181
Mutation Re search , 42 ( 19 77 ) 181-190
© Elsevier/North-Holland Biomedical Press
SULPHYDRYL.MEDIATED DNA BREAKAGE BY PHLEOMYCIN IN ESCHERICHIA COLI
MERILYN J. SLEIGH and GEOFFREY W. GRIGG
C.S.l.R .O., Molecular and Cellular Biology Unit, P.O. Box 90, Epping, N .S .W . 2121 (Australia) (Received August 8th, 1976) (A ccepted October 20th, 1976)
Summary Sulphydryl-mediated DNA breakage, which is induced by the antibiotic phleomycin in vitro, has been found to contribute significantly to the DNA damage produced by phleomycin in Escherichia coli. The effect of pleomycin was inhibited in vivo, as in vitro, by chelating agents, sulphydryl blocking agents and antioxidants. An increase in the intracellular concentration of free sulphydryl resulted in an increased response to phleomycin, while mutants containing very low levels of free sulphydryl due to a defect in glutathione synthesis showed greatly reduced DNA breakage , particularly at low phleomycin concentrations. In spheroplasts of these gshA mutants, restoration of the response to phleomycin did not require glutathione specifically, but could be achieved by the addition of dithiothreitol. Sulphydryl-mediated breakage appears to be the principal mechanism for DNA damage in E. coli at low phleomycin concentrations, while some other mechanism, possibly enzymic , operates at higher phleomycin concentrations.
Introduction The glycopeptide antibiotic phleomycin inhibits replication and induces DNA degradation in a wide range of organisms, e.g. viruses, bacterial cells and tumour cells [4---{),15] . In vitro, phleomycin sensitises DNA to breakage by both enzymic [17] and non-enzymic mechanisms [16,19]. The enzymic mechanism depends on the ability of relatively high concentrations of phleomycin to produce local denaturation in DNA, which is then susceptible to the action of single strand-specific endonucleases [17] . Non-enzymic DNA breakage occurs in the presence of sulphydryl compounds. The proposed mechanism involves metal ion/Ov-catalysed oxidation of sulphydry l to its free radical form RS , fol-
182
lowed by reaction of RS' with phleomycin bound to DNA, leading to regeneration of RSH and DNA breakage [16]. We have studied the contribution of this non-enzymic mechanism, which occurs at very low phleomycin concentrations in vitro [16], to DNA breakage produced by phleomycin in Escherichia coli. We find that sulphydryl-mediated breakage occurs in vivo, and appears to be the principal mechanism for DNA breakage at low phleomycin concentrations (';;;;2 ,ug/ml). Materials and methods Bacterial strains E. coli B, obtained from the late Dr. Ruth Hill, is lon, sur and is filamentforming. A thymine-requiring mutant was prepared from the parent strain [13]. The isogenic E. coli K12 strains 129 (pyr, thy, thi, lac, gshA) and KMBL 54 (pyr, thy, thi, lac, gshA +) were kindly provided by Professor W. Berends, Technological University of Delft (Delft, The Netherlands). Materials Phleomycin (batch A9331-648) was supplied by Dr. W. Bradner, Bristol Laboratories, Syracuse, N.Y., U.S.A. All other materials were obtained from commercial sources and were of the purest grade available. Growth of cells The growth medium used was a modified Gray and Tatum (GT) glucose-salts medium [4]. Cultures were grown to stationary phase (16 h at 37°C) with addition of thymidine, thiamine, cytosine, and uracil (each at 20,ug/ml) as required. DNA was labelled in thymine-requiring ,strains by the addition of 20 pg of thymidine per ml, either 4 C] thymidine (60 mCi/mM undiluted with cold thymidine) or [3H] thymidine (1.0 Ci/mmol, diluted 100-fold with unlabelled thymidine). Both were from The Radiochemical Centre (Amersham, Bucks., U.K.). Stationary phase cells were harvested by filtration on pre-washed Millipore membrane filters (pore size, 0.45 ,urn), washed with GT medium containing unlabelled thymidine and resuspended in the same medium. Exponentiallygrowing cells were obtained by incubating the cells for 2 h at 37° C after resuspension.
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Measurement of DNA damage in vitro The method for analysis of single strand breakage in native T2 phage DNA by separation of DNA fragments on a column of agarose beads (Biegel A-50M) has been described previously [16]. Measurement of DNA damage in E. coli The appearance of single strand breaks and/or alkali-labile sites in cellular 14 [ C] thymidine-labelled DNA was measured by alkaline sucrose density gradient centrifugation [10]. E. coli B samples containing approx. 10 8 cells in a volume of 0.1 ml were incubated at room temperature with 10 ,ul of lysozyme (2 rng/ml) (Calbiochem, Spring Valley, N.Y., U.S.A.) and 10,u1 of 0.01 M Tris/
183
0.01 M EDTA, pH 8.3. After 1 min, 10 pI (l08 cells) of 3H-Iabelled control cells was added. After a further 10 min, the spheroplasts were transferred to a layer (0.4 ml) of 0.5 M NaOH and 0.5% sodium lauryl sarcosinate on 5-20% alkaline sucrose gradients (11 ml on a 1 ml cushion of 2 M sucrose, prepared in 0.1 M NaOH/0.9 M NaCI/1 mM EDTA). After 20 min at room temperature, gradients were centrifuged in a Beckman SW41 rotor at 30,000 r.p.m. for 90 min at 21°C. Fractions (0.22 ml) were pumped from the bottom of the tube onto glass fibre paper (Whatman CF187). The papers were dried, transferred to vials containing 10 ml of scintillator (5.5 g of Packard Permablend/1 litre of toluene) and counted for radioactivity in a Packard liquid scintillation spectrometer. E. coli K12 samples were prepared for centrifugation by EDT A/lysozyme treatment as described above, followed by incubation for 10 min with 10 pI pronase (7 mg/ml) (Calbiochem, Spring Valley, N.Y., U.S.A.), and 50 pI sodium dodecyl sulphate (0.2% w/v). Samples were then transferred to a layer (0.4 ml) of NaOH (0.5 M) on top of 5-20% alkaline sucrose gradients. Subsequent treatment was as described above. Preparation of E. coli spheroplasts Growing cells (approx. 10 9 ) were suspended in 16 ml 0.05 M Tris-HCI buffer, pH 8.0, containing 0.5 M sucrose, 0.1 M KCI, 0.025 M NaCl, 0.001 M EDTA. The cells were spun at 8000 g for 10 min, and resuspended in 0.7 ml of the same buffer but without EDTA. After 6 min, an equal volume of 0.05 M TrisHCI, pH 8.0, containing pg/ml lysozyme, was added. The mixture was held for 5 min at room temperature and then 10 min at 4°C. MgSO,! (1 mM), glucose (1 mg/rnl) and thymidine (20 pg/ml) were added to the final preparation and 0.2 ml samples were used for phleomycin treatment. Each spheroplast was checked by observing cell lysis on addition of distilled water.
Results and discussion Requirements for DNA breakage in vivo Sulphydryl-mediated, phleomycin-induced DNA breakage in vitro requires oxygen, and phleomycin-bound metal ions, which catalyse the oxidation of free thiol to its radical form, an intermediate in the breakage pathway. DNA breakage is inhibited by 8-hydroxyquinoline, which removes copper ions from phleomycin, and EDTA, which blocks the catalytic activity of the metal ions [16]. Breakage is also inhibited by the sulphydryl blocking agent N-ethylmaleimide, and by KI [16], which inhibits free radical reactions such as lipid autoxidation [8] and DNA breakage after X-irradiation [20]. DNA breakage by phleomycin in E. coli B had similar requirements. The response to phleomycin was blocked by EDTA (Fig. 1), 8-hydroxyquinoline (Fig. 1), N-ethylmaleimide (Fig. 2) and KI (Fig. 2). The inhibition by KI was not a non-specific effect of high salt concentration, since an equivalent concentration of NaCI had only a slight inhibitory effect on the DNA breakage induced by phleomycin. Inhibition of breakage by KI, EDT A, 8-hydroxyquinoline and N-ethylmaleimide would be expected if non-enzymic sulphydrylmediated breakage was contributing significantly to the response to phleo-
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Fig. 1. Inhibition of phleornvcin-Induced DNA breakage by chelating agents. Exponentially-growing E. coli B thy were treated for 30 min with phleom ycin (0.5 I-lg{ml) alone (A), or in the presence of 50 mM 8-hydroxyquinoline (B), or 50 mM EDTA (C). (e) 1 4C-labelled DNA from phleornvcln-treated cells; (0) 3H.labelled marker DNA. Sedimentation is from right to left. Fig. 2. Inhibition of phleomvcin-Induced DNA breakage by KI and N-ethylmaleimide. Exponentially growing E. coli B thy cells were treated for 30 min with phlcomycin (1 Ilg{ml) alone (A) or in the presence of 0.1 M KI (B) or 0.2 M N-ethylmakimide (C). (-) 14C-labelled DNA from phleomv cin-treatcd cells; (0) 3H-Iabelled marker DNA. Sedimentation is from right to left.
mycin in vivo. However, the results do not exclude an enzymic breakage mechanism e.g. N-ethylmaleimide may be reacting with a sulphydryl group essential for enzyme activity. The DNA sedimentation patterns from phleomycin-treated cells showed a range of sizes of DNA fragments, with some DNA apparently resistant to breakage. The effect of increasing the phleomycin concentration was to reduce the amount of this resistant DNA (e.g. compare Figs. 1A and 2A). The "unbroken" DNA in the sedimentation patterns actually contained 8-10 breaks per chromosome single strand (calculated as described in [21], using DNA from T2 bacteriophage as a size marker). Since binding sites for phleomycin are distributed non-randomly in DNA [2,14], the simplest explanation for the apparent resistance of some DNA species to phleomycin-induced breakage is that they contain binding sites which are occupied only at high levels of phleomycin. Variations in the metabolic state of the cells are unlikely to explain the variation in sensitivity to phleomycin [18] since all the experiments described here used exponentially-growing cells. Since the methods available for estimating breakage frequency from this sort of data assume randomness of DNA strand break-
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Fig. 3. Addition of dithiothreitol to phleomycin-treated cells and spheroplasts of E. coli B thy. Exponent. ially growing E. coli B thy cells were treated for 15 min with phleomycin (2 IJgjml) (A). or with d ithiothreitol (5 mM) added 1 min after phleomycin (2 IJg/ml) (B). Spheroplasts of E. coli B thy were treated for 15 min with phleomvcin (0.5 IJgjml) alone (C) or with dithiothreitol (5 rn M) added 1 min after phleomycin (D). (-) 14C-labelled DN A from phleom vctn-tre ated cells; (el) 3H-labelled marker DNA. Sedimentation is from right to left.
age, no attempt has been made to express results in terms of numbers of breaks per chromosome. Effect of increasing the intracellular concentration of free sulphydryl Simultaneous addition of phleomycin and sulphydryl compounds to E. coli cells resulted in a decrease in the amount of DNA breakage observed (Fig. 3 and [12]). In spheroplasts of E. coli B, the response to phleomycin was greater than in whole cells, and the effect of added dithiothreitol was to increase DNA breakage by phleomycin (Fig. 3). In both spheroplasts and whole cells, dithiothreitol was added 1 min after phleomycin. In vitro, incubation of dithiothreitol with phleomycin before the addition of DNA resulted in inhibition of the DNA breaking effect, while addition of dithiothreitol and phleomycin to DNA at the same time, resulted in considerable DNA damage [16]. The effects of dithiothreitol on phleomycin-induced DNA breakage in whole cells and spheroplasts can be explained if the penetration by phleomycin into E. coli is a relatively slow process. Certainly the cell envelope presents a barrier to phleomycin since an increase in membrane permeability as a result of EDT A treatment [9] resulted in an increase in the response to phleomycin. Slow penetration of the cell envelope may allow time for dithiothreitol to inactivate phleomycin, while in spheroplasts, rapid entry into the cell and binding to the DNA may protect phleomycin from dithiothreitol inactivation. From the results, we conclude that DNA treated with phleomycin in vivo (at least in spheroplasts) is susceptible to sulphydryl-mediated breakage. Since an increase in free sulphydryl levels results in an increase in DNA breakage, the amount of sulphydryl normally available in the cell to catalyse breakage must be rate-limiting.
186
Effect of decreasing the intracellular concentration of free sulph ydryl In E. coli, at least 95% of low molecular weight free sulphydryl is found as reduced glutathione [7J. Apontoweil and Berends [1] have recently isolated an E. coli K12 mutant, gshA, which is deficient in glutathione synthesis, and contains no detectable glutathione. Phleomycin-induced DNA breakage in this mutant was greatly reduced, compared with the response in the parent, gshA +, strain (Fig. 4). Note that the E. coli K12 gshA + strain was rather more sensitive to phleomycin-induced DNA breakage than the E. coli B strain (cf. Figs lA, 2A, 4D and 4E). Since glutathione has been implicated in some amino acid transport pro cesses [3], it was possible that the reduced response to phleomycin in the glutathione-deficient strain 129 was due to a decrease in permeability to the antibiotic. However, spheroplasts of E. coli strain 129 (gshA -), showed the same reduction in phleomycin-induced DNA breakage, compared with wild type, as was observed in whole cells (compare Figs. 5A and 5D). Addition of an excess of dithiothreitol to phleomycin-treated spheroplasts of the gshA - strain 129 resulted in an increase in DNA breakage to the level observed in wild type spheroplasts under the same conditions (Fig. 5), demonstrating that in the mutant strain there was no change in the sensitivity of the DNA to sulphydryl-mediated breakage. Glutathione itself did not increase DNA
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Fig. 4 . Phelomycin-induced DNA breakage in a glutathione-deficlent strain of E. coli. Exponentiall y gr owing E, coli 129 (gshA) (A-{;) or KMBL 54 (gshA +) (D-F) were treated for 30 min with 0.5, 1.0, 2 .0 .ug/ml phleomycin respectively. (.) ) 4 C-l ab elled DNA from phteom y ci n -t re a t ed cells ; ( 0) 3 H-labelled mark er DNA . Seodim entation is from right t o left.
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Fig. 5. Effect of dithiothreitol and glutathione on phleomycin-induced DNA breakage in spheroplasts of E . coli 129 (gshA) and KMBL 54 ( gshA ~). Spheroplasts of E. coli 129 (A-C) or KMBL 54 (D-F) were treated for 30 min with phlcorn ycin (0.2 ,ug/ml) (A,D), phleornvcin added 1 min before glutathione (10 mM) (B,E), or phleomvctn added 1 min before dithiothreitol (5 mM) (C,F). ( ...) 14C·labelled DNA from phleomycin·treated cells; ( v) 3H-labelled marker DNA. Sedimentation is from right to left.
188
breakage in phleomycin-treated spheroplasts of strain 129 (Fig. 5) and completely inhibited DNA breakage both in wild type spheroplasts (Fig. 5) and in spheroplasts of strain 129 treated with sufficient phleomycin to produce DNA breakage. This result may be explained by the fact that glutathione is a poor stimulator of DNA breakage by phleomycin, but a good phleomycin inactivator. Comparison of the ability of glutathione and dithiothreitol to introduce single strand breaks into native T2 phage DNA in vitro, in the presence of phleomycin (0.5 t-tg/mI), revealed that 1 mM dithiothreitol had the same activity as 5 mM glutathione. In these experiments, phleomycin was incubated with DNA for 2 min before glutathione or dithiothreitol was added. However, incubation of phleomycin (0.5 pg/ml) with glutathione (10 mM) for 5 min before addition of DNA, reduced subsequent DNA breakage during 30 min incubation by half, while 10 min incubation with 10 mM dithiothreitol was required to inactivate phleomycin to the same extent. Consequently, the ability of glutathione to inactivate phleomycin may predominate over its DNA breakage-stimulating activity, unless phleomycin is protected from glutathione until after it has bound to DNA. For example, in E. coli, phleomycin appears to induce DNA breakage predominantly in membrane-bound DNA [18]. Since glutathione occurs in a bound form in the cell [7] it may be that phleomycin is not exposed to glutathione until after it is attached to membrane-bound DNA. Miyaki et al. [11J have reported that the sensitivity of rat ascites tumour cells to bleomycin (an antibiotic which is structurally related to phleomycin, and seems to break DNA by a similar mechanism [16]) was related to the free sulphydryl concentration in the cells. Our results demonstrate that phleomycininduced DNA breakage in E. coli shows a similar sensitivity to intracellular sulphydryl levels. In addition, we have found that the requirements for DNA breakage in vivo are similar to those for sulphydryl-mediated breakage in vitro. This suggests that sulphydryl-mediated breakage makes a substantial contribution to the response to phleomycin in E. coli. Our results, however, do not exclude the possibility that DNA breakage is occurring by an enzymic mechanism which has the same requirements and sensitivity to free sulphydryl concentration. Sulphydryl-dependent DNA breakage appears to be of greatest importance in E. coli at low phleomycin concentrations «2 pg/ml). At higher concentrations DNA breakage was seen even in glutathione-deficient cells. This breakage, while still inhibited by chelating agents and sulphydryl-blocking agents, is resistant to inhibition by KI (Sleigh and Grigg, unpublished). This suggests that a second breakage mechanism operates at high phleomycin concentrations, as suggested by Grigg [4]. References 1 Apontoweil, P. and W. Berends, Isolation and initial characterization of glutathione-deficient mutants of Escherichia coli K12, Biochim. Biophvs, Acta, 399 (1975) 10-22. 2 Falaschi , A. and A. Kornberg, Phleomycin, an inhibitor of DNA polymerase, Fed. Pro c., 23 (1964) 940--945. 3 Fuchs, J.A. and H.R. Warner, Isolation of Escherichia coli mutant deficient in glutathione sv nth ..sis, J. Bacterio l.; 124 (1975) 140-148. 4 Grigg, G.W., Induction of.DNA breakdown and death in Escherichia coli by phleomycin. Its association with dark repair processes, Mol. Gen. Gene t., 104 (1969) 1-11.
189 5 Iwata, A. and R.A. Consiglt, Effect of phleomycin on polyoma virus synthesis in mouse embryo cells, J. Virol., 7 (1971) 29-40. 6 Jacobs, N .F., R.L. Neu and L.I. Gardner, Phleomycin-induced mitotic inhibition and chromosomal abnormalities in cultured human leucocytes, Mutation Res., 7 (1969) 251-253. 7 Jocelyn, P .C., Biochemistry of the SH group. Academic Press, London, 1972. 8 Kanazawa, K., G. Danno and M. Natake, Stabilisation of linoleic acid at the process of the autoxidation by potassium iodide, Agr, BioI. Chern., 39 (1975) 1177-1186. 9 Lieve , L., A non-specific increase in permeability in Escherichia coli produced by EDT A, Proc. Nat!. Acad. Sci. (U.S.A.), 53 (1965) 747-750. 10 McGrath, R.A. and R.W. Willams, Reconstruction in vivo of irradiated Escherichia coli deoxyribonucleic acid; the rejoining of broken pieces, Nature, 212 (1966) 534-535. 11 Mivaki, Moo T. Ono, S. Hori and H. Urnezaw a, Binding of bleomycin to DNA in bleomycin-sensitive and -resistant rat ascites hepatoma cells, Cancer Res., 35 (1975) 2015-2019. 12 Nakayama, H., Phleornycin induced lethality and DNA degradation in Escherichia coli K12, Mutation Res .. 29 (1975) 21-33. 13 Okada, F.J., K. Yanagisaw a and F.J. Ryan, Elective production of thymine-less mutants, Nature, 188 (1960) 340-341. 14 Pietsch, P. and H. Garrett, Phleornv cin: evidence of in vivo binding to DNA, Cv tobios, lA (1969) 7-15. 15 Reiter, H., M. Milewskiy and P. Kelley, Mode of action of phleornycin on Bacillus sub til is, J. Bacteriol., 111 (1972) 586-592. 16 Sleigh, M.J., The mechanism of DNA breakage by phleomvcin in vitro, Nucl, Acids. Res., 3 (1976) 891-901. 17 Sleigh, M.J. and G.W. Grigg, Induction of local denaturation in DNA in vitro by phleomycin and caffeine, FEBS Let t., 39 (1974) 35-38. 18 Sleigh, M.J. and G.W. Grigg, The mechanism of sensitivity to phleomycin in growing Escherichia coli cells, Biochem , J., 155 (1976) 87-99. 19 Stern, Roo J.A. Rose and R.M. Friedman, Phle omvcin-induced cleavage of deoxyribonucleic acid, Biochemistry, 13 (1974) 307-312. 20 Strniste, G.F. and S.S, Wallace, Endonucleolytic incision of X-irradiated deoxycibonucleic acid by extracts of Escherichia coli, Proc. Natl. Acad. Sci. (U.S.A.), 72 (1975) 1997-2001. 21 Woodcock, E.A. and G.W. Grigg, Repair of thermally induced DNA breakage in Escherichia coli, Nature, 237 (1972) 76-79.
Mutation Re search , 42 ( 19 77 ) 181-190
© Elsevier/North-Holland Biomedical Press
SULPHYDRYL.MEDIATED DNA BREAKAGE BY PHLEOMYCIN IN ESCHERICHIA COLI
MERILYN J. SLEIGH and GEOFFREY W. GRIGG
C.S.l.R .O., Molecular and Cellular Biology Unit, P.O. Box 90, Epping, N .S .W . 2121 (Australia) (Received August 8th, 1976) (A ccepted October 20th, 1976)
Summary Sulphydryl-mediated DNA breakage, which is induced by the antibiotic phleomycin in vitro, has been found to contribute significantly to the DNA damage produced by phleomycin in Escherichia coli. The effect of pleomycin was inhibited in vivo, as in vitro, by chelating agents, sulphydryl blocking agents and antioxidants. An increase in the intracellular concentration of free sulphydryl resulted in an increased response to phleomycin, while mutants containing very low levels of free sulphydryl due to a defect in glutathione synthesis showed greatly reduced DNA breakage , particularly at low phleomycin concentrations. In spheroplasts of these gshA mutants, restoration of the response to phleomycin did not require glutathione specifically, but could be achieved by the addition of dithiothreitol. Sulphydryl-mediated breakage appears to be the principal mechanism for DNA damage in E. coli at low phleomycin concentrations, while some other mechanism, possibly enzymic , operates at higher phleomycin concentrations.
Introduction The glycopeptide antibiotic phleomycin inhibits replication and induces DNA degradation in a wide range of organisms, e.g. viruses, bacterial cells and tumour cells [4---{),15] . In vitro, phleomycin sensitises DNA to breakage by both enzymic [17] and non-enzymic mechanisms [16,19]. The enzymic mechanism depends on the ability of relatively high concentrations of phleomycin to produce local denaturation in DNA, which is then susceptible to the action of single strand-specific endonucleases [17] . Non-enzymic DNA breakage occurs in the presence of sulphydryl compounds. The proposed mechanism involves metal ion/Ov-catalysed oxidation of sulphydry l to its free radical form RS , fol-
182
lowed by reaction of RS' with phleomycin bound to DNA, leading to regeneration of RSH and DNA breakage [16]. We have studied the contribution of this non-enzymic mechanism, which occurs at very low phleomycin concentrations in vitro [16], to DNA breakage produced by phleomycin in Escherichia coli. We find that sulphydryl-mediated breakage occurs in vivo, and appears to be the principal mechanism for DNA breakage at low phleomycin concentrations (';;;;2 ,ug/ml). Materials and methods Bacterial strains E. coli B, obtained from the late Dr. Ruth Hill, is lon, sur and is filamentforming. A thymine-requiring mutant was prepared from the parent strain [13]. The isogenic E. coli K12 strains 129 (pyr, thy, thi, lac, gshA) and KMBL 54 (pyr, thy, thi, lac, gshA +) were kindly provided by Professor W. Berends, Technological University of Delft (Delft, The Netherlands). Materials Phleomycin (batch A9331-648) was supplied by Dr. W. Bradner, Bristol Laboratories, Syracuse, N.Y., U.S.A. All other materials were obtained from commercial sources and were of the purest grade available. Growth of cells The growth medium used was a modified Gray and Tatum (GT) glucose-salts medium [4]. Cultures were grown to stationary phase (16 h at 37°C) with addition of thymidine, thiamine, cytosine, and uracil (each at 20,ug/ml) as required. DNA was labelled in thymine-requiring ,strains by the addition of 20 pg of thymidine per ml, either 4 C] thymidine (60 mCi/mM undiluted with cold thymidine) or [3H] thymidine (1.0 Ci/mmol, diluted 100-fold with unlabelled thymidine). Both were from The Radiochemical Centre (Amersham, Bucks., U.K.). Stationary phase cells were harvested by filtration on pre-washed Millipore membrane filters (pore size, 0.45 ,urn), washed with GT medium containing unlabelled thymidine and resuspended in the same medium. Exponentiallygrowing cells were obtained by incubating the cells for 2 h at 37° C after resuspension.
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Measurement of DNA damage in vitro The method for analysis of single strand breakage in native T2 phage DNA by separation of DNA fragments on a column of agarose beads (Biegel A-50M) has been described previously [16]. Measurement of DNA damage in E. coli The appearance of single strand breaks and/or alkali-labile sites in cellular 14 [ C] thymidine-labelled DNA was measured by alkaline sucrose density gradient centrifugation [10]. E. coli B samples containing approx. 10 8 cells in a volume of 0.1 ml were incubated at room temperature with 10 ,ul of lysozyme (2 rng/ml) (Calbiochem, Spring Valley, N.Y., U.S.A.) and 10,u1 of 0.01 M Tris/
183
0.01 M EDTA, pH 8.3. After 1 min, 10 pI (l08 cells) of 3H-Iabelled control cells was added. After a further 10 min, the spheroplasts were transferred to a layer (0.4 ml) of 0.5 M NaOH and 0.5% sodium lauryl sarcosinate on 5-20% alkaline sucrose gradients (11 ml on a 1 ml cushion of 2 M sucrose, prepared in 0.1 M NaOH/0.9 M NaCI/1 mM EDTA). After 20 min at room temperature, gradients were centrifuged in a Beckman SW41 rotor at 30,000 r.p.m. for 90 min at 21°C. Fractions (0.22 ml) were pumped from the bottom of the tube onto glass fibre paper (Whatman CF187). The papers were dried, transferred to vials containing 10 ml of scintillator (5.5 g of Packard Permablend/1 litre of toluene) and counted for radioactivity in a Packard liquid scintillation spectrometer. E. coli K12 samples were prepared for centrifugation by EDT A/lysozyme treatment as described above, followed by incubation for 10 min with 10 pI pronase (7 mg/ml) (Calbiochem, Spring Valley, N.Y., U.S.A.), and 50 pI sodium dodecyl sulphate (0.2% w/v). Samples were then transferred to a layer (0.4 ml) of NaOH (0.5 M) on top of 5-20% alkaline sucrose gradients. Subsequent treatment was as described above. Preparation of E. coli spheroplasts Growing cells (approx. 10 9 ) were suspended in 16 ml 0.05 M Tris-HCI buffer, pH 8.0, containing 0.5 M sucrose, 0.1 M KCI, 0.025 M NaCl, 0.001 M EDTA. The cells were spun at 8000 g for 10 min, and resuspended in 0.7 ml of the same buffer but without EDTA. After 6 min, an equal volume of 0.05 M TrisHCI, pH 8.0, containing pg/ml lysozyme, was added. The mixture was held for 5 min at room temperature and then 10 min at 4°C. MgSO,! (1 mM), glucose (1 mg/rnl) and thymidine (20 pg/ml) were added to the final preparation and 0.2 ml samples were used for phleomycin treatment. Each spheroplast was checked by observing cell lysis on addition of distilled water.
Results and discussion Requirements for DNA breakage in vivo Sulphydryl-mediated, phleomycin-induced DNA breakage in vitro requires oxygen, and phleomycin-bound metal ions, which catalyse the oxidation of free thiol to its radical form, an intermediate in the breakage pathway. DNA breakage is inhibited by 8-hydroxyquinoline, which removes copper ions from phleomycin, and EDTA, which blocks the catalytic activity of the metal ions [16]. Breakage is also inhibited by the sulphydryl blocking agent N-ethylmaleimide, and by KI [16], which inhibits free radical reactions such as lipid autoxidation [8] and DNA breakage after X-irradiation [20]. DNA breakage by phleomycin in E. coli B had similar requirements. The response to phleomycin was blocked by EDTA (Fig. 1), 8-hydroxyquinoline (Fig. 1), N-ethylmaleimide (Fig. 2) and KI (Fig. 2). The inhibition by KI was not a non-specific effect of high salt concentration, since an equivalent concentration of NaCI had only a slight inhibitory effect on the DNA breakage induced by phleomycin. Inhibition of breakage by KI, EDT A, 8-hydroxyquinoline and N-ethylmaleimide would be expected if non-enzymic sulphydrylmediated breakage was contributing significantly to the response to phleo-
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Fig. 1. Inhibition of phleornvcin-Induced DNA breakage by chelating agents. Exponentially-growing E. coli B thy were treated for 30 min with phleom ycin (0.5 I-lg{ml) alone (A), or in the presence of 50 mM 8-hydroxyquinoline (B), or 50 mM EDTA (C). (e) 1 4C-labelled DNA from phleornvcln-treated cells; (0) 3H.labelled marker DNA. Sedimentation is from right to left. Fig. 2. Inhibition of phleomvcin-Induced DNA breakage by KI and N-ethylmaleimide. Exponentially growing E. coli B thy cells were treated for 30 min with phlcomycin (1 Ilg{ml) alone (A) or in the presence of 0.1 M KI (B) or 0.2 M N-ethylmakimide (C). (-) 14C-labelled DNA from phleomv cin-treatcd cells; (0) 3H-Iabelled marker DNA. Sedimentation is from right to left.
mycin in vivo. However, the results do not exclude an enzymic breakage mechanism e.g. N-ethylmaleimide may be reacting with a sulphydryl group essential for enzyme activity. The DNA sedimentation patterns from phleomycin-treated cells showed a range of sizes of DNA fragments, with some DNA apparently resistant to breakage. The effect of increasing the phleomycin concentration was to reduce the amount of this resistant DNA (e.g. compare Figs. 1A and 2A). The "unbroken" DNA in the sedimentation patterns actually contained 8-10 breaks per chromosome single strand (calculated as described in [21], using DNA from T2 bacteriophage as a size marker). Since binding sites for phleomycin are distributed non-randomly in DNA [2,14], the simplest explanation for the apparent resistance of some DNA species to phleomycin-induced breakage is that they contain binding sites which are occupied only at high levels of phleomycin. Variations in the metabolic state of the cells are unlikely to explain the variation in sensitivity to phleomycin [18] since all the experiments described here used exponentially-growing cells. Since the methods available for estimating breakage frequency from this sort of data assume randomness of DNA strand break-
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Fig. 3. Addition of dithiothreitol to phleomycin-treated cells and spheroplasts of E. coli B thy. Exponent. ially growing E. coli B thy cells were treated for 15 min with phleomycin (2 IJgjml) (A). or with d ithiothreitol (5 mM) added 1 min after phleomycin (2 IJg/ml) (B). Spheroplasts of E. coli B thy were treated for 15 min with phleomvcin (0.5 IJgjml) alone (C) or with dithiothreitol (5 rn M) added 1 min after phleomycin (D). (-) 14C-labelled DN A from phleom vctn-tre ated cells; (el) 3H-labelled marker DNA. Sedimentation is from right to left.
age, no attempt has been made to express results in terms of numbers of breaks per chromosome. Effect of increasing the intracellular concentration of free sulphydryl Simultaneous addition of phleomycin and sulphydryl compounds to E. coli cells resulted in a decrease in the amount of DNA breakage observed (Fig. 3 and [12]). In spheroplasts of E. coli B, the response to phleomycin was greater than in whole cells, and the effect of added dithiothreitol was to increase DNA breakage by phleomycin (Fig. 3). In both spheroplasts and whole cells, dithiothreitol was added 1 min after phleomycin. In vitro, incubation of dithiothreitol with phleomycin before the addition of DNA resulted in inhibition of the DNA breaking effect, while addition of dithiothreitol and phleomycin to DNA at the same time, resulted in considerable DNA damage [16]. The effects of dithiothreitol on phleomycin-induced DNA breakage in whole cells and spheroplasts can be explained if the penetration by phleomycin into E. coli is a relatively slow process. Certainly the cell envelope presents a barrier to phleomycin since an increase in membrane permeability as a result of EDT A treatment [9] resulted in an increase in the response to phleomycin. Slow penetration of the cell envelope may allow time for dithiothreitol to inactivate phleomycin, while in spheroplasts, rapid entry into the cell and binding to the DNA may protect phleomycin from dithiothreitol inactivation. From the results, we conclude that DNA treated with phleomycin in vivo (at least in spheroplasts) is susceptible to sulphydryl-mediated breakage. Since an increase in free sulphydryl levels results in an increase in DNA breakage, the amount of sulphydryl normally available in the cell to catalyse breakage must be rate-limiting.
186
Effect of decreasing the intracellular concentration of free sulph ydryl In E. coli, at least 95% of low molecular weight free sulphydryl is found as reduced glutathione [7J. Apontoweil and Berends [1] have recently isolated an E. coli K12 mutant, gshA, which is deficient in glutathione synthesis, and contains no detectable glutathione. Phleomycin-induced DNA breakage in this mutant was greatly reduced, compared with the response in the parent, gshA +, strain (Fig. 4). Note that the E. coli K12 gshA + strain was rather more sensitive to phleomycin-induced DNA breakage than the E. coli B strain (cf. Figs lA, 2A, 4D and 4E). Since glutathione has been implicated in some amino acid transport pro cesses [3], it was possible that the reduced response to phleomycin in the glutathione-deficient strain 129 was due to a decrease in permeability to the antibiotic. However, spheroplasts of E. coli strain 129 (gshA -), showed the same reduction in phleomycin-induced DNA breakage, compared with wild type, as was observed in whole cells (compare Figs. 5A and 5D). Addition of an excess of dithiothreitol to phleomycin-treated spheroplasts of the gshA - strain 129 resulted in an increase in DNA breakage to the level observed in wild type spheroplasts under the same conditions (Fig. 5), demonstrating that in the mutant strain there was no change in the sensitivity of the DNA to sulphydryl-mediated breakage. Glutathione itself did not increase DNA
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Fig. 4 . Phelomycin-induced DNA breakage in a glutathione-deficlent strain of E. coli. Exponentiall y gr owing E, coli 129 (gshA) (A-{;) or KMBL 54 (gshA +) (D-F) were treated for 30 min with 0.5, 1.0, 2 .0 .ug/ml phleomycin respectively. (.) ) 4 C-l ab elled DNA from phteom y ci n -t re a t ed cells ; ( 0) 3 H-labelled mark er DNA . Seodim entation is from right t o left.
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Fig. 5. Effect of dithiothreitol and glutathione on phleomycin-induced DNA breakage in spheroplasts of E . coli 129 (gshA) and KMBL 54 ( gshA ~). Spheroplasts of E. coli 129 (A-C) or KMBL 54 (D-F) were treated for 30 min with phlcorn ycin (0.2 ,ug/ml) (A,D), phleornvcin added 1 min before glutathione (10 mM) (B,E), or phleomvctn added 1 min before dithiothreitol (5 mM) (C,F). ( ...) 14C·labelled DNA from phleomycin·treated cells; ( v) 3H-labelled marker DNA. Sedimentation is from right to left.
188
breakage in phleomycin-treated spheroplasts of strain 129 (Fig. 5) and completely inhibited DNA breakage both in wild type spheroplasts (Fig. 5) and in spheroplasts of strain 129 treated with sufficient phleomycin to produce DNA breakage. This result may be explained by the fact that glutathione is a poor stimulator of DNA breakage by phleomycin, but a good phleomycin inactivator. Comparison of the ability of glutathione and dithiothreitol to introduce single strand breaks into native T2 phage DNA in vitro, in the presence of phleomycin (0.5 t-tg/mI), revealed that 1 mM dithiothreitol had the same activity as 5 mM glutathione. In these experiments, phleomycin was incubated with DNA for 2 min before glutathione or dithiothreitol was added. However, incubation of phleomycin (0.5 pg/ml) with glutathione (10 mM) for 5 min before addition of DNA, reduced subsequent DNA breakage during 30 min incubation by half, while 10 min incubation with 10 mM dithiothreitol was required to inactivate phleomycin to the same extent. Consequently, the ability of glutathione to inactivate phleomycin may predominate over its DNA breakage-stimulating activity, unless phleomycin is protected from glutathione until after it has bound to DNA. For example, in E. coli, phleomycin appears to induce DNA breakage predominantly in membrane-bound DNA [18]. Since glutathione occurs in a bound form in the cell [7] it may be that phleomycin is not exposed to glutathione until after it is attached to membrane-bound DNA. Miyaki et al. [11J have reported that the sensitivity of rat ascites tumour cells to bleomycin (an antibiotic which is structurally related to phleomycin, and seems to break DNA by a similar mechanism [16]) was related to the free sulphydryl concentration in the cells. Our results demonstrate that phleomycininduced DNA breakage in E. coli shows a similar sensitivity to intracellular sulphydryl levels. In addition, we have found that the requirements for DNA breakage in vivo are similar to those for sulphydryl-mediated breakage in vitro. This suggests that sulphydryl-mediated breakage makes a substantial contribution to the response to phleomycin in E. coli. Our results, however, do not exclude the possibility that DNA breakage is occurring by an enzymic mechanism which has the same requirements and sensitivity to free sulphydryl concentration. Sulphydryl-dependent DNA breakage appears to be of greatest importance in E. coli at low phleomycin concentrations «2 pg/ml). At higher concentrations DNA breakage was seen even in glutathione-deficient cells. This breakage, while still inhibited by chelating agents and sulphydryl-blocking agents, is resistant to inhibition by KI (Sleigh and Grigg, unpublished). This suggests that a second breakage mechanism operates at high phleomycin concentrations, as suggested by Grigg [4]. References 1 Apontoweil, P. and W. Berends, Isolation and initial characterization of glutathione-deficient mutants of Escherichia coli K12, Biochim. Biophvs, Acta, 399 (1975) 10-22. 2 Falaschi , A. and A. Kornberg, Phleomycin, an inhibitor of DNA polymerase, Fed. Pro c., 23 (1964) 940--945. 3 Fuchs, J.A. and H.R. Warner, Isolation of Escherichia coli mutant deficient in glutathione sv nth ..sis, J. Bacterio l.; 124 (1975) 140-148. 4 Grigg, G.W., Induction of.DNA breakdown and death in Escherichia coli by phleomycin. Its association with dark repair processes, Mol. Gen. Gene t., 104 (1969) 1-11.
189 5 Iwata, A. and R.A. Consiglt, Effect of phleomycin on polyoma virus synthesis in mouse embryo cells, J. Virol., 7 (1971) 29-40. 6 Jacobs, N .F., R.L. Neu and L.I. Gardner, Phleomycin-induced mitotic inhibition and chromosomal abnormalities in cultured human leucocytes, Mutation Res., 7 (1969) 251-253. 7 Jocelyn, P .C., Biochemistry of the SH group. Academic Press, London, 1972. 8 Kanazawa, K., G. Danno and M. Natake, Stabilisation of linoleic acid at the process of the autoxidation by potassium iodide, Agr, BioI. Chern., 39 (1975) 1177-1186. 9 Lieve , L., A non-specific increase in permeability in Escherichia coli produced by EDT A, Proc. Nat!. Acad. Sci. (U.S.A.), 53 (1965) 747-750. 10 McGrath, R.A. and R.W. Willams, Reconstruction in vivo of irradiated Escherichia coli deoxyribonucleic acid; the rejoining of broken pieces, Nature, 212 (1966) 534-535. 11 Mivaki, Moo T. Ono, S. Hori and H. Urnezaw a, Binding of bleomycin to DNA in bleomycin-sensitive and -resistant rat ascites hepatoma cells, Cancer Res., 35 (1975) 2015-2019. 12 Nakayama, H., Phleornycin induced lethality and DNA degradation in Escherichia coli K12, Mutation Res .. 29 (1975) 21-33. 13 Okada, F.J., K. Yanagisaw a and F.J. Ryan, Elective production of thymine-less mutants, Nature, 188 (1960) 340-341. 14 Pietsch, P. and H. Garrett, Phleornv cin: evidence of in vivo binding to DNA, Cv tobios, lA (1969) 7-15. 15 Reiter, H., M. Milewskiy and P. Kelley, Mode of action of phleornycin on Bacillus sub til is, J. Bacteriol., 111 (1972) 586-592. 16 Sleigh, M.J., The mechanism of DNA breakage by phleomvcin in vitro, Nucl, Acids. Res., 3 (1976) 891-901. 17 Sleigh, M.J. and G.W. Grigg, Induction of local denaturation in DNA in vitro by phleomycin and caffeine, FEBS Let t., 39 (1974) 35-38. 18 Sleigh, M.J. and G.W. Grigg, The mechanism of sensitivity to phleomycin in growing Escherichia coli cells, Biochem , J., 155 (1976) 87-99. 19 Stern, Roo J.A. Rose and R.M. Friedman, Phle omvcin-induced cleavage of deoxyribonucleic acid, Biochemistry, 13 (1974) 307-312. 20 Strniste, G.F. and S.S, Wallace, Endonucleolytic incision of X-irradiated deoxycibonucleic acid by extracts of Escherichia coli, Proc. Natl. Acad. Sci. (U.S.A.), 72 (1975) 1997-2001. 21 Woodcock, E.A. and G.W. Grigg, Repair of thermally induced DNA breakage in Escherichia coli, Nature, 237 (1972) 76-79.
