INT . J . RADIAT . BIOL .,
1976,
VOL .
30,
NO .
1, 1 3 -24
The genetic control of liquid-holding recovery and U.V .-induced repair resistance in the yeast, Saccharomyces cerevisiae ELIZABETH M . PARRY and JAMES M . PARRY Department of Genetics, University College of Swansea, Singleton Park, Swansea SA2 8PP, West Glamorgan, U .K .
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(Received 5 May 1976 ; accepted 24 June 1976)
Liquid-holding treatment in non-nutrient solutions after U .V .-exposure results in an increased resistance of wild-type (RAD) yeast cells to a second U .V .treatment (repair resistance) . U .V .-sensitive (rad) mutants of yeast which show variation in their response to liquid-holding treatment and a second U .V .-dose-range have been classified into two groups . Mutants of Group 1 show increased viability after post-U .V .liquid-holding treatment and show repair resistance . In contrast, mutants of Group 2 which show reduced viability during post-U .V .-liquid-holding treatment have the same U .V.-sensitivity, both before and after liquid-holding treatment . Genetic analysis of crosses of the rad mutants to wild-type cultures indicate that the phenotype of repair resistance to UN .-treatment is under genetic control and depends on the presence of alleles of radiation sensitivity genes, which also confer the property of liquid-holding recovery .
1 . Introduction Wild-type (RAD) cultures of the yeast, Saccharomyces cerevisiae show partial recovery from the lethal effects of U .V .-light when exposed to post-irradiation treatments such as photoreactivating light and liquid holding in the dark in non-nutrient solutions before plating on nutrient agar (Kelner 1949, Patrick, Haynes and Uretz 1964) . The U .V .-sensitive mutants of yeast (Cox and Parry 1968) show considerable variation in their response to post-U .V .-liquid-holding treatment (Parry and Parry 1969, Parry, Parry and Waters 1972) . The various strains may be classified into four groups on the basis of positive or negative responses to liquid holding and photoreactivating light after liquid-holding treatment . An additional property of liquid-holding treatment in yeast was demonstrated by Patrick and Haynes (1968)-that yeast cultures exposed to liquid-holding conditions after U .V .-irradiation show increased resistance to a second range of UN .-light exposures . After a further study of this property of ' repair resistance ' (Parry and Parry 1972), we have suggested that the repair of U .V .-damage in yeast involves the action of at least two repair processes and that increased resistance to the second U .V .-exposure results from an increase in the proportion of U .V .induced lesions repaired by the excision system as opposed to the post-replication repair system . In this paper we report the effects of liquid-holding treatment after U .V .exposure and a second U .V .-treatment after the liquid-holding treatment in a wide range of UN .-sensitive mutants of yeast . Our results show that ` repair
14
E . M . Parry and J. M. Parry
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resistance ' was demonstrable only in cultures which showed a positive increase in cell viability after post-U .V .-liquid-holding treatment . Genetic analysis indicated that in mutants which do not show ' repair resistance ', this lack of resistance to a second UN .-exposure segregated with the phenotype of negative liquid-holding . 2. Materials and methods 2 .1 . Strains RAD a and a haploid cultures of Saccharomyes cerevisiae, which show wildtype sensitivity to U .V .-irradiation. rad2_e32, radio_e2, radg_eu, radb_11, rad22_e41, rad2_ ee , rad3 _ es, rad15_ e7 and rad 5p _ e43 are all U .V .-sensitive mutants of yeast isolated by Cox and Parry (1968) of genotype a ade rad. The prefix rad indicates the appropriate radiationsensitive locus, and the second term the isolation number of each particular allele used in this study . 2 .2 . Medium The complete medium used (YC) was described by Cox and Bevan (1962) . It is a yeast extract and peptone medium with 2 per cent (w/v) glucose, pH 6 . 7, solidified with Lab M agar . 2.3 . U. V.-treatment The source of U .V.-light was a Hanovia lla mercury discharge tube generating almost monochromatic radiation at 254 nM . The dose-rate was determined by means of a calibrated photocell . Cultures were grown for 3 days on solid YC, washed off and suspended in saline at a titre of 2 x 10 7 cells/ml and shaken on a vortex shaker. These cultures are at the end of log phase and contain fewer than 5 per cent budded cells. 50 ml portions were exposed to U .V . in open glass Petri dishes . All UN.-exposures were made at dose-rates of 2 and 1 J/M 2 /sec . All manipulations were performed in red light to prevent uncontrolled photoreactivation. 2 .4 . Liquid-holding treatment Samples of U .V .-treated suspensions and untreated controls were stored in saline in the dark at 28°C for 7 days . During this treatment, there was a complete cessation of growth and macromolecular synthesis . 2.5 . Incubation and scoring All plated cultures were incubated at 28°C in the dark and scored after 5 days' growth . Survival was scored by counting colony-forming units on YC medium . All survival experiments were performed at least twice and typical results are presented . 3.
Results and conclusions After U .V .-irradiation, the incubation of yeast cells in saline in the dark at 28 ° C results in increased cell viability (see Parry and Parry 1969) . After liquidholding treatment, wild-type (RAD) cells showed increased resistance to a second dose of U .V .-radiation . The results of a typical experiment using a haploid RAD strain are shown in table 1 and demonstrate that after 264 J/M 2
15
Liquid holding recovery
Strain
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RAD
rad21(e32)
Treatment given to cultures before final series of U .V.-exposures
Sensitivity expressed as U .V .-dose in J/M2 required to give 1 log cycle reduction- in cell viability
Control 132 J UN. + liquid-holding 264 J UN. + liquid holding 396 J U .V . + liquid holding
70 135 210 200
Control 120 J U .V. + liquid holding 240 J U .V. + liquid holding 360 J U .V . + liquid holding
108 114 216 228
Control 99 J U.V . + liquid holding 132 J U.V . + liquid holding
43 61 . 8 62 . 7
Control 90 J U .V . + liquid holding 120 J UN . + liquid holding 135 J U .V .+liquid holding
22 . 5 69 . 8 72 . 8 82 . 8
rad5(ul)
Control 180 J U .V . + liquid holding 240 J UN . + liquid holding
60 82 80
rad22(e41)
Control 60 J U .V . + liquid holding 180 J U .V .+liquid holding
72 108 135
rad10 (e2)
rad 0(ul1)
rad2(e9)
rad3(e5)
Control 5 . 05 J/M 2 U .V . + liquid holding 7 . 5 J/M2 U .V. + liquid holding 10 . 0 J/M2 U .V. + liquid holding 15 . 0 J/M 2 U .V. + liquid holding
5.0 5 .63 5 . 63 5 . 75 5 . 75
Control 0 .8 J/M 2 U.V .+liquid holding 2 . 5 J/M 2 U.V . + liquid holding 7 .5 J /M2 U.V . + liquid holding
6.2 6. 9 7. 8 7. 8
rad15 (e7)
Control 60 J/M 2 U .V. +liquid holding 120 J/M 2 U .V. + liquid holding
45 . 0 48 . 0 45 .0
rad50 (e43)
Control 120 J/M 2 U .V . + liquid holding 240 J/M 2 U .V . + liquid holding
88 . 8 60 .0 57 . 6
Table 1 .
The effects of liquid-holding treatment on U .V.-survival in the wild-type (Each result describes the characteristics of a U .V .-survival curve produced by irradiation after the treatment listed in column 2 . The results listed are based upon the final slope of each survival curve .)
RAD and U .V.-sensitive rad mutants .
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16
E . M. Parry and j. M . Parry
• Direct Figure 1 . The sensitivity to U .V.-light of cultures of the yeast mutant radio . plating . A U .V.-survival curve of radio culture exposed to a prior treatment with 99 J/M2 U .V., followed by liquid-holding treatment . 0 U .V .-survival curve of radio culture exposed to a prior treatment with 132 J/M 2 U .V., followed by liquid-holding treatment .
of U .V . followed by liquid-holding treatment the U .V .-dose required to produce a one log-cycle reduction in viability was increased from 70 to 210 J/M 2 compared with direct plating . The U .V .-survival curve for haploid wild-type cultures with and without liquid-holding treatment have been published in detail elsewhere (Parry and Parry 1972) . We have examined further the phenomenon of ' repair resistance ' by a study of the effects of liquid-holding treatment in yeast cultures carrying defective alleles of a number of the RAD genes of yeast . The mutant cultures used show both positive and negative liquid holding after exposure to U .V . (Parry and Parry 1969, Parry 1972) . The mutant cultures rad21 , radio , rads , rads and rad22 are more sensitive to U .V .-light than the RAD parent . Each of these five cultures also show increased cell viability when stored in saline for 7 days before plating upon nutrient medium . The results of a typical culture radio are shown in figure 1 . The presence of a resistant ' tail ' on the survival curve of the radio culture are characteristic of stationary-phase cultures of many radiation-sensitive mutants of yeast (Parry 1972) . All of the mutants which showed increased survival after liquid-holding treatment also showed increased resistance to a second U .V .-exposure after U .V .- and liquid-holding treatment .
Liquid holding recovery
17
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A second group of yeast strains carrying defective alleles of the genes rad2 and rad3 were exposed to U .V.-radiation followed by liquid-holding treatment . Both rad2 (figure 2) and rad3 (figure 3) cultures show extreme sensitivity to U .V.-light, and it has been demonstrated both indirectly and directly that they are defective in the process of excision repair (Parry and Parry 1969, Unrau, Wheatcroft and Cox 1971) . During post-U .V.-liquid-holding treatment, rad2 and rads cultures show increased cell lethality, whereas a photoreactivating light treatment after liquid holding increased cell viability, indicating the persistence in their DNA of U .V.-induced pyrimidine dimers during the holding period (Parry and Parry 1969, Parry, Parry and Waters 1972) . 0 100
UM-dose J/m 2 10 15
20
10
01
001
0 . 001
0 . 0001 Figure 2 . The sensitivity to U .V .-light of cultures of the yeast mutant rad2. ∎ Direct plating. 0 U .V .-survival curve of rad 2 culture exposed to a prior treatment with 2. 5 J/M 2 U .V., followed by liquid-holding treatment . • U .V .-survival curve of rad2 culture exposed to a prior treatment with 5 . 0 J/M 2 U .V., followed by liquidholding treatment . A UN.-survival curve of rad 2 culture exposed to a prior treatment with 10 . 0 J/M2 U.V ., followed by liquid-holding treatment . 0 U.V .survival curve of rad2 culture exposed to a prior treatment with 15 J/M 2 U.V ., followed by liquid-holding treatment . R .B .
B
18
E. M. Parry and J. M. Parry
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The results in figures 2 and 3 demonstrate that the final slopes of the U .V .survival curves of the rad2 and rad3 cultures after liquid-holding treatment were identical to that of the cultures after direct plating . The identical sensitivities of the rad2 and rad3 cultures before and after post-U .V .-liquid-holding treatment were confirmed by calculation of the U .V.-doses required to produce a one log-cycle reduction in cell viability . As shown in table 1, only small differences in sensitivities before and after liquid-holding treatment were detectable . The absence of ' repair resistance ' after post-U .V .-liquid-holding treatment in the excision deficient mutants rad2 and rad3 implicates the process of excision repair in the increased resistance of RAD cultures and the five mutants of Group 1 .
A Direct Figure 3 . The sensitivity to U .V.-light of cultures of the yeast mutant rad.. . plating. 0 U .V .-survival curve of rad3 culture exposed to a prior treatment with 0 . 8 J/M 3 U .V ., followed by liquid-holding treatment . 11 U .V.-survival curve of rad3 culture exposed to a prior treatment with 2 . 5 J/M3 U .V., followed by liquido U .V .-survival curve of rad3 culture exposed to a prior holding treatment . treatment with 75 J/M3 U .V ., followed by liquid-holding treatment .
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Liquid holding recovery
19
Figure 4 . The sensitivity to U .V.-light of cultures of the yeast mutant rad,,. o Direct plating. o U .V.-survival curve of a rad,,, culture exposed to a prior treatment with. 60 J/M' U .V ., followed by liquid-holding treatment. 0 U .V .-survival curve of a radlb culture exposed to a prior treatment with 120 J/M' U .V., followed by liquid-holding treatment . A further pair of yeast strains carrying defective alleles of the genes rad,,, and rad bo were exposed to U .V .-radiation followed by liquid-holding treatment . Both radbo (figure 4) and radbo (figure 5) are sensitive to U .V.-light, and after a liquid-holding treatment they show increased cell lethality . However, in these mutant cultures photoreactivating light treatment after liquid holding produced no increase in cell viability in either culture, indicating the removal of U .V .induced pyrimidine dimers (Parry and Parry 1969) . The results presented in figures 4 and 5 and table 1 demonstrate that no increase in resistances to a second dose-range of U .V . was detectable in either mutant culture. In the case of cultures of radbo post-U .V .-liquid-holding treatment led to increased sensitivity to the second UN .-dose-range . The absence of photoreactivating-light-sensitive lesions after liquid-holding treatment in radio and radbo cultures indicates that the first step of repair, i .e . the excision of a dimer, is completed . The failure of such excision to lead to increased cell viability indicates that radio and radbo may be defective in the
20
E . M. Parry and Y. M . Parry
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E. M. Parry and ,J. M . Parry
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resynthesis of the excised region . The absence of ` repair resistance ' in such cultures suggests that the increased resistance of RAD yeast culture to a second U .V .-dose is dependent upon the completion of the whole process of excision repair . Each of the U .V.-sensitive rad mutants used in this study was mated to a wild-type RAD culture of opposite mating type to produce a heterozygous diploid culture . After sporulation, the spore tetrads were separated and the products of a single tetrad derived from each cross were studied in detail . Each
Figure 5 . The sensitivity to U .V.-light of cultures of the yeast mutant radso . ∎ Direct plating. • U .V.-survival curve of a radso culture exposed to a prior treatment A U .V.-survival with 120 J/M 2 U .V ., followed by liquid-holding treatment . curve of a rad, culture exposed to a prior treatment with 240 J/M 2 U .V ., followed by liquid-holding treatment.
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Liquid holding recovery
23
ascospore culture was analysed for mating type, adenine requirement, U .V.sensitivity, response to post-U .V.-liquid-holding treatment and response to a second U .V .-treatment . The results obtained from the genetic analyses are shown in table 2 . The five mutants rad21i radio , rad s, rads, and rad22 shown in table 2 show a 2 : 2 segregation for the phenotypes : mating type, adenine requirement and U .V.-sensitivity. All the spores derived from the five mutants showed increased viability after post-U.V.-liquid-holding treatment and the presence of repair resistance, i .e . 4 : 0 segregation . There was no evidence for the separation of these two phenotypes in our limited genetic data . In contrast, the spores derived from the four mutant cultures which did not show repair resistance showed 2 : 2 segregation of the phenotypes of liquid holding and repair resistance . The most important observation in table 2 was the correlation between negative liquid holding and the lack of ` repair resistance ' together with positive liquid holding and the presence of ` repair resistance ' . Although the genetic data are necessarily limited, i .e . only one tetrad per mutant culture analysed, the results indicate that the phenotype of ' repair resistance ' is under genetic control and depends upon the presence of alleles which also confer the property of liquid-holding recovery . ACKNOWLEDGMENTS
The work was supported by Euratom Grant B10 E 1199-72-1 . We are grateful to Mrs . Margaret Clatworthy for the preparation of the figures and to Drs . E . Moustacchi and F . Zimmerman for French and German translations . Si a la suite d'une irradiation aux ultraviolets (U .V .) de levures de type sauvage (RAD) les cellules sont conservees a l'obscurite en milieu liquide non nutritif, on observe une resistance accrue a un second traitement par les U .V . (resistance reparative) . Des mutants U.V .-sensibles (rad) de levure qui montrent des reponses differentes vis-a-vis du post-traitement de conservation a l'obscurite et une seconde irradiation ont ete classes en deux groupes . Les mutants du groupe I montrent une augmentation de la survie a la fois apres la conservation post-U .V . en milieu liquide et la resistance reparative au second traitement par les U .V . En revanche, les mutants du groupe II ont une viabilite reduite apres conservation post-U .V. en milieu liquide et presentent la meme sensibilite aux U .V . avant ou apres cette conservation . L'analyse genetique de croisement des mutants rad et du type sauvage montre que l'aptitude a la resistance reparative aux U .V. est sous controle genetique . Celle-ci depend de la presence d'alleles de genes de sensibilite aux radiations qui conferent egalement la propriete de restauration de la survie par la conservation de post-irradiation en millieu liquide non nutritif. Werden Hefezellen nach Bestrahlung mit U .V . in einem nahrstofffreien Medium gehalten, so kommt es zu einer erhohten Resistenz gegen ein zweite U .V.-Bestrahlung bei Wildtypzellen (RAD) : Reparatur-Resistenz . U .V.-empfindliche Hefemutanten (rad) zeigen unterschiedliche Reaktionen auf eine Nachinkubation in nahrstofffreiem Medium and eine zweite U .V.-Bestrahlung and wurden hiernach in zwei Gruppen gegliedert . Mutanten der Gruppe 1 zeigen eine erhohte Uberlebensrate nach Nachinkubation and Reparatur-Resistenz . Im Gegensatz hierzu ergeben die Mutanten der Gruppe 2 eine niedrigere Uberlebensrate bei der Nachinkubation nach U .V .-Bestrahlung, wobei die U .V.-Empfindlichkeit vor and nach der Nachbehandlung gleich bleibt .
24
Liquid holding recovery
Eine genetische Analyse von Kreuzungen zwischen rad-Mutanten and dem Wildtyp ergab, daI3 die Eigenschaft ` Reparatur-Resistenz gegen U .V.-Bestrahlung' genetisch bedingt ist and von der Anwesenheit von denjenigen Allelen abhangt, welche die Strahlenempfindlichkeit kontrollieren and die Eigenschaft des liquid holding recovery bedingen.
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REFERENCES
Cox, B . S ., and BEVAN, E . A ., 1962, New Phytol., 61, 342 . Cox, B . S ., and PARRY, J . M., 1968, Mutation Res., 6, 37 . KELNER, A., 1949, Y. Bact., 58, 511 . PARRY, J . M ., 1972, Molec . gen. Genet ., 118, 33 . PARRY, J . M ., and PARRY, E . M ., 1969, Mutation Res ., 8, 545 ; 1972, Genet. Res ., 19, 1 . PARRY, J . M ., PARRY, E . M ., and WATERS, R., 1972, Mutation Res ., 15, 135 . PATRICK, M. H ., and HAYNES, R ., 1968, Y . Bact ., 95, 1350. PATRICK, M. H ., HAYNES, R. H ., and URETZ, R . B., 1964, Radiat. Res ., 21, 144 . UNRAU, P ., WHEATCROFT, R ., and Cox, B . S ., 1971, Molec . gen . Genet., 113, 359 .
1976,
VOL .
30,
NO .
1, 1 3 -24
The genetic control of liquid-holding recovery and U.V .-induced repair resistance in the yeast, Saccharomyces cerevisiae ELIZABETH M . PARRY and JAMES M . PARRY Department of Genetics, University College of Swansea, Singleton Park, Swansea SA2 8PP, West Glamorgan, U .K .
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(Received 5 May 1976 ; accepted 24 June 1976)
Liquid-holding treatment in non-nutrient solutions after U .V .-exposure results in an increased resistance of wild-type (RAD) yeast cells to a second U .V .treatment (repair resistance) . U .V .-sensitive (rad) mutants of yeast which show variation in their response to liquid-holding treatment and a second U .V .-dose-range have been classified into two groups . Mutants of Group 1 show increased viability after post-U .V .liquid-holding treatment and show repair resistance . In contrast, mutants of Group 2 which show reduced viability during post-U .V .-liquid-holding treatment have the same U .V.-sensitivity, both before and after liquid-holding treatment . Genetic analysis of crosses of the rad mutants to wild-type cultures indicate that the phenotype of repair resistance to UN .-treatment is under genetic control and depends on the presence of alleles of radiation sensitivity genes, which also confer the property of liquid-holding recovery .
1 . Introduction Wild-type (RAD) cultures of the yeast, Saccharomyces cerevisiae show partial recovery from the lethal effects of U .V .-light when exposed to post-irradiation treatments such as photoreactivating light and liquid holding in the dark in non-nutrient solutions before plating on nutrient agar (Kelner 1949, Patrick, Haynes and Uretz 1964) . The U .V .-sensitive mutants of yeast (Cox and Parry 1968) show considerable variation in their response to post-U .V .-liquid-holding treatment (Parry and Parry 1969, Parry, Parry and Waters 1972) . The various strains may be classified into four groups on the basis of positive or negative responses to liquid holding and photoreactivating light after liquid-holding treatment . An additional property of liquid-holding treatment in yeast was demonstrated by Patrick and Haynes (1968)-that yeast cultures exposed to liquid-holding conditions after U .V .-irradiation show increased resistance to a second range of UN .-light exposures . After a further study of this property of ' repair resistance ' (Parry and Parry 1972), we have suggested that the repair of U .V .-damage in yeast involves the action of at least two repair processes and that increased resistance to the second U .V .-exposure results from an increase in the proportion of U .V .induced lesions repaired by the excision system as opposed to the post-replication repair system . In this paper we report the effects of liquid-holding treatment after U .V .exposure and a second U .V .-treatment after the liquid-holding treatment in a wide range of UN .-sensitive mutants of yeast . Our results show that ` repair
14
E . M . Parry and J. M. Parry
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resistance ' was demonstrable only in cultures which showed a positive increase in cell viability after post-U .V .-liquid-holding treatment . Genetic analysis indicated that in mutants which do not show ' repair resistance ', this lack of resistance to a second UN .-exposure segregated with the phenotype of negative liquid-holding . 2. Materials and methods 2 .1 . Strains RAD a and a haploid cultures of Saccharomyes cerevisiae, which show wildtype sensitivity to U .V .-irradiation. rad2_e32, radio_e2, radg_eu, radb_11, rad22_e41, rad2_ ee , rad3 _ es, rad15_ e7 and rad 5p _ e43 are all U .V .-sensitive mutants of yeast isolated by Cox and Parry (1968) of genotype a ade rad. The prefix rad indicates the appropriate radiationsensitive locus, and the second term the isolation number of each particular allele used in this study . 2 .2 . Medium The complete medium used (YC) was described by Cox and Bevan (1962) . It is a yeast extract and peptone medium with 2 per cent (w/v) glucose, pH 6 . 7, solidified with Lab M agar . 2.3 . U. V.-treatment The source of U .V.-light was a Hanovia lla mercury discharge tube generating almost monochromatic radiation at 254 nM . The dose-rate was determined by means of a calibrated photocell . Cultures were grown for 3 days on solid YC, washed off and suspended in saline at a titre of 2 x 10 7 cells/ml and shaken on a vortex shaker. These cultures are at the end of log phase and contain fewer than 5 per cent budded cells. 50 ml portions were exposed to U .V . in open glass Petri dishes . All UN.-exposures were made at dose-rates of 2 and 1 J/M 2 /sec . All manipulations were performed in red light to prevent uncontrolled photoreactivation. 2 .4 . Liquid-holding treatment Samples of U .V .-treated suspensions and untreated controls were stored in saline in the dark at 28°C for 7 days . During this treatment, there was a complete cessation of growth and macromolecular synthesis . 2.5 . Incubation and scoring All plated cultures were incubated at 28°C in the dark and scored after 5 days' growth . Survival was scored by counting colony-forming units on YC medium . All survival experiments were performed at least twice and typical results are presented . 3.
Results and conclusions After U .V .-irradiation, the incubation of yeast cells in saline in the dark at 28 ° C results in increased cell viability (see Parry and Parry 1969) . After liquidholding treatment, wild-type (RAD) cells showed increased resistance to a second dose of U .V .-radiation . The results of a typical experiment using a haploid RAD strain are shown in table 1 and demonstrate that after 264 J/M 2
15
Liquid holding recovery
Strain
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RAD
rad21(e32)
Treatment given to cultures before final series of U .V.-exposures
Sensitivity expressed as U .V .-dose in J/M2 required to give 1 log cycle reduction- in cell viability
Control 132 J UN. + liquid-holding 264 J UN. + liquid holding 396 J U .V . + liquid holding
70 135 210 200
Control 120 J U .V. + liquid holding 240 J U .V. + liquid holding 360 J U .V . + liquid holding
108 114 216 228
Control 99 J U.V . + liquid holding 132 J U.V . + liquid holding
43 61 . 8 62 . 7
Control 90 J U .V . + liquid holding 120 J UN . + liquid holding 135 J U .V .+liquid holding
22 . 5 69 . 8 72 . 8 82 . 8
rad5(ul)
Control 180 J U .V . + liquid holding 240 J UN . + liquid holding
60 82 80
rad22(e41)
Control 60 J U .V . + liquid holding 180 J U .V .+liquid holding
72 108 135
rad10 (e2)
rad 0(ul1)
rad2(e9)
rad3(e5)
Control 5 . 05 J/M 2 U .V . + liquid holding 7 . 5 J/M2 U .V. + liquid holding 10 . 0 J/M2 U .V. + liquid holding 15 . 0 J/M 2 U .V. + liquid holding
5.0 5 .63 5 . 63 5 . 75 5 . 75
Control 0 .8 J/M 2 U.V .+liquid holding 2 . 5 J/M 2 U.V . + liquid holding 7 .5 J /M2 U.V . + liquid holding
6.2 6. 9 7. 8 7. 8
rad15 (e7)
Control 60 J/M 2 U .V. +liquid holding 120 J/M 2 U .V. + liquid holding
45 . 0 48 . 0 45 .0
rad50 (e43)
Control 120 J/M 2 U .V . + liquid holding 240 J/M 2 U .V . + liquid holding
88 . 8 60 .0 57 . 6
Table 1 .
The effects of liquid-holding treatment on U .V.-survival in the wild-type (Each result describes the characteristics of a U .V .-survival curve produced by irradiation after the treatment listed in column 2 . The results listed are based upon the final slope of each survival curve .)
RAD and U .V.-sensitive rad mutants .
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16
E . M. Parry and j. M . Parry
• Direct Figure 1 . The sensitivity to U .V.-light of cultures of the yeast mutant radio . plating . A U .V.-survival curve of radio culture exposed to a prior treatment with 99 J/M2 U .V., followed by liquid-holding treatment . 0 U .V .-survival curve of radio culture exposed to a prior treatment with 132 J/M 2 U .V., followed by liquid-holding treatment .
of U .V . followed by liquid-holding treatment the U .V .-dose required to produce a one log-cycle reduction in viability was increased from 70 to 210 J/M 2 compared with direct plating . The U .V .-survival curve for haploid wild-type cultures with and without liquid-holding treatment have been published in detail elsewhere (Parry and Parry 1972) . We have examined further the phenomenon of ' repair resistance ' by a study of the effects of liquid-holding treatment in yeast cultures carrying defective alleles of a number of the RAD genes of yeast . The mutant cultures used show both positive and negative liquid holding after exposure to U .V . (Parry and Parry 1969, Parry 1972) . The mutant cultures rad21 , radio , rads , rads and rad22 are more sensitive to U .V .-light than the RAD parent . Each of these five cultures also show increased cell viability when stored in saline for 7 days before plating upon nutrient medium . The results of a typical culture radio are shown in figure 1 . The presence of a resistant ' tail ' on the survival curve of the radio culture are characteristic of stationary-phase cultures of many radiation-sensitive mutants of yeast (Parry 1972) . All of the mutants which showed increased survival after liquid-holding treatment also showed increased resistance to a second U .V .-exposure after U .V .- and liquid-holding treatment .
Liquid holding recovery
17
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A second group of yeast strains carrying defective alleles of the genes rad2 and rad3 were exposed to U .V.-radiation followed by liquid-holding treatment . Both rad2 (figure 2) and rad3 (figure 3) cultures show extreme sensitivity to U .V.-light, and it has been demonstrated both indirectly and directly that they are defective in the process of excision repair (Parry and Parry 1969, Unrau, Wheatcroft and Cox 1971) . During post-U .V.-liquid-holding treatment, rad2 and rads cultures show increased cell lethality, whereas a photoreactivating light treatment after liquid holding increased cell viability, indicating the persistence in their DNA of U .V.-induced pyrimidine dimers during the holding period (Parry and Parry 1969, Parry, Parry and Waters 1972) . 0 100
UM-dose J/m 2 10 15
20
10
01
001
0 . 001
0 . 0001 Figure 2 . The sensitivity to U .V .-light of cultures of the yeast mutant rad2. ∎ Direct plating. 0 U .V .-survival curve of rad 2 culture exposed to a prior treatment with 2. 5 J/M 2 U .V., followed by liquid-holding treatment . • U .V .-survival curve of rad2 culture exposed to a prior treatment with 5 . 0 J/M 2 U .V., followed by liquidholding treatment . A UN.-survival curve of rad 2 culture exposed to a prior treatment with 10 . 0 J/M2 U.V ., followed by liquid-holding treatment . 0 U.V .survival curve of rad2 culture exposed to a prior treatment with 15 J/M 2 U.V ., followed by liquid-holding treatment . R .B .
B
18
E. M. Parry and J. M. Parry
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The results in figures 2 and 3 demonstrate that the final slopes of the U .V .survival curves of the rad2 and rad3 cultures after liquid-holding treatment were identical to that of the cultures after direct plating . The identical sensitivities of the rad2 and rad3 cultures before and after post-U .V .-liquid-holding treatment were confirmed by calculation of the U .V.-doses required to produce a one log-cycle reduction in cell viability . As shown in table 1, only small differences in sensitivities before and after liquid-holding treatment were detectable . The absence of ' repair resistance ' after post-U .V .-liquid-holding treatment in the excision deficient mutants rad2 and rad3 implicates the process of excision repair in the increased resistance of RAD cultures and the five mutants of Group 1 .
A Direct Figure 3 . The sensitivity to U .V.-light of cultures of the yeast mutant rad.. . plating. 0 U .V .-survival curve of rad3 culture exposed to a prior treatment with 0 . 8 J/M 3 U .V ., followed by liquid-holding treatment . 11 U .V.-survival curve of rad3 culture exposed to a prior treatment with 2 . 5 J/M3 U .V., followed by liquido U .V .-survival curve of rad3 culture exposed to a prior holding treatment . treatment with 75 J/M3 U .V ., followed by liquid-holding treatment .
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Liquid holding recovery
19
Figure 4 . The sensitivity to U .V.-light of cultures of the yeast mutant rad,,. o Direct plating. o U .V.-survival curve of a rad,,, culture exposed to a prior treatment with. 60 J/M' U .V ., followed by liquid-holding treatment. 0 U .V .-survival curve of a radlb culture exposed to a prior treatment with 120 J/M' U .V., followed by liquid-holding treatment . A further pair of yeast strains carrying defective alleles of the genes rad,,, and rad bo were exposed to U .V .-radiation followed by liquid-holding treatment . Both radbo (figure 4) and radbo (figure 5) are sensitive to U .V.-light, and after a liquid-holding treatment they show increased cell lethality . However, in these mutant cultures photoreactivating light treatment after liquid holding produced no increase in cell viability in either culture, indicating the removal of U .V .induced pyrimidine dimers (Parry and Parry 1969) . The results presented in figures 4 and 5 and table 1 demonstrate that no increase in resistances to a second dose-range of U .V . was detectable in either mutant culture. In the case of cultures of radbo post-U .V .-liquid-holding treatment led to increased sensitivity to the second UN .-dose-range . The absence of photoreactivating-light-sensitive lesions after liquid-holding treatment in radio and radbo cultures indicates that the first step of repair, i .e . the excision of a dimer, is completed . The failure of such excision to lead to increased cell viability indicates that radio and radbo may be defective in the
20
E . M. Parry and Y. M . Parry
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E. M. Parry and ,J. M . Parry
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resynthesis of the excised region . The absence of ` repair resistance ' in such cultures suggests that the increased resistance of RAD yeast culture to a second U .V .-dose is dependent upon the completion of the whole process of excision repair . Each of the U .V.-sensitive rad mutants used in this study was mated to a wild-type RAD culture of opposite mating type to produce a heterozygous diploid culture . After sporulation, the spore tetrads were separated and the products of a single tetrad derived from each cross were studied in detail . Each
Figure 5 . The sensitivity to U .V.-light of cultures of the yeast mutant radso . ∎ Direct plating. • U .V.-survival curve of a radso culture exposed to a prior treatment A U .V.-survival with 120 J/M 2 U .V ., followed by liquid-holding treatment . curve of a rad, culture exposed to a prior treatment with 240 J/M 2 U .V ., followed by liquid-holding treatment.
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Liquid holding recovery
23
ascospore culture was analysed for mating type, adenine requirement, U .V.sensitivity, response to post-U .V.-liquid-holding treatment and response to a second U .V .-treatment . The results obtained from the genetic analyses are shown in table 2 . The five mutants rad21i radio , rad s, rads, and rad22 shown in table 2 show a 2 : 2 segregation for the phenotypes : mating type, adenine requirement and U .V.-sensitivity. All the spores derived from the five mutants showed increased viability after post-U.V.-liquid-holding treatment and the presence of repair resistance, i .e . 4 : 0 segregation . There was no evidence for the separation of these two phenotypes in our limited genetic data . In contrast, the spores derived from the four mutant cultures which did not show repair resistance showed 2 : 2 segregation of the phenotypes of liquid holding and repair resistance . The most important observation in table 2 was the correlation between negative liquid holding and the lack of ` repair resistance ' together with positive liquid holding and the presence of ` repair resistance ' . Although the genetic data are necessarily limited, i .e . only one tetrad per mutant culture analysed, the results indicate that the phenotype of ' repair resistance ' is under genetic control and depends upon the presence of alleles which also confer the property of liquid-holding recovery . ACKNOWLEDGMENTS
The work was supported by Euratom Grant B10 E 1199-72-1 . We are grateful to Mrs . Margaret Clatworthy for the preparation of the figures and to Drs . E . Moustacchi and F . Zimmerman for French and German translations . Si a la suite d'une irradiation aux ultraviolets (U .V .) de levures de type sauvage (RAD) les cellules sont conservees a l'obscurite en milieu liquide non nutritif, on observe une resistance accrue a un second traitement par les U .V . (resistance reparative) . Des mutants U.V .-sensibles (rad) de levure qui montrent des reponses differentes vis-a-vis du post-traitement de conservation a l'obscurite et une seconde irradiation ont ete classes en deux groupes . Les mutants du groupe I montrent une augmentation de la survie a la fois apres la conservation post-U .V . en milieu liquide et la resistance reparative au second traitement par les U .V . En revanche, les mutants du groupe II ont une viabilite reduite apres conservation post-U .V. en milieu liquide et presentent la meme sensibilite aux U .V . avant ou apres cette conservation . L'analyse genetique de croisement des mutants rad et du type sauvage montre que l'aptitude a la resistance reparative aux U .V. est sous controle genetique . Celle-ci depend de la presence d'alleles de genes de sensibilite aux radiations qui conferent egalement la propriete de restauration de la survie par la conservation de post-irradiation en millieu liquide non nutritif. Werden Hefezellen nach Bestrahlung mit U .V . in einem nahrstofffreien Medium gehalten, so kommt es zu einer erhohten Resistenz gegen ein zweite U .V.-Bestrahlung bei Wildtypzellen (RAD) : Reparatur-Resistenz . U .V.-empfindliche Hefemutanten (rad) zeigen unterschiedliche Reaktionen auf eine Nachinkubation in nahrstofffreiem Medium and eine zweite U .V.-Bestrahlung and wurden hiernach in zwei Gruppen gegliedert . Mutanten der Gruppe 1 zeigen eine erhohte Uberlebensrate nach Nachinkubation and Reparatur-Resistenz . Im Gegensatz hierzu ergeben die Mutanten der Gruppe 2 eine niedrigere Uberlebensrate bei der Nachinkubation nach U .V .-Bestrahlung, wobei die U .V.-Empfindlichkeit vor and nach der Nachbehandlung gleich bleibt .
24
Liquid holding recovery
Eine genetische Analyse von Kreuzungen zwischen rad-Mutanten and dem Wildtyp ergab, daI3 die Eigenschaft ` Reparatur-Resistenz gegen U .V.-Bestrahlung' genetisch bedingt ist and von der Anwesenheit von denjenigen Allelen abhangt, welche die Strahlenempfindlichkeit kontrollieren and die Eigenschaft des liquid holding recovery bedingen.
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REFERENCES
Cox, B . S ., and BEVAN, E . A ., 1962, New Phytol., 61, 342 . Cox, B . S ., and PARRY, J . M., 1968, Mutation Res., 6, 37 . KELNER, A., 1949, Y. Bact., 58, 511 . PARRY, J . M ., 1972, Molec . gen. Genet ., 118, 33 . PARRY, J . M ., and PARRY, E . M ., 1969, Mutation Res ., 8, 545 ; 1972, Genet. Res ., 19, 1 . PARRY, J . M ., PARRY, E . M ., and WATERS, R., 1972, Mutation Res ., 15, 135 . PATRICK, M. H ., and HAYNES, R ., 1968, Y . Bact ., 95, 1350. PATRICK, M. H ., HAYNES, R. H ., and URETZ, R . B., 1964, Radiat. Res ., 21, 144 . UNRAU, P ., WHEATCROFT, R ., and Cox, B . S ., 1971, Molec . gen . Genet., 113, 359 .
