Molec. gen. Genet. 145, 287--291 (1976) © by Springer-Verlag 1976
Division Delay and DNA Degradation after Mutagen Treatment of the Yeast, Saccharomyces cerevisiae P.J. Wilmore and James M. Parry Department of Genetics, UniversityCollegeof Swansea, SingletonPark, SwanseaSA2 8PP, U.K.
Summary. The treatment of the yeast mutant T M P I - 1 , which is capable of incorporating low levels of 3Hthymidine-5'- monophosphate with UV light and ethyl methane sulphonate resulted in division delay when cultures were reinnoculated into fresh medium. The initiation of cell division was accompanied by the degradation of up to 20% of the nuclear DNA fraction. The period of DNA degradation correlates closely with the time at which yeast cultures undergo mitotic recombination and appears to represent the degradation of DNA during a post-replication repair process.
Introduction Yeast cells exposed to radiation and chemical mutagens show dose dependent delays in cell division (see Mitchison, 1971). After UV irradiation of yeast cells, this division delay appears to depend upon UV induced damage to a limited number of essential genes (Johnson et al., 1973). As well as inducing division delay the radiation exposure of several organisms has been shown to result in the partial breakdown of DNA after subsequent incubation in nutrient medium. The initial observation by Stuy (1960) demonstrated that a number of bacterial species degrade their DNA after X-irradiation. In Escherichia coli, wild type cultures show between 2 to 20% degradation after X-irradiation (Pollard and Achey, 1964), whereas the excision deficient mutants uvr A , B and C show little breakdown following treatment with UV light and mitomycin C (Howard-Flanders and Boyce, 1966). Rec A mutants of Escherichia coli degrade more of their DNA after both UV light and X-irradiation than do rec + cells (up to 90% degradation), however rec B and rec C mutants showed considerably less degradation
than rec + cells (Smith, 1971). DNA degradation has been extensively studied in the radiation resistant bacterium, Micrococcus radiodurans. After an X-ray dose of 220 Krads, resulting in 100% survival there was considerable DNA degradation, but only if cellular growth occurred during post-irradiation incubation (Dean et al., 1966). In mammalian cells, DNA degradation is generally reported to be small or negligible (Painter, 1968; Looney and Chang, 1969; Mattern et al., 1973). However, as was pointed out by Hatzfield (1973) in those experiments the post-incubation times were generally only a fraction of the cell division cycle, so that all the degradative processes recurring during the replicatire cycle have not been tested. Studies involving the measurement of DNA degradation after radiation treatment in fungi have been restricted in the past by the absence of specific DNA labelling techniques. However, Holliday and Resnick (1970), using an indirect biological assay demonstrated radiation-induced DNA degradation in the smut fungus Ustilago maydis. Wild type strains of Ustilago maydis were shown to degrade up to 20% of their DNA 3 to 4h after irradiation, whereas no breakdown was detectable in the recombination deficient strain UVS1. Holliday and Resnick (1970) have suggested that the degradation was part of the recombination process and that recombination repair was induced by the products of irradiation. Hatzfield (1973) has investigated DNA breakdown in yeast using a non-specific radioactive uracil label. After removal of RNA he measured radiation induced changes in the insoluble DNA fraction. In spite of the considerable technical problems involved in using a non-specific DNA label, Hatzfield (1973) was able to demonstrate the degradation of up to 15% of the DNA of irradiated yeast cells when incubated in nutrient medium. The availability of mutants of yeast, auxotrophic
288
P.J. Wilmore and J.M. Parry: Division Delay after Mutagen Treatment of S. cerevisiae
for thymidine monophosphate allow for the specific labelling of yeast DNA (Brendel and Haynes, 1974; Fath and Brendel, 1974a and b). We have utilized such a mutant T M P 1-I, to investigate the effects of UV irradiation and exposure to ethyl methane sulphonate upon cell division and DNA degradation in yeast cultures.
filters, washed five times with HzO and once with 96% ethanol, b) added to an excess o f 10% Trichloroacetic acid (TCA) : Bovine serum albumin (BSA) 20 m g / m l at 0 ° C for at least 30 minutes, then passed through G F / C filters, washed four times with ice cold 5% T C A and twice with 96% ethanol. Filters were dried overnight in a h o t oven and placed in vials containing 5 ml. scintillation fluid (2 (4'-t-Butylphenyl)-5-(4"-biphenylyl)-l, 3, 4-oxadiazole) 5 g m / L and vials counted in a Beckman LS-230 scintillation counter. The results presented are the m e a n s of three separate experiments.
Materials and Methods Results a) Strain Culture TMPI-1 of Saceharomyees eerevisiae: haploid, auxotrophic for isoleucine, valine and thymidine monophosphate. This strain has been described by Fath and Brendel (1974) and was kindly supplied by Dr. Martin Brendel. The culture was petite and lacked the characteristic mitochondrial D N A c o m p o n e n t on a cesium chloride gradient (Wilmore unpublished observations).
b) Media The complete m e d i u m (YC) was a yeast extract, peptone m e d i u m with 4% (w/v) glucose, pH 6.7 and solidified with Lab-M agar No. 1. M e d i u m N contains per ml: 6.7 m g Difco Yeast nitrogen base w/o a m i n o acids, 2 m g Difco casamino acids w/o vitamins, 20 m g D (+)-glucose, 100 gg isoleucine and 100 gg valine.
c) Labelling of Cells Cells were added to 10 ml m e d i u m N plus 0.1 ml ((methyl)-3H) thymidiiae-5'-monophosphate (Amersham, specific activity approx 1 Ci/mmole) and " c o l d " T M P (Sigma) to 20 gg/ml approx, a n d grown overnight in a Griffin orbital incubator at 28 ° C. Uptakes of a b o u t 1.5% were obtained and with suitable chase experiments, incorporation of label into D N A of about 90% was obtained.
d) Mutagen Treatment UV treatment. 10 ml portions of the culture suspended in saline were irradiated in agitated open petri dishes. The source of U V light was a Hanovia 11 a low pressure mercury discharge tube generating almost m o n o c h r o m a t i c radiation at 254 nM. Dose rate was determined by the use of a calibrated photocell. All manipulations were performed in red light to avoid uncontrolled photoreactiration. F o r survival estimates suitable dilutions were spread on agar plates and incubated at 28 ° C. For estimates of D N A degradation, labelled cells were transferred to growth m e d i u m a n d incubated in a covered flask in the orbital incubator at 28 ° C. EMS treatment. Cultures suspended in p H 7.0 buffer were exposed to EMS solution at 2 8 ° C in the orbital incubator set at high speed. Doses and periods of exposure are given in parentheses. Reactions were stopped by diluting samples 1 : 10 in 10% sodium thiosulphate. Cells were spun down and washed 5 x by centrifugation before dilutions were made for survival plating, or labelled cells were resuspended in nutrient medium.
e) Estimation of DNA Degradation At various intervals, samples were withdrawn from the growth m e d i u m and equal volumes a) passed through W h a t m a n G F / C
The exposure of stationary phase cells of the yeast strain T M P I - 1 to UV light and the chemical mutagen EMS results in cell death as shown in Figures 1 and 2. Both survival curves are characterised by the presence of a resistant shoulder followed by an exponential decline in cell viability. In order to determine the effects of the two mutagens upon division delay and DNA degradation exposures were selected which gave only low levels of cell death i.e. 44 JIM z UV giving 70% survival, 110 J/ M 2 UV giving 45% survival, 15 min. exposure to 1% EMS giving 60% survival and 15 min. exposure to 2% EMS giving 50% survival. Under these conditions the majority of the treated cells undergo 1 to 2 cell divisions when innoculated into nutrient medium. The effects of UV exposure upon cell division and the % of radioactive material present in the insoluble DNA fraction are shown in Figure 3. Control cultures of T M P I - 1 innoculated into fresh medium start to divide approximately 3a/z h after transfer and reach a maximum cell density of 7 to 8 x 10 7 cells/ml after 18 h growth. During this period of growth the percentage of 3H labelled material found in the DNA remains constant in each cell sample. UV exposure at a fluence of 440 JIM 2 results in the complete inhibition of cell division as shown in Figure 3. During this period of growth inhibition no change could be detected in the percentage of 3H labelled material in the insoluble DNA fraction. At a dose of 600 JIM 2 Fergeson and Cox (1974) were able to demonstrate the excision of UV induced pyrimidine dimers in a culture of the yeast mutant TMP-1. However, the small changes in DNA structure produced by excision are not detectable by the techniques utilized in our experiments. UV exposure of cultures of T M P I - 1 at 44 and 110 J/M 2 produce delays in cell division of 2 h and 8 h respectively as shown in Figure 3. However in the cultures showing only a temporary delay in cell division the period of the first doubling in cell numbers was accompanied by a reduction in the percentage of 3H labelled material found in the insoluble
P.J. Wilmore and J.M. Parry: Division Delay after Mutagen Treatment of S. cerevisiae
D N A fraction. The degradation of D N A detected in our experiments varied from 15% to 20% and was dependent upon the initiation of cell division. In all cases the period of D N A degradation extended from the initiation of cell division and was completed by the end of the second doubling in cell numbers. Figure 4 demonstrates the effects of the exposure of cultures of T M P I - 1 to 1 and 2% EMS for a period
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Fig. 3. Cell growth and D N A degradation of stationary phase cultures of TMPI-1 incubated at 28 ° C in nutrient medium following exposure to UV light. • • control; n - - i 44 j/M2; o - - 0 110 j/M2; [] [] 440 J/M 2
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Fig.2. The effects of ethyl methane sulphonate upon the cell viability of stationary phase cells of strain TMPI-1. o - - o 1% EMS: • - - o 2% EMS
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Fig. 4. Cell growth and D N A degradation of stationary phase cultures of TMPI-1 incubated at 28 ° C in nutrient medium following exposure to ethyl methane sulphonate, o - - o 1% EMS treatment for 15 rain; o - - o 2% EMS treatment for 15 min
290
P.J. Wilmore and J.M. Parry: Division Delay after Mutagen Treatment of S. cerevisiae
of 15 min EMS treatment resulted in delays in the initiation of cell division of 3 h and 7 h for 1% and 2% EMS respectively. The percentage of 3H material found in the insoluble DNA fraction shows a similar pattern of response as after UV light. Both EMS treatments produced a reduction in the insoluble fraction of 17 to 20% during the period of the first and second doubling of cell numbers. The results thus demonstrated that for both UV light and EMS treatments producing delays in cell division the reinitiation of cell growth was accompanied by the degradation of up to 20% of the insoluble nuclear DNA fraction.
Discussion
The exposure of stationary phase cultures of yeast to the mutagens UV light and ethyl methane sulphonate resulted in a dose dependent division delay before growth commenced in nutrient media. During the subsequent division up to 20% of the DNA was degraded with a loss of radioactive material from the insoluble fraction. This DNA degradation was dependent upon cell division and was completed during the first and second cell doublings after the mutagen treatments (see also Hatzfield, 1973 and Evans, 1974). Because the yeast strain TMPI-1 utilized in this study was sensitive to UV light compared with the wild type RAD strain the experiments described were performed at relatively low UV exposures. However, Evans (1974) has demonstrated a similar pattern of DNA degradation at UV exposures of up to 660 J/M 2 in the more UV resistant strain, tup-2. Hatzfield (1973) also demonstrated that UV induced DNA degradation became saturated at high doses of UV exposure until UV treatment resulted in the complete cessation of growth with no detectable degradation. In Micrococcus radiodurans, X-ray exposure resulting in 100% cell survival produced up to 50% DNA degradation (Dean, Feldschreiber and Lett, 1966) and it seems unlikely that DNA degradation depends upon the disintegration of a few of the cells in the irradiated population. In our own experiments and in the work of Evans (1974) and Hatzfield (1973) the majority of the mutagen treated cells undergo at least one cell division, indicating that the DNA degradation observed was a property of the whole cell population and not due to the loss of large quantities of insoluble material from a few cells. The absence of a mitochondrial DNA fraction in the cells of the TMPI-1 mutant used indicates that changes in the insoluble DNA fraction detected here result from the loss of nuclear DNA material alone.
After UV treatment, yeast cells incubated in nutrient medium excise a percentage of the UV induced pyrimidine dimers from their DNA (Fergeson and Cox, 1974). These authors have demonstrated that the excision process was completed within 2 to 4 h after irradiation in the yeast strain TMP-1, at UV exposures which prevent cell division. Clearly the degradation process observed in our experiments took place later in the cell cycle than excision repair. Yeast cells exposed to a wide range of mutagens undergo intragenic recombination by the process of gene conversion. The detection of recombinants has been shown to be dependent upon growth for 1 to 2 divisions before prototrophs may be detected on selective media (Davies, Evans and Parry, 1975). The correlation between the timing of DNA degradation in the cell cycle and the conditions necessary for the detection of recombination suggests that DNA degradation represents the biochemical expression of a post-replication repair process presumably involving recombination. Acknowledgements. The work was supported by Euratom Grant No. B10 E 1199-1.
References Brendel, M., Haynes, R.H.: Kinetics and the genetic control of the incorporation of thymidine monophosphate in yeast DNA. Molec. gen. Genet. 117, 3 9 4 4 (1974) Dean, C.J., Feldschreiber, P., Lett, J.T.: Repair of X-ray damage to DNA in Micrococcus radiodurans. Nature (Lond.) 209, 49-52 (1966) Davies, P.J., Evans, W.E., Parry, J.M.: Mitotic recombination induced by chemical and physical agents in the yeast Saccharomyees cerevisiae. Mutation Res. 29, 301 314 (1975) Evans, W.E.: Ph.D. thesis 1974: Studies upon the inactivation and repair of cellular damage induced by chemical and physical agents. University of Wales Fath, W.W., Brendel, M. : Specific DNA labelling by exogenous thymidine-5'-monophosphate in Saecharomyces eerevisiae. Molec. gen. Genet. 131, 57-67 (1974) Fath, W.W., Brendel, M. : Economizing DNA-specific labelling by deoxythymidine-5'-monophosphate in Saceharomyces eerevisiae. Molec. gen. Genet. 132, 335-345 (1974) Ferguson, L.R., Cox, B.S. : Excision of bases accompanying the excision of dimers from DNA of UV-irradiated yeast. Molec. gen. Genet. 135, 87-90 (1974) Hatzfield, J. : Correlation between degradation, replication and repair of yeast DNA irradiated by UV or X-rays. Bioehim. biophys. Acta (Amst.) 299, 43 53 (1973) Holliday, R., Resnick, M.A. : Allelic recombination and DNA degradation in Ustilago. Heredity 25, 494 (abstract) (1970) Hofii, Z.I., Suzuki, K. : Degradation of the DNA of Escherichia coli K-12 Rec (JC 1569b) after irradiation with ultraviolet light. Photochem. Photobiol. 8, 93 105 (1968) Horii, Z.I., Suzuki, K. : Degradation of the DNA of recA mutants ofEseherichia coli K-12 after irradiation with ultraviolet l i g h t 11. Further studies including a recA uvrA double mutant. Photochem. Photobiol. 11, 99 107 (1970)
P.J. Wilmore and J.M. Parry: Division Delay after Mutagen Treatment of S. cerevisiae Howard-Flanders, P., Boyce, R.P.: DNA repair and genetic recombination studies on mutants of Escherichia coli defective in these processes. Radiat. Res., Suppl. 6, 156 184 (1966) Johnson, R.G., Walmsley, J.G., Goodwin, L.J., Morrison, H.G.: Ultraviolet-induced delay of budding in synchronized yeast cells. Radiat. Res. 53, 134 143 (1973) Looney, N.B., Chang, L.D. : The effect of X-irradiation on thymidine labelled DNA of regenerating rat liver. Radiat. Res. 37, 525-530 (1969) Mattern, M.R., Hariharan, P.V., Dunlop, B.E., Cerruti, P.A.: DNA degradation and excision repair in Chinese hamster ovary cells. Nature (Lond.) New Biol. 245, 230-233 (1972) Mitchison, T.M. : Biology of the cell cycle. Cambridge Univ. Press pp. 201-250 (1971) Painter, R.B. : DNA degradation in Hela cells after UV or Xirradiation. Radiat. Res. 35, 513 (abstract) (1968)
291
Pollard, E.C., Achey, P.M. : Radiation action on DNA in bacteria: Effect of oxygen Science 146, 71-73 (1964) Smith, K.C. : The roles of genetic recombination and DNA polymerase in the repair of damaged DNA. Photophysiology 4, 209 278 (1971) Stuy, T.H. : Studies on the radiation inactivation of microorganisms. VI. X-ray induced breakdown of DNA in Haemophilus influenzae and other bacteria. J. Baeteriol. 79, 707-715 (1960)
Communicated by H. B6hme
Received February 10, 1976
Division Delay and DNA Degradation after Mutagen Treatment of the Yeast, Saccharomyces cerevisiae P.J. Wilmore and James M. Parry Department of Genetics, UniversityCollegeof Swansea, SingletonPark, SwanseaSA2 8PP, U.K.
Summary. The treatment of the yeast mutant T M P I - 1 , which is capable of incorporating low levels of 3Hthymidine-5'- monophosphate with UV light and ethyl methane sulphonate resulted in division delay when cultures were reinnoculated into fresh medium. The initiation of cell division was accompanied by the degradation of up to 20% of the nuclear DNA fraction. The period of DNA degradation correlates closely with the time at which yeast cultures undergo mitotic recombination and appears to represent the degradation of DNA during a post-replication repair process.
Introduction Yeast cells exposed to radiation and chemical mutagens show dose dependent delays in cell division (see Mitchison, 1971). After UV irradiation of yeast cells, this division delay appears to depend upon UV induced damage to a limited number of essential genes (Johnson et al., 1973). As well as inducing division delay the radiation exposure of several organisms has been shown to result in the partial breakdown of DNA after subsequent incubation in nutrient medium. The initial observation by Stuy (1960) demonstrated that a number of bacterial species degrade their DNA after X-irradiation. In Escherichia coli, wild type cultures show between 2 to 20% degradation after X-irradiation (Pollard and Achey, 1964), whereas the excision deficient mutants uvr A , B and C show little breakdown following treatment with UV light and mitomycin C (Howard-Flanders and Boyce, 1966). Rec A mutants of Escherichia coli degrade more of their DNA after both UV light and X-irradiation than do rec + cells (up to 90% degradation), however rec B and rec C mutants showed considerably less degradation
than rec + cells (Smith, 1971). DNA degradation has been extensively studied in the radiation resistant bacterium, Micrococcus radiodurans. After an X-ray dose of 220 Krads, resulting in 100% survival there was considerable DNA degradation, but only if cellular growth occurred during post-irradiation incubation (Dean et al., 1966). In mammalian cells, DNA degradation is generally reported to be small or negligible (Painter, 1968; Looney and Chang, 1969; Mattern et al., 1973). However, as was pointed out by Hatzfield (1973) in those experiments the post-incubation times were generally only a fraction of the cell division cycle, so that all the degradative processes recurring during the replicatire cycle have not been tested. Studies involving the measurement of DNA degradation after radiation treatment in fungi have been restricted in the past by the absence of specific DNA labelling techniques. However, Holliday and Resnick (1970), using an indirect biological assay demonstrated radiation-induced DNA degradation in the smut fungus Ustilago maydis. Wild type strains of Ustilago maydis were shown to degrade up to 20% of their DNA 3 to 4h after irradiation, whereas no breakdown was detectable in the recombination deficient strain UVS1. Holliday and Resnick (1970) have suggested that the degradation was part of the recombination process and that recombination repair was induced by the products of irradiation. Hatzfield (1973) has investigated DNA breakdown in yeast using a non-specific radioactive uracil label. After removal of RNA he measured radiation induced changes in the insoluble DNA fraction. In spite of the considerable technical problems involved in using a non-specific DNA label, Hatzfield (1973) was able to demonstrate the degradation of up to 15% of the DNA of irradiated yeast cells when incubated in nutrient medium. The availability of mutants of yeast, auxotrophic
288
P.J. Wilmore and J.M. Parry: Division Delay after Mutagen Treatment of S. cerevisiae
for thymidine monophosphate allow for the specific labelling of yeast DNA (Brendel and Haynes, 1974; Fath and Brendel, 1974a and b). We have utilized such a mutant T M P 1-I, to investigate the effects of UV irradiation and exposure to ethyl methane sulphonate upon cell division and DNA degradation in yeast cultures.
filters, washed five times with HzO and once with 96% ethanol, b) added to an excess o f 10% Trichloroacetic acid (TCA) : Bovine serum albumin (BSA) 20 m g / m l at 0 ° C for at least 30 minutes, then passed through G F / C filters, washed four times with ice cold 5% T C A and twice with 96% ethanol. Filters were dried overnight in a h o t oven and placed in vials containing 5 ml. scintillation fluid (2 (4'-t-Butylphenyl)-5-(4"-biphenylyl)-l, 3, 4-oxadiazole) 5 g m / L and vials counted in a Beckman LS-230 scintillation counter. The results presented are the m e a n s of three separate experiments.
Materials and Methods Results a) Strain Culture TMPI-1 of Saceharomyees eerevisiae: haploid, auxotrophic for isoleucine, valine and thymidine monophosphate. This strain has been described by Fath and Brendel (1974) and was kindly supplied by Dr. Martin Brendel. The culture was petite and lacked the characteristic mitochondrial D N A c o m p o n e n t on a cesium chloride gradient (Wilmore unpublished observations).
b) Media The complete m e d i u m (YC) was a yeast extract, peptone m e d i u m with 4% (w/v) glucose, pH 6.7 and solidified with Lab-M agar No. 1. M e d i u m N contains per ml: 6.7 m g Difco Yeast nitrogen base w/o a m i n o acids, 2 m g Difco casamino acids w/o vitamins, 20 m g D (+)-glucose, 100 gg isoleucine and 100 gg valine.
c) Labelling of Cells Cells were added to 10 ml m e d i u m N plus 0.1 ml ((methyl)-3H) thymidiiae-5'-monophosphate (Amersham, specific activity approx 1 Ci/mmole) and " c o l d " T M P (Sigma) to 20 gg/ml approx, a n d grown overnight in a Griffin orbital incubator at 28 ° C. Uptakes of a b o u t 1.5% were obtained and with suitable chase experiments, incorporation of label into D N A of about 90% was obtained.
d) Mutagen Treatment UV treatment. 10 ml portions of the culture suspended in saline were irradiated in agitated open petri dishes. The source of U V light was a Hanovia 11 a low pressure mercury discharge tube generating almost m o n o c h r o m a t i c radiation at 254 nM. Dose rate was determined by the use of a calibrated photocell. All manipulations were performed in red light to avoid uncontrolled photoreactiration. F o r survival estimates suitable dilutions were spread on agar plates and incubated at 28 ° C. For estimates of D N A degradation, labelled cells were transferred to growth m e d i u m a n d incubated in a covered flask in the orbital incubator at 28 ° C. EMS treatment. Cultures suspended in p H 7.0 buffer were exposed to EMS solution at 2 8 ° C in the orbital incubator set at high speed. Doses and periods of exposure are given in parentheses. Reactions were stopped by diluting samples 1 : 10 in 10% sodium thiosulphate. Cells were spun down and washed 5 x by centrifugation before dilutions were made for survival plating, or labelled cells were resuspended in nutrient medium.
e) Estimation of DNA Degradation At various intervals, samples were withdrawn from the growth m e d i u m and equal volumes a) passed through W h a t m a n G F / C
The exposure of stationary phase cells of the yeast strain T M P I - 1 to UV light and the chemical mutagen EMS results in cell death as shown in Figures 1 and 2. Both survival curves are characterised by the presence of a resistant shoulder followed by an exponential decline in cell viability. In order to determine the effects of the two mutagens upon division delay and DNA degradation exposures were selected which gave only low levels of cell death i.e. 44 JIM z UV giving 70% survival, 110 J/ M 2 UV giving 45% survival, 15 min. exposure to 1% EMS giving 60% survival and 15 min. exposure to 2% EMS giving 50% survival. Under these conditions the majority of the treated cells undergo 1 to 2 cell divisions when innoculated into nutrient medium. The effects of UV exposure upon cell division and the % of radioactive material present in the insoluble DNA fraction are shown in Figure 3. Control cultures of T M P I - 1 innoculated into fresh medium start to divide approximately 3a/z h after transfer and reach a maximum cell density of 7 to 8 x 10 7 cells/ml after 18 h growth. During this period of growth the percentage of 3H labelled material found in the DNA remains constant in each cell sample. UV exposure at a fluence of 440 JIM 2 results in the complete inhibition of cell division as shown in Figure 3. During this period of growth inhibition no change could be detected in the percentage of 3H labelled material in the insoluble DNA fraction. At a dose of 600 JIM 2 Fergeson and Cox (1974) were able to demonstrate the excision of UV induced pyrimidine dimers in a culture of the yeast mutant TMP-1. However, the small changes in DNA structure produced by excision are not detectable by the techniques utilized in our experiments. UV exposure of cultures of T M P I - 1 at 44 and 110 J/M 2 produce delays in cell division of 2 h and 8 h respectively as shown in Figure 3. However in the cultures showing only a temporary delay in cell division the period of the first doubling in cell numbers was accompanied by a reduction in the percentage of 3H labelled material found in the insoluble
P.J. Wilmore and J.M. Parry: Division Delay after Mutagen Treatment of S. cerevisiae
D N A fraction. The degradation of D N A detected in our experiments varied from 15% to 20% and was dependent upon the initiation of cell division. In all cases the period of D N A degradation extended from the initiation of cell division and was completed by the end of the second doubling in cell numbers. Figure 4 demonstrates the effects of the exposure of cultures of T M P I - 1 to 1 and 2% EMS for a period
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Fig. 3. Cell growth and D N A degradation of stationary phase cultures of TMPI-1 incubated at 28 ° C in nutrient medium following exposure to UV light. • • control; n - - i 44 j/M2; o - - 0 110 j/M2; [] [] 440 J/M 2
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Fig. 4. Cell growth and D N A degradation of stationary phase cultures of TMPI-1 incubated at 28 ° C in nutrient medium following exposure to ethyl methane sulphonate, o - - o 1% EMS treatment for 15 rain; o - - o 2% EMS treatment for 15 min
290
P.J. Wilmore and J.M. Parry: Division Delay after Mutagen Treatment of S. cerevisiae
of 15 min EMS treatment resulted in delays in the initiation of cell division of 3 h and 7 h for 1% and 2% EMS respectively. The percentage of 3H material found in the insoluble DNA fraction shows a similar pattern of response as after UV light. Both EMS treatments produced a reduction in the insoluble fraction of 17 to 20% during the period of the first and second doubling of cell numbers. The results thus demonstrated that for both UV light and EMS treatments producing delays in cell division the reinitiation of cell growth was accompanied by the degradation of up to 20% of the insoluble nuclear DNA fraction.
Discussion
The exposure of stationary phase cultures of yeast to the mutagens UV light and ethyl methane sulphonate resulted in a dose dependent division delay before growth commenced in nutrient media. During the subsequent division up to 20% of the DNA was degraded with a loss of radioactive material from the insoluble fraction. This DNA degradation was dependent upon cell division and was completed during the first and second cell doublings after the mutagen treatments (see also Hatzfield, 1973 and Evans, 1974). Because the yeast strain TMPI-1 utilized in this study was sensitive to UV light compared with the wild type RAD strain the experiments described were performed at relatively low UV exposures. However, Evans (1974) has demonstrated a similar pattern of DNA degradation at UV exposures of up to 660 J/M 2 in the more UV resistant strain, tup-2. Hatzfield (1973) also demonstrated that UV induced DNA degradation became saturated at high doses of UV exposure until UV treatment resulted in the complete cessation of growth with no detectable degradation. In Micrococcus radiodurans, X-ray exposure resulting in 100% cell survival produced up to 50% DNA degradation (Dean, Feldschreiber and Lett, 1966) and it seems unlikely that DNA degradation depends upon the disintegration of a few of the cells in the irradiated population. In our own experiments and in the work of Evans (1974) and Hatzfield (1973) the majority of the mutagen treated cells undergo at least one cell division, indicating that the DNA degradation observed was a property of the whole cell population and not due to the loss of large quantities of insoluble material from a few cells. The absence of a mitochondrial DNA fraction in the cells of the TMPI-1 mutant used indicates that changes in the insoluble DNA fraction detected here result from the loss of nuclear DNA material alone.
After UV treatment, yeast cells incubated in nutrient medium excise a percentage of the UV induced pyrimidine dimers from their DNA (Fergeson and Cox, 1974). These authors have demonstrated that the excision process was completed within 2 to 4 h after irradiation in the yeast strain TMP-1, at UV exposures which prevent cell division. Clearly the degradation process observed in our experiments took place later in the cell cycle than excision repair. Yeast cells exposed to a wide range of mutagens undergo intragenic recombination by the process of gene conversion. The detection of recombinants has been shown to be dependent upon growth for 1 to 2 divisions before prototrophs may be detected on selective media (Davies, Evans and Parry, 1975). The correlation between the timing of DNA degradation in the cell cycle and the conditions necessary for the detection of recombination suggests that DNA degradation represents the biochemical expression of a post-replication repair process presumably involving recombination. Acknowledgements. The work was supported by Euratom Grant No. B10 E 1199-1.
References Brendel, M., Haynes, R.H.: Kinetics and the genetic control of the incorporation of thymidine monophosphate in yeast DNA. Molec. gen. Genet. 117, 3 9 4 4 (1974) Dean, C.J., Feldschreiber, P., Lett, J.T.: Repair of X-ray damage to DNA in Micrococcus radiodurans. Nature (Lond.) 209, 49-52 (1966) Davies, P.J., Evans, W.E., Parry, J.M.: Mitotic recombination induced by chemical and physical agents in the yeast Saccharomyees cerevisiae. Mutation Res. 29, 301 314 (1975) Evans, W.E.: Ph.D. thesis 1974: Studies upon the inactivation and repair of cellular damage induced by chemical and physical agents. University of Wales Fath, W.W., Brendel, M. : Specific DNA labelling by exogenous thymidine-5'-monophosphate in Saecharomyces eerevisiae. Molec. gen. Genet. 131, 57-67 (1974) Fath, W.W., Brendel, M. : Economizing DNA-specific labelling by deoxythymidine-5'-monophosphate in Saceharomyces eerevisiae. Molec. gen. Genet. 132, 335-345 (1974) Ferguson, L.R., Cox, B.S. : Excision of bases accompanying the excision of dimers from DNA of UV-irradiated yeast. Molec. gen. Genet. 135, 87-90 (1974) Hatzfield, J. : Correlation between degradation, replication and repair of yeast DNA irradiated by UV or X-rays. Bioehim. biophys. Acta (Amst.) 299, 43 53 (1973) Holliday, R., Resnick, M.A. : Allelic recombination and DNA degradation in Ustilago. Heredity 25, 494 (abstract) (1970) Hofii, Z.I., Suzuki, K. : Degradation of the DNA of Escherichia coli K-12 Rec (JC 1569b) after irradiation with ultraviolet light. Photochem. Photobiol. 8, 93 105 (1968) Horii, Z.I., Suzuki, K. : Degradation of the DNA of recA mutants ofEseherichia coli K-12 after irradiation with ultraviolet l i g h t 11. Further studies including a recA uvrA double mutant. Photochem. Photobiol. 11, 99 107 (1970)
P.J. Wilmore and J.M. Parry: Division Delay after Mutagen Treatment of S. cerevisiae Howard-Flanders, P., Boyce, R.P.: DNA repair and genetic recombination studies on mutants of Escherichia coli defective in these processes. Radiat. Res., Suppl. 6, 156 184 (1966) Johnson, R.G., Walmsley, J.G., Goodwin, L.J., Morrison, H.G.: Ultraviolet-induced delay of budding in synchronized yeast cells. Radiat. Res. 53, 134 143 (1973) Looney, N.B., Chang, L.D. : The effect of X-irradiation on thymidine labelled DNA of regenerating rat liver. Radiat. Res. 37, 525-530 (1969) Mattern, M.R., Hariharan, P.V., Dunlop, B.E., Cerruti, P.A.: DNA degradation and excision repair in Chinese hamster ovary cells. Nature (Lond.) New Biol. 245, 230-233 (1972) Mitchison, T.M. : Biology of the cell cycle. Cambridge Univ. Press pp. 201-250 (1971) Painter, R.B. : DNA degradation in Hela cells after UV or Xirradiation. Radiat. Res. 35, 513 (abstract) (1968)
291
Pollard, E.C., Achey, P.M. : Radiation action on DNA in bacteria: Effect of oxygen Science 146, 71-73 (1964) Smith, K.C. : The roles of genetic recombination and DNA polymerase in the repair of damaged DNA. Photophysiology 4, 209 278 (1971) Stuy, T.H. : Studies on the radiation inactivation of microorganisms. VI. X-ray induced breakdown of DNA in Haemophilus influenzae and other bacteria. J. Baeteriol. 79, 707-715 (1960)
Communicated by H. B6hme
Received February 10, 1976
