This possibility was eliminated by the experiment in which mildly acid-hydrolysed, alkylated DNA was dialysed. Since virtually all of the radioactivity was dialysable, it cou!d not have been associated with apurinic acid. Second, it was conceivable that the diguaninyl compound could have been selectively excised but remained trapped in the DNA helix resulting in erroneously high values for covalently bound diguaninyl compound. This possibility was eliminated by counting samples of iasSJsulfur-mustard-treated DNA with and without alkaline denaturation and neutralization. The purpose of the denaturation step was to cause the helix to unfold, thus allowing any diguaninyl compounds trapped in the helix by physical forces to be freed into the surrounding medium. The results of this experiment showed no difference in the total counts with or without denaturation and neutralization. Therefore, no diguaninyl products were physically trapped in the DNA helix, producing false values, and all the radioactivity measured nmst have been due to covalently bound sulfur mustard. Third, the size of the DNA molecules isolated would affect the methylated albumin kieselguhr column or ultracentrifuge results, since the proportion of renaturable DNA is dependent upon the molecular weight of the DNA isolated 22. If a long DNA molecule containing a limited number of cross-links is broken into several pieces, only those pieces containing cross-links would be capable of renaturation. As the amount of shearing or enzyme-mediated breakage of the molecule increased, the proportion of renaturable DNA would decrease. The measurements on DNA from unBiochim. Biophys. dcla. 179 (I969) 179 188

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187

treated and treated cells, showed that there was no change in the molecular weight of the DNA isolated due either to treatment with sulfur mustard or to the loss of alkylation products from the DNA. It could be argued that alkylation and subsequent nuclease activity could lead to single-strand breaks which would not be detected in the above analysis, and which would result in a lowered recovery of renaturable DNA. However, i h after treatment the extent of alkylation was one mole of sulfur mustard per 6.104 nucleotides. This corresponded to approx, one alkylation per molecule of DNA isolated. It is unlikely that the introduction of such a small number of alkylations would lead to the large number of single-strand breaks necessary to account for the decrease in the renaturable fraction of DNA. That the band widths of the denatured fraction of DNA centrifuged in CsC1 were the same at each observation time supports this conclusion. Fourth, it could be argued that the majority of the diguaninyl compounds measured chemically were derived from intrastrand rather than interstrand crosslinks and that a preferential loss of the latter would not have been detected by the analyses. This argument fails when it is considered that the average molecular weight of the DNA isolated was 2" I07 and that the degree of alkylation was about one alkyl group per 105 nucleotides which comprise a molecular weight of 3" IOV. Of these alkylation products only about one quarter is a diguaninyl compound. In order to achieve the degree of renaturation observed, most of these diguaninyl compounds must have participated in interstrand cross-links. The wholle question of interstrand versus intrastrand cross-links is still unresolved in the literature. LAWLEY AND BROOKES3 showed that native salmon sperm DNA alkylated with sulfur mustard in aqueous buffer contained 22 % of the total alkylated bases as the diguaninyl derivative, whereas the same DNA alkylated in 9° % methanol, where strand association was absent, contained only the monoguaninyl derivative. VENITT° treated E. coli B/r with sulfur mustard, and after an 80-rain incubation he found that both cross-links and diguaninyl compound had been removed. YAMAMOTO, NAITO AND SHIMKIN24 found that bacteriophages that contained single-stranded RNA and DNA were inactivated readily by bifunctional mustards but not by monofunctional alkylating agents. They inferred from this that intrastrand cross-links had been formed in the nucleic acid but pointed out that crosslinks between nucleic acid and protein could also have been responsible for the effect. Until more evidence is available about the formation of intrastrand cross-links in vivo, particularly within mammalian cells, we shall continue to assume that the diguaninyl compound arises from an interstrand cross-link. We propose the following explanation for our results. There exists within the cell some mechanism, probably enzymic, which 'unhooks' one arm of the cross-link. This would leave the DNA non-renaturable but since the other arm of the crosslink is still covalently attached to the DNA molecule, it would be detected as a diguaninyl compound after acid hydrolysis and paper chromatography. Eventually, the other arm of the cross-link would be excised in the same manner as any monoalkylation product. Our results and this explanation are diagrammed in Fig. 2. HANAWALT AND HAYNES 25, using E. coli and nitrogen mustard, and ROBERTS, CRATHORN AND BRENT 26, using HeLa cells and very large doses of sulfur mustard, demonstrated nonconservative DNA replication following treatment with the musBiochim. Biophys Acla, 179 (1969) 179-188

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tard. These findings suggest an excision and replacement process, however, the exact nucleic derivative removed by the cell, nucleotide, free base, etc., remains unknown. KOHN, STEIGBIGEL AND SPEARS7 have suggested that in the case of alkylation of E. coli by nitrogen mustard, 'a strand segment containing one end of the cross-link could be excised, allowing it to swing out and permit replacement of the segment by new DNA, synthesized using the intact strand as template'. Our results support this idea. However, the template strand still bears an alkylated positively charged guanine, albeit displaced vertically by one base unit from the gap on the other strand. The influence of this alkylated guanine could make it difficult for correct replacement of the excised guanine. This could be part of the explanation of the much greater toxicity of difunctional alkylating agents relative to monofunctional agents. Finally, it remains to be established what relation these excision studies have to the repair process. At the alkylation levels used in the present and other studies with mammalian cells, very few cells survive. The fraction surviving after a dose of sulfur mustard of I #g/ml is between Io -4 and IO 5. Thus it cannot be said yet, whether these excision processes are only pathological events occurring in the dying cell or whether they are truly part of a repair process that allows survival. REFERENCES 1 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

P. D. LAWLEY AND P. BROOKES, Exptl. Cell Res. suppl., 9 (I963) 512. P. BROOKES AND P. D. LAWLEY, Biochem. J., 80 (1961) 496. P. D. LA~VLEY AND P. BROOKES, J. Mol. Biol., 26 (1967) 143. E. P. GEIDUSCHEK, Proc. Natl. Acad. Sci. U.S., 47 (1961) 95 o. P- BROOKES AND P. D. LAWLEY, Exptl. Cell Res. Suppl., 9 (1963) 521. K. W. KOHN, C. L. SPEARS AND P. DOTY, J. Mol. Biol., 19 (1966) 266. K . W . KOHN, N. H. STEIGBIGEL AND C. L. SPEARS, Proc. Natl. Acad. Sci. U.S., 53 (1965) 1154. P. D. LAWLEY AND P. BROOKES,Nature, 206 (1965) 480. S. VENITT, Biochim. Biophys. Acta, 31 (1968) 355. A. R. CRATHORN AND J. J. ROBERTS, Nature, 2 i i (1966) 15o. J. E. TILL, G. F. WHITMORE AND S. GULYAS, Biochim. Biophys. Acta, 72 (1963) 277. L. DIAMOND, V. DEFENDI AND P. BROOKES, Cancer Res., 27 (1967) 890. K. BURTON, Biochem. J., 62 (1956) 315 . M. JEFFAY, F. O. OLUBAJO AND W. R. JEWEL, Anal. Biochem., 32 (196o) 3o6. H. BRAUNSBERG AND A. GUYVES, Anal. Biochem., IO (1965) 86. J. D. MANDELL AND A. D. HERSHEY, Anal. Biochem., i (196o) 66. R. SHEININ, Virology, 28 (1966) 621. J. EIGNER AND P. DOTY, J. Mol. Biol., 12 (1965) 549. P. D. LAWLEY, The Molecular Basis o[ Neoplasia, University of Texas Press, Austin, 1962, p. 123. I. G. WALKER AND C. J. THATCHER, Radiation Res., 34 (1968) i i o . C. TAMM, M. E. HODES AND E. CHARGAFF,J. Biol. Chem., 195 (1952) 49. V. N. EYER AND W. SZYBALSKI, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 355. E. H. L. CHUN AND J. W. LITTLEFIELD, J. Mol. Biol., 7 (1963) 245N. YAMAMOTO, S. NAITO AND lX~. B. SHIMKIN, Cancer Res., 26 (1966) 23Ol. P. HANAWALT AND R. H. HAYNES, Biochem. Biophys. Res. Commun., 19 (1965) 462J. J. ROBERTS, A. R. CRATHORN AND T. P. BRENT, Nature, 218 (1968) 97 o.

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