INT . J . RADIAT . BIOL .,
1975,
VOL .
28,
NO .
6, 5 1 1 -518
Determination of the number of photoreactivating enzyme molecules per haploid Saccharomyces cell AKIRA YASUI and WOLFGANG LASKOWSKI Zentralinstitut fur Biochemie and Biophysik, der Freien Universitat Berlin, Berlin, Germany
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(Received 8 July 1975 ; accepted 9 October 1975)
Two haploid radiation-sensitive mutants of Saccharomyces were studied to investigate the formation of complex between photoreactivating-enzyme and substrate after ultra-violet irradiation . Using photo-flashes, the time necessary for maximum complex formation has been determined . Within 1 min, 70 per cent of the complexes have been formed . To determine the number of photoreactivating enzyme molecules per cell, the maximum dose decrement obtained after one photo-flash was determined and corrected for the effects of non-photoreactivable lesions . The corrected maximum dose decrement was found to be identical for both strains (8 . 5 erg mm -2) . The number of photoreactivating-enzyme molecules involved in the photorepair of nuclear DNA damage was calculated as 272 ± 27 .
1 . Introduction Photoreactivating-enzyme (PRE) repairs pyrimidine dimers that have been formed in DNA irradiated with ultra-violet light (U .V .) according to the following reaction scheme (Rupert 1964) k3 kl E+S . ES > E+P, light k2 where E is the PRE, S the pyrimidine dimer (substrate), ES the PRE-substrate complex and P the repaired product . Light flash experiments have recently been proved to be very useful in the study of photoenzymatic repair (Harm, and Rupert 1968, Harm, Rupert and Harm 1971) . After a short intense flash, only those substrates can be repaired that are complexed with PRE at the moment of the flash . The aim of this paper is to apply this technique to yeast cells in an investigation of the time-dependence of the formation of PRE-substrate complexes and to determine the number of PRE molecules per cell . The method is based on the fact that, as in E . coli (Harm et al . 1971), in yeast (Waters and Moustacchi 1975) pyrimidine dimers are induced proportionally to the U .V .-dose, the numbers of pyrimidine dimers induced per genome per erg mm -2 U .V .-light is known (Unrau, Wheatcroft, Cox and Oliver 1973, Fath and Brendel 1975) . The effects of non-photoreparable damage, which were neglected by the calculation of the number of PRE per E. coli cell (Harm et al. 1971), are taken into account in this paper . Suitable radiation-sensitive mutants of Saccharomyces were used, in which the ratio between PRE molecules and pyrimidine dimers at any measurable survival level is large enough to produce a recognizable flash effect and small enough to see the maximum complex.
512
A . Yasui and W. Laskowski
Materials and methods The following haploid Saccharomyces strains were used : MB 1030-1A (a, radl-18, rad2-17, radl8-2), S 24-12c (a, rad2-20) . The symbols in brackets indicate mating type alleles and mutated genes causing radiation sensitivity (Game and Cox 1971) . radl and rad2 mutants are known to block the excision-repair mechanism (Unrau et al . 1971), whereas radl8 blocks another not clearly identified dark-repair mechanism (Game and Mortimer 1974) . Strain MB1030-1A was obtained from M . Brendel . Strain S24-12c from D . Averbeck .
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2.
2.1 . Medium Yeast extract peptone medium (YEP) containing 1 per cent yeast extract (Difco), 0 . 5 per cent peptone (Merck), 2 per cent glucose and 2 per cent agar (Merck), was used throughout . 2.2 . Growth conditions and cell preparation The strains were stored on YEP agar slants at 4 ° C . Cell samples were inoculated on fresh YEP agar slants and incubated for 48 hours at 30°C . The cells were then suspended in buffer (0 . 05 M KH2PO 4 i pH 4 . 8), washed three times in buffer, and resuspended in buffer for further treatment . After irradiation and photoreactivation, appropriate cell-samples were plated on YEP medium and incubated for 72 hours at 30°C . 2.3 . U. V .-irradiation The U .V .-source was a low-pressure mercury lamp (Osram HNS 12) with a maximum emission at 254 nm wave-length . The dose rate was 3 erg sec -I mm -2 , measured by a photon flux counter (Schaarschmidt 1970) . 2.4 . Photoreactivation (Phl?) For photoreactivating light flashes, an electronic flash unit (Rollei E 36 RE) was used, delivering a light flash of 1/800 sec duration . This light flash was filtered by a Jenaer Filter Glas WG 320, transmitting only wave-lengths longer than 320 nm . The U .V .-irradiated cell suspension, placed in a small glass dish, was positioned 5 cm from the surface of the flash unit and exposed to one light flash . More photons reached the cells when the distance was less than 5 cm (as measured with a photodiode), but the amount of photoreactivation did not increase . Maximum photoreactivation was obtained by continuous illumination with photoreactivating light of an Osram-L-20W-70 lamp . 2.5 . Dose decrement The difference in the U .V .-doses applied with and without subsequent photoreactivation, which leads to the same survival level, is called the dose decrement (Harm et al. 1971) . 2.6 . Experimental conditions All experiments were performed at room temperature of approximately 22°C under yellow light to prevent uncontrolled PhR . The results depicted in the figures are average values obtained from three to five independent identical experiments .
Photoreactivation o f Saccharomyces
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The cell concentration during irradiation and photoreactivation was 5 x 10 6 cells per ml buffer . Before plating, the cell suspensions were diluted appropriately to obtain from 100 to 400 colonies per plate .
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3.
Results Figure 1 shows the survival of the strain MB1030-1A after U .V .-irradiation (left curve) and the results of PhR by single light flashes at various times after U .V .-irradiation (right curves) . These data show that by 1 min after U .V .-irradiation, 70 per cent of the maximum survival can be obtained by one flash, whereas about 20 min are necessary to achieve maximum survival . The same kind of experiment have been performed with the less-U .V .-sensitive haploid strain S24-12c, which carries only the mutated rad2 gene . The results were similar to those described above . This time interval is considered to be necessary for maximum complex formation of PRE molecules and substrate UV-dose (erg/mm 2 ) 5 10 15 20 10°
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Figure 1 . Survival of strain MB1030-1A after U .V .-irradiation (left curve), and after U .V .-irradiation plus application of a single light-flash given at various times after U .V .-irradiation (right curves) .
514
A . Yasui and W. Laskowski
(Harm et al. 1971) . These results were considered in the procedure of the following experiments, in which flash photoreactivation was applied 40 min after U . V .-irradiation . Figure 2 depicts the survival curve after U .V .-irradiation and U .V .-irradiation plus photoflash 40 min after U .V .-irradiation for strain MB1030-1A (left curves) and the less-sensitive strain S24-12c (right curves) . UV- dose (erg/mm2 )
10 20 3,0 40 590 6,0
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Figure 2 . Survival after U .V .-irradiation (closed symbols) and U.V .-irradiation plus single photo-flash (open symbols) of MB1030-1A (A A) and S 24-12c ( • 0) . To determine the number of PRE molecules per cell, the U .V .-dosedependence of the dose decrement by a single light flash at the time of maximum complex formation was investigated for both strains . Dark repair between U .V .-irradiation and PhR may be neglected, because of the short time-interval . The maximum dose decrement obtained for strain MB1030-1A was 6 erg mm -2 , whereas that obtained for S24-12c was 7 . 2 erg mm-2 (figure 3) . This difference may either indicate a different number of PRE molecules per cell in the two strains or it may be produced by a different capacity to remove non-photoreactivable lesions in the two strains . It was therefore necessary to test whether the non-photoreactivable lesions affect determination of the dose decrement .
Photoreactivation o f
Saccharomyces
5 15
The increase in photoreactivable and non-photoreactivable lesions with increasing U .V .-dose is shown schematically in figure 4 . The number of photoreactivable lesions per cell after U .V .-irradiation is given by N 1 . After application of a single photo-flash, Nl decreases to N2. The dose decrement U2- U, can be determined from the survival curve if the application of U2 and 0
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30
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Figure 3 .
Dose decrement of photoreactivation by a single photo-flash 40 min after U .V .-irradiation . MB1030-1A 0 . S24-12c o .
Figure 4 . Schematic representation of the amounts of photoreactivable and nonphotoreactivable lesions and the effect of photoreactivation (see text for further explanation) .
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A . Yasui and W. Laskowski
one photo-flash results in the same survival frequency as the application of U, without photo-flash . Owing to a possible increase in non-photoreactivable lesions with increasing U .V .-dose, however, a different number of non-photoreactivable lesions per cell, indicated by N3 and N4 , can be produced . The survival frequency after application of the U .V.-dose U2 plus photo-flash may therefore not equal that after application of U, without photo-flash . Based on the assumption that the number of non-photoreactivable lesions increases linearly with increasing U .V .-dose, a measure for the real dose decrement can be deduced . It is evident from figure 4 that, if the cells are irradiated with a U .V .-dose U2-U,, maximally photoreactivated and thereafter U .V .-irradiated with U1 , the survival frequency should be the same as that obtained after application of the U .V .-dose U2 and one photo-flash . Figure 5 depicts the effect of U .V . -irradiation and additional maximum photoreactivation applied continuously for 30 min . After the maximum photoreactivation, cells were irradiated again with a U .V .-dose U,-U (postirradiation) . By changing the dose U, that dose was determined, which results in survival frequencies coinciding with the survival frequency by a single flash photoreactivation after U .V .-irradiation with U2 . According to figure 4, this dose should correspond to the dose decrement U2 U1 at the dose U2 . 10
0
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10
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Figure 5 . Survival after U .V .-irradiation (closed symbols) and U .V.-irradiation plus maximum photoreactivation (open symbols) of MB1030-1A (A ,) and S 24-12c ( • 0) . Figure 6 shows the corrected dose decrement (upper curves) compared with the uncorrected dose decrement depicted in figure 3 . After this correction, the two strains show the same maximum dose decrement of 8 . 5 erg mm -2 . The corrected maximum dose decrement does not increase, although the substrate concentration increases with increasing U .V .-dose . The numbers of PREsubstrate complexes should therefore be equal to the number of PRE molecules in the corresponding cells. The number of PRE molecules per cell involved with the photorepair of nuclear DNA can be calculated from the corrected dose
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Figure 6 .
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Uncorrected (square) and corrected (star) dose decrements for MB1030-1A (left side) and S 24-12c (right side) .
decrement if the number of pyrimidine dimers produced per erg mm -2 is known . Unrau et al . (1973) have found 24 dimers per haploid genome per erg MM -2 in U .V .-irradiated Saccharomyces cells using 14 C marking followed by purification of DNA from RNA . Fath and Brendel (1975) found 32 ± 3 dimers per haploid genome and erg mm-2 in recently isolated Saccharomyces mutants auxotrophic for deoxythymidinemonophosphate . Using the latter value and the corrected maximum dose decrement of 8 . 5 erg mm -2, we determined the number of PRE molecules per haploid cell involved with the photorepair of nuclear DNA to be 272 + 27 . 4 . Discussion The results in figure 1 were interpreted as indicating that approximately 70 per cent of maximum complexes have been formed 1 min after U .V .-irradiation, and the remaining 30 per cent require about 20 min for formation . The time needed to obtain maximum PRE-substrate complex formation is two to three times longer in Saccharomyces than in E . coli (Harm et al . 1968) . This may indicate a wide distribution of PRE molecules in the cell . The dose decrement obtained with one photo-flash was corrected for the effects of non-photoreactivable lesions . To achieve the same effect as with photoreactivation by one photo-flash after U .V .-irradiation, cells were first pre-irradiated with U .V ., closely followed by maximum photoreactivation and then U .V .-post-irradiated . The U .V .-doses for pre-irradiation and postirradiation were varied, so that the total U .V .-dose remained constant . The corrected dose decrement eliminating the effect of non-photoreactivable lesions was determined from that pre-irradiation U .V .-dose, which results in a surviving fraction after maximum photoreactivation followed by immediate postirradiation, that coincides with the surviving fraction after photoreactivation with one photo-flash after application of a U .V .-dose totalling the pre-and post-irradiation dose . This argument is based on the assumption that the splitting of the U .V .-dose and the irradiation with photoreactivating light before U .V .-irradiation has no effect on the surviving fraction of the two strains used . This has been shown to be the case in control experiments . R.B .
2 N
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518
Photoreactivation o f Saccharomyces
A further assumption is the linear increase of non-photoreactivable lesions with increasing UN .-dose . It is supported by the following results : The photoreactivable lesions increase linearly with increasing U .V .-dose (Waters and Moustacchi 1975), and the inactivation curves of the strains used exhibit a linear increase of lethal lesions in the dose-range . It is evident from figure 5 that at all U .V .-doses tested the maximum photoreactivation achieved in strain MB1030-1A is smaller than that in strain S24-12c . This indicates that MB1030-1A has less dark-repair capacity of non-photoreactivable lesions than S24-12c . The fact that the difference between the dose decrement of these two strains disappears after correction for the effects of non-photoreactivable lesions is taken as evidence that the two strains possess the same number of PRE molecules per haploid cell, i .e . 272 ± 27 . This is rather high compared with the corresponding value obtained by Harm et al . (1968) for E . coli, which is about 20 . The different cell-.ize of E . coli and S . cerevisiae must be taken into account when these data are compared . ACKNOWLEDGMENTS Financial support by the Deutsche Forschungsgemeinschaft is acknowledged . La formation d'un complexe entre 1'enzyme photoreactivant (PRE) et le substrat apres une U .V . -irradiation a ete recherchee pour deux mutants de Saccharomyces, qui sont haploides et sensibles a 1'irradiation. En utilisant des flashs le temps necessaire a une formation maximale de complexe a pu titre determine . Apres 1 min, 70 pour cent de complexes sont formes . Pour la determination du nombre de molecules d'enzyme photoreactivant par cellule, la reduction maximale de la dose produite par un flash a ete recherchee et l'influence de lesions non-photoreactivables eliminee par correction . La reduction maximale de la dose apres cette correction etait la meme pour les deux souches (8,5 erg mm-2) . Le nombre de molecules de l'enzyme photoreactivant qui produisent la photoreparation de lesions dans 1'ADN nucleaire a ete calcule a 272 ± 27 . Bei zwei haploiden, strahlensensiblen Saccharomyces Mutanten wurde die Komplexbildung zwischen Photoreaktivierungsenzymen and Substrat nach U .V . Bestrahlung untersucht . Durch Anwendung von Lichtblitzen konnte die zur maximalen Komplexbildung notwendige Zeit bestimmt werden . Nach 1 Min sind 70 Prozent der Komplexe gebildet . Zur Bestimmung der Anzahl der Photoreaktivierungsenzymmolekule pro Zelle wurde die durch einen Lichtblitz zu erzielende maximale Dosisverringerung ermittelt and die Wirkung nicht photoreaktivierbarer Schaden durch Korrektur ausgeschaltet . Die korrigierte maximale Dosisverringerung war fur beide Stamme gleich (8,5 erg mm-2) . Die Anzahl der Photoreaktiverungsenzymmolekule, die die Photoreparatur nuklearer DNS-Schaden bewirken, wurde zu 272 ± 27 bestimmt . REFERENCES FATH, W . W ., and BRENDEL, M ., 1975, Z. Naturf. (in the press) . GAME, J . C ., and Cox, B . S ., 1971, Mutation Res ., 12, 328 . GAME, J . C ., and MORTIMER, R . L., 1974, Mutation Res ., 24, 281 . HARM, W ., HARM, H ., and RuPERT, C . S ., 1968, Mutation Res., 6, 371 . HARM, W., RuPERT, C . S ., and HARM, H ., 1971, Photophysiology, Vol . VI, edited by A . C . Giese (New York, London : Academic Press), pp. 279-324 . RUPERT, C . S ., 1964, Photophysiology, Vol . II, edited by A . C . Giese (New York, London : Academic Press), pp. 283-327 . SCHAARSCHMIDT, B ., 1970, Z . Naturf. 25 b, 330 . UNRAU, P ., WHEATCROFT, R ., and Cox, B . S ., 1971, Molec . gen . Genetics, 113, 359 . UNRAU, P., WHEATCROFT, R ., Cox, B . S ., OLIVE, T., 1973, Biochim . biophys. Acta, 312, 626 . WATERS, R ., and MOUSTACCHI, E ., 1975, J . Bact ., 121, 901 .
1975,
VOL .
28,
NO .
6, 5 1 1 -518
Determination of the number of photoreactivating enzyme molecules per haploid Saccharomyces cell AKIRA YASUI and WOLFGANG LASKOWSKI Zentralinstitut fur Biochemie and Biophysik, der Freien Universitat Berlin, Berlin, Germany
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(Received 8 July 1975 ; accepted 9 October 1975)
Two haploid radiation-sensitive mutants of Saccharomyces were studied to investigate the formation of complex between photoreactivating-enzyme and substrate after ultra-violet irradiation . Using photo-flashes, the time necessary for maximum complex formation has been determined . Within 1 min, 70 per cent of the complexes have been formed . To determine the number of photoreactivating enzyme molecules per cell, the maximum dose decrement obtained after one photo-flash was determined and corrected for the effects of non-photoreactivable lesions . The corrected maximum dose decrement was found to be identical for both strains (8 . 5 erg mm -2) . The number of photoreactivating-enzyme molecules involved in the photorepair of nuclear DNA damage was calculated as 272 ± 27 .
1 . Introduction Photoreactivating-enzyme (PRE) repairs pyrimidine dimers that have been formed in DNA irradiated with ultra-violet light (U .V .) according to the following reaction scheme (Rupert 1964) k3 kl E+S . ES > E+P, light k2 where E is the PRE, S the pyrimidine dimer (substrate), ES the PRE-substrate complex and P the repaired product . Light flash experiments have recently been proved to be very useful in the study of photoenzymatic repair (Harm, and Rupert 1968, Harm, Rupert and Harm 1971) . After a short intense flash, only those substrates can be repaired that are complexed with PRE at the moment of the flash . The aim of this paper is to apply this technique to yeast cells in an investigation of the time-dependence of the formation of PRE-substrate complexes and to determine the number of PRE molecules per cell . The method is based on the fact that, as in E . coli (Harm et al . 1971), in yeast (Waters and Moustacchi 1975) pyrimidine dimers are induced proportionally to the U .V .-dose, the numbers of pyrimidine dimers induced per genome per erg mm -2 U .V .-light is known (Unrau, Wheatcroft, Cox and Oliver 1973, Fath and Brendel 1975) . The effects of non-photoreparable damage, which were neglected by the calculation of the number of PRE per E. coli cell (Harm et al. 1971), are taken into account in this paper . Suitable radiation-sensitive mutants of Saccharomyces were used, in which the ratio between PRE molecules and pyrimidine dimers at any measurable survival level is large enough to produce a recognizable flash effect and small enough to see the maximum complex.
512
A . Yasui and W. Laskowski
Materials and methods The following haploid Saccharomyces strains were used : MB 1030-1A (a, radl-18, rad2-17, radl8-2), S 24-12c (a, rad2-20) . The symbols in brackets indicate mating type alleles and mutated genes causing radiation sensitivity (Game and Cox 1971) . radl and rad2 mutants are known to block the excision-repair mechanism (Unrau et al . 1971), whereas radl8 blocks another not clearly identified dark-repair mechanism (Game and Mortimer 1974) . Strain MB1030-1A was obtained from M . Brendel . Strain S24-12c from D . Averbeck .
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2.
2.1 . Medium Yeast extract peptone medium (YEP) containing 1 per cent yeast extract (Difco), 0 . 5 per cent peptone (Merck), 2 per cent glucose and 2 per cent agar (Merck), was used throughout . 2.2 . Growth conditions and cell preparation The strains were stored on YEP agar slants at 4 ° C . Cell samples were inoculated on fresh YEP agar slants and incubated for 48 hours at 30°C . The cells were then suspended in buffer (0 . 05 M KH2PO 4 i pH 4 . 8), washed three times in buffer, and resuspended in buffer for further treatment . After irradiation and photoreactivation, appropriate cell-samples were plated on YEP medium and incubated for 72 hours at 30°C . 2.3 . U. V .-irradiation The U .V .-source was a low-pressure mercury lamp (Osram HNS 12) with a maximum emission at 254 nm wave-length . The dose rate was 3 erg sec -I mm -2 , measured by a photon flux counter (Schaarschmidt 1970) . 2.4 . Photoreactivation (Phl?) For photoreactivating light flashes, an electronic flash unit (Rollei E 36 RE) was used, delivering a light flash of 1/800 sec duration . This light flash was filtered by a Jenaer Filter Glas WG 320, transmitting only wave-lengths longer than 320 nm . The U .V .-irradiated cell suspension, placed in a small glass dish, was positioned 5 cm from the surface of the flash unit and exposed to one light flash . More photons reached the cells when the distance was less than 5 cm (as measured with a photodiode), but the amount of photoreactivation did not increase . Maximum photoreactivation was obtained by continuous illumination with photoreactivating light of an Osram-L-20W-70 lamp . 2.5 . Dose decrement The difference in the U .V .-doses applied with and without subsequent photoreactivation, which leads to the same survival level, is called the dose decrement (Harm et al. 1971) . 2.6 . Experimental conditions All experiments were performed at room temperature of approximately 22°C under yellow light to prevent uncontrolled PhR . The results depicted in the figures are average values obtained from three to five independent identical experiments .
Photoreactivation o f Saccharomyces
513
The cell concentration during irradiation and photoreactivation was 5 x 10 6 cells per ml buffer . Before plating, the cell suspensions were diluted appropriately to obtain from 100 to 400 colonies per plate .
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3.
Results Figure 1 shows the survival of the strain MB1030-1A after U .V .-irradiation (left curve) and the results of PhR by single light flashes at various times after U .V .-irradiation (right curves) . These data show that by 1 min after U .V .-irradiation, 70 per cent of the maximum survival can be obtained by one flash, whereas about 20 min are necessary to achieve maximum survival . The same kind of experiment have been performed with the less-U .V .-sensitive haploid strain S24-12c, which carries only the mutated rad2 gene . The results were similar to those described above . This time interval is considered to be necessary for maximum complex formation of PRE molecules and substrate UV-dose (erg/mm 2 ) 5 10 15 20 10°
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•
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Figure 1 . Survival of strain MB1030-1A after U .V .-irradiation (left curve), and after U .V .-irradiation plus application of a single light-flash given at various times after U .V .-irradiation (right curves) .
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(Harm et al. 1971) . These results were considered in the procedure of the following experiments, in which flash photoreactivation was applied 40 min after U . V .-irradiation . Figure 2 depicts the survival curve after U .V .-irradiation and U .V .-irradiation plus photoflash 40 min after U .V .-irradiation for strain MB1030-1A (left curves) and the less-sensitive strain S24-12c (right curves) . UV- dose (erg/mm2 )
10 20 3,0 40 590 6,0
100
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Figure 2 . Survival after U .V .-irradiation (closed symbols) and U.V .-irradiation plus single photo-flash (open symbols) of MB1030-1A (A A) and S 24-12c ( • 0) . To determine the number of PRE molecules per cell, the U .V .-dosedependence of the dose decrement by a single light flash at the time of maximum complex formation was investigated for both strains . Dark repair between U .V .-irradiation and PhR may be neglected, because of the short time-interval . The maximum dose decrement obtained for strain MB1030-1A was 6 erg mm -2 , whereas that obtained for S24-12c was 7 . 2 erg mm-2 (figure 3) . This difference may either indicate a different number of PRE molecules per cell in the two strains or it may be produced by a different capacity to remove non-photoreactivable lesions in the two strains . It was therefore necessary to test whether the non-photoreactivable lesions affect determination of the dose decrement .
Photoreactivation o f
Saccharomyces
5 15
The increase in photoreactivable and non-photoreactivable lesions with increasing U .V .-dose is shown schematically in figure 4 . The number of photoreactivable lesions per cell after U .V .-irradiation is given by N 1 . After application of a single photo-flash, Nl decreases to N2. The dose decrement U2- U, can be determined from the survival curve if the application of U2 and 0
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0
10
2b
30
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UV- dose (erq/mm 2 )
Figure 3 .
Dose decrement of photoreactivation by a single photo-flash 40 min after U .V .-irradiation . MB1030-1A 0 . S24-12c o .
Figure 4 . Schematic representation of the amounts of photoreactivable and nonphotoreactivable lesions and the effect of photoreactivation (see text for further explanation) .
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A . Yasui and W. Laskowski
one photo-flash results in the same survival frequency as the application of U, without photo-flash . Owing to a possible increase in non-photoreactivable lesions with increasing U .V .-dose, however, a different number of non-photoreactivable lesions per cell, indicated by N3 and N4 , can be produced . The survival frequency after application of the U .V.-dose U2 plus photo-flash may therefore not equal that after application of U, without photo-flash . Based on the assumption that the number of non-photoreactivable lesions increases linearly with increasing U .V .-dose, a measure for the real dose decrement can be deduced . It is evident from figure 4 that, if the cells are irradiated with a U .V .-dose U2-U,, maximally photoreactivated and thereafter U .V .-irradiated with U1 , the survival frequency should be the same as that obtained after application of the U .V .-dose U2 and one photo-flash . Figure 5 depicts the effect of U .V . -irradiation and additional maximum photoreactivation applied continuously for 30 min . After the maximum photoreactivation, cells were irradiated again with a U .V .-dose U,-U (postirradiation) . By changing the dose U, that dose was determined, which results in survival frequencies coinciding with the survival frequency by a single flash photoreactivation after U .V .-irradiation with U2 . According to figure 4, this dose should correspond to the dose decrement U2 U1 at the dose U2 . 10
0
10 1
10
2
Figure 5 . Survival after U .V .-irradiation (closed symbols) and U .V.-irradiation plus maximum photoreactivation (open symbols) of MB1030-1A (A ,) and S 24-12c ( • 0) . Figure 6 shows the corrected dose decrement (upper curves) compared with the uncorrected dose decrement depicted in figure 3 . After this correction, the two strains show the same maximum dose decrement of 8 . 5 erg mm -2 . The corrected maximum dose decrement does not increase, although the substrate concentration increases with increasing U .V .-dose . The numbers of PREsubstrate complexes should therefore be equal to the number of PRE molecules in the corresponding cells. The number of PRE molecules per cell involved with the photorepair of nuclear DNA can be calculated from the corrected dose
Photoreactivation o f Saccharomyces
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Figure 6 .
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UV-dose (erg/mm 2)
Uncorrected (square) and corrected (star) dose decrements for MB1030-1A (left side) and S 24-12c (right side) .
decrement if the number of pyrimidine dimers produced per erg mm -2 is known . Unrau et al . (1973) have found 24 dimers per haploid genome per erg MM -2 in U .V .-irradiated Saccharomyces cells using 14 C marking followed by purification of DNA from RNA . Fath and Brendel (1975) found 32 ± 3 dimers per haploid genome and erg mm-2 in recently isolated Saccharomyces mutants auxotrophic for deoxythymidinemonophosphate . Using the latter value and the corrected maximum dose decrement of 8 . 5 erg mm -2, we determined the number of PRE molecules per haploid cell involved with the photorepair of nuclear DNA to be 272 + 27 . 4 . Discussion The results in figure 1 were interpreted as indicating that approximately 70 per cent of maximum complexes have been formed 1 min after U .V .-irradiation, and the remaining 30 per cent require about 20 min for formation . The time needed to obtain maximum PRE-substrate complex formation is two to three times longer in Saccharomyces than in E . coli (Harm et al . 1968) . This may indicate a wide distribution of PRE molecules in the cell . The dose decrement obtained with one photo-flash was corrected for the effects of non-photoreactivable lesions . To achieve the same effect as with photoreactivation by one photo-flash after U .V .-irradiation, cells were first pre-irradiated with U .V ., closely followed by maximum photoreactivation and then U .V .-post-irradiated . The U .V .-doses for pre-irradiation and postirradiation were varied, so that the total U .V .-dose remained constant . The corrected dose decrement eliminating the effect of non-photoreactivable lesions was determined from that pre-irradiation U .V .-dose, which results in a surviving fraction after maximum photoreactivation followed by immediate postirradiation, that coincides with the surviving fraction after photoreactivation with one photo-flash after application of a U .V .-dose totalling the pre-and post-irradiation dose . This argument is based on the assumption that the splitting of the U .V .-dose and the irradiation with photoreactivating light before U .V .-irradiation has no effect on the surviving fraction of the two strains used . This has been shown to be the case in control experiments . R.B .
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Photoreactivation o f Saccharomyces
A further assumption is the linear increase of non-photoreactivable lesions with increasing UN .-dose . It is supported by the following results : The photoreactivable lesions increase linearly with increasing U .V .-dose (Waters and Moustacchi 1975), and the inactivation curves of the strains used exhibit a linear increase of lethal lesions in the dose-range . It is evident from figure 5 that at all U .V .-doses tested the maximum photoreactivation achieved in strain MB1030-1A is smaller than that in strain S24-12c . This indicates that MB1030-1A has less dark-repair capacity of non-photoreactivable lesions than S24-12c . The fact that the difference between the dose decrement of these two strains disappears after correction for the effects of non-photoreactivable lesions is taken as evidence that the two strains possess the same number of PRE molecules per haploid cell, i .e . 272 ± 27 . This is rather high compared with the corresponding value obtained by Harm et al . (1968) for E . coli, which is about 20 . The different cell-.ize of E . coli and S . cerevisiae must be taken into account when these data are compared . ACKNOWLEDGMENTS Financial support by the Deutsche Forschungsgemeinschaft is acknowledged . La formation d'un complexe entre 1'enzyme photoreactivant (PRE) et le substrat apres une U .V . -irradiation a ete recherchee pour deux mutants de Saccharomyces, qui sont haploides et sensibles a 1'irradiation. En utilisant des flashs le temps necessaire a une formation maximale de complexe a pu titre determine . Apres 1 min, 70 pour cent de complexes sont formes . Pour la determination du nombre de molecules d'enzyme photoreactivant par cellule, la reduction maximale de la dose produite par un flash a ete recherchee et l'influence de lesions non-photoreactivables eliminee par correction . La reduction maximale de la dose apres cette correction etait la meme pour les deux souches (8,5 erg mm-2) . Le nombre de molecules de l'enzyme photoreactivant qui produisent la photoreparation de lesions dans 1'ADN nucleaire a ete calcule a 272 ± 27 . Bei zwei haploiden, strahlensensiblen Saccharomyces Mutanten wurde die Komplexbildung zwischen Photoreaktivierungsenzymen and Substrat nach U .V . Bestrahlung untersucht . Durch Anwendung von Lichtblitzen konnte die zur maximalen Komplexbildung notwendige Zeit bestimmt werden . Nach 1 Min sind 70 Prozent der Komplexe gebildet . Zur Bestimmung der Anzahl der Photoreaktivierungsenzymmolekule pro Zelle wurde die durch einen Lichtblitz zu erzielende maximale Dosisverringerung ermittelt and die Wirkung nicht photoreaktivierbarer Schaden durch Korrektur ausgeschaltet . Die korrigierte maximale Dosisverringerung war fur beide Stamme gleich (8,5 erg mm-2) . Die Anzahl der Photoreaktiverungsenzymmolekule, die die Photoreparatur nuklearer DNS-Schaden bewirken, wurde zu 272 ± 27 bestimmt . REFERENCES FATH, W . W ., and BRENDEL, M ., 1975, Z. Naturf. (in the press) . GAME, J . C ., and Cox, B . S ., 1971, Mutation Res ., 12, 328 . GAME, J . C ., and MORTIMER, R . L., 1974, Mutation Res ., 24, 281 . HARM, W ., HARM, H ., and RuPERT, C . S ., 1968, Mutation Res., 6, 371 . HARM, W., RuPERT, C . S ., and HARM, H ., 1971, Photophysiology, Vol . VI, edited by A . C . Giese (New York, London : Academic Press), pp. 279-324 . RUPERT, C . S ., 1964, Photophysiology, Vol . II, edited by A . C . Giese (New York, London : Academic Press), pp. 283-327 . SCHAARSCHMIDT, B ., 1970, Z . Naturf. 25 b, 330 . UNRAU, P ., WHEATCROFT, R ., and Cox, B . S ., 1971, Molec . gen . Genetics, 113, 359 . UNRAU, P., WHEATCROFT, R ., Cox, B . S ., OLIVE, T., 1973, Biochim . biophys. Acta, 312, 626 . WATERS, R ., and MOUSTACCHI, E ., 1975, J . Bact ., 121, 901 .
