Photochemistry and Photobiology Vol. 40, No. 2, pp. 221 - 230, 1984 Printed in Great Britain. All rights reserved

0031-8655184 $03.(H)+O.O() Copyright @ 1984 Pergamon Prcas Ltd

MODIFYING FACTORS OF THE CELLULAR CONCENTRATION OF PHOTOLYASE MOLECULES IN

Saccharomyces cerevisiae-11. EFFECTS OF PREILLUMINATION WITH LIGHT FLASHES

ATSUSHI FUKUI and WOLFGANG LASKOWSKI* Institute of Biophysics, Free University Berlin D-1000 Berlin 33, W. Germany

(Received 21 September 1983; accepted 7 February 1984)

Abstract-The effects of preillumination with photoreactivating light flashes before UV-irradiation on the number of photoreactivable complexes consisting of UV-induced DNA-damages and active photolyase molecules (NpREac1), on the fluence decrements, ADPRE, that are obtained from two UV-survival curves without and with 1 flash photoreactivation and proportional to NpREacr were determined in haploid Saccharomyces cerevisiae cells. ADPRE increased by preillurnination from 0.1 15 J m-’ to 0.460 Jrn-‘ and from 0.376 Jm-’ to 0.494 Jm-* in cells in logarithmic growth phase and in stationary growth phase, respectively. ADPRE in log-cells that were preilluminated before and after resuspension in buffer at 40°C for 60 min was larger than ADPRE in log-cells preilluminated only after resuspension in buffer.

INTRODUCTION

For investigation of the photoreactivation (PR)t process on cell survival an effective method is the light flash analysis used by Harm et al. (1971). For yeast cells, the number of photoreactivating enzyme molecules per cell (NPRE) has been determined by flash analysis in cells of stationary growth phase (stat-cells) (Yasui and Laskowski, 1975). It was also shown by this method that NpRE for cells in logarithmic growth phase (log-cells) is smaller than that for stat-cells(Fukui etal . , 1978). The latter result agreed with results of in vitro experiments using Hoernophilus injluenzae transforming DNA and PRE extract from yeast cells (Boling and Setlow, 1967). In addition Harm and Rupert (1976) have reported that the thermal stability and the number of active PRE molecules present in extracts from yeast cells increased by illumination with near-UV or short wavelength of visible light. The illumination effects may be due to direct photochemical alteration within PRE molecules changing those with low activity in the light-dependent step of the reaction to a higher activity, that is, alteration of “inactive” PRE to “active” PRE (Harm and Rupert, 1976). Since N p R E

determined by using a light flash equals the number of photoreactivable complexes consisting of pyrimidine dimers and active photoreactivating enzyme (PRE) molecules (NPREact), NpREact should increase by preillumination of cells before UV-

“To whom correspondence should be addressed. +Abbreviations: ADpRB, fluence decrement; log-cells,

cells in logarithmic growth phase; N,,,, number of PRE molecules per cell; NPREacf, number of active PRE molecules per cell; NPREinacI, number of inactive PRE molecules per cell; PR, photoreactivation; PRE, photo- reactivating enzyme; stat-cells, cells in stationary growth phase.

irradiation, when the increase in the number of active PRE molecules shown in vitro by Harm and Rupert (1976) occurs also in vivo.

In a previous paper (Fukui and Laskowski, 1984), it was shown that NPREact was affected by light- illumination during cell culture and resuspension in buffer and also by temperature during cell culture. It was proposed that a portion of the variation of NpREact in cells illuminated with light was due to the activation of inactive PRE molecules by light illumi- nation. In order to know the contribution of the activation of inactive PRE molecules to NPREact in illuminated cells, the increase of N ~ R E ~ ~ ~ by preillum- ination with photoreactivating light must be deter- mined in stat-cells grown in the dark. In the present work, the effect of preillumination on NpREitct in stat-cells as well as log-cells was investigated. The temperature stability of the preillumination effect on N P ~ p a c t as well as the conditions necessary for an increase of N ~ R E ~ ~ ~ were checked. By this means, information about the effects of preillumination and temperature on PR ability of cells was obtained.

MATERIALS AND METHODS

Sfrains. The haploid strain XS 774-6A (a , radl-I) of Saccharomyces cerevisiae (Nakai and Matsumoto, 1967) which is excision repair-defective (Unrau er a[ . , 1971) and an isolated photoreactivationless mutant (phrl) of this strain were used.

Cells were cultivated by shaking at 30°C in the dark in 20 me liquid growth medium (pH 5 ) containing 0.5% Difco yeast extract, 0.3% Difco bacto- peptone and 1% glucose. Stationary-phase cells (stat-cells) were harvested after 3 days of incubation. The cell suspension obtained was diluted about lo3 times in 20 mZ fresh medium, and after 6 7 h of incubation, logarithmic growing cells (log-cells) were harvested. During this incubation time cells divided 3-4 times. Harvested cells were washed and resuspended in phosphate buffer (0.05 M KH2P0,, pH 4.8) with a concentration of 0.5-1 x 10‘ cells per mP.

Cell preparation.

221

228 ATSUSHI FUKUI and WOLFGANG LASKOWSKI

Illumination. For preillumination (illumination before UV-irradiation) repeated light flashes applied in intervals of 10 s produced by a simultaneous discharge of 2 strobos (National PE 563, Japan) were used. The time interval between the last preillumination flash and PR with 1 light flash after UV-irradiation was shorter than 1 h except in buffer holding experiments. The conditions of UV- irradiation were described by Fukui and Laskowski (1984).

Determination of the number of active photoreactivating enzyme molecules per cell (NpREact). In order to deter- mine NpREact UV-irradiated cells were photoreactivated with a single light flash after keeping them for more than 15 rnin in the dark for maximum complex formation. The light flash was produced from the two strobos mentioned above and intense enough to photolyze all the complexes present (Fukui et al., 1978).

The NpREact was calculated using a ADpRE, where a is the number of pyrimidine dimers produced by a unit fluence of UV in nuclear DNA and assumed to be 240 (pyrimidine dimers)/(J m-2) per haploid cell (Unrau ef al., 1973) and AD,,, is the fluence decrement defined by Harm et al. (1968).

Cells were generally manipulated in the dark or under a red fluor- escent lamp (Philips TL 40W/15, FRG) to exclude any uncontrolled PR and to minimize uncontrolled activation of inactive PRE molecules during the experiments. The treated cells were plated on complete agar medium. After 4 days of incubation at 30°C in the dark, colonies were counted.

Cell manipulation during the experiment.

RESULTS

Increase of the jlwnce akcremnt by pre-illumination

Figure 1 shows the UV-survival curves without P R or with PR in stat-cells (a-1 and a-2) or in log-cells (b-1 and b-2) in strain XS 774-6A. The cells either not preilluminated (a-1 , b-1) or after preillumination with 20 flashes applied in intervals of 10 s (a-2, b-2) were irradiated with UV, and then photoreactivated with a single light flash, in order to determine NpREact. Preillumination with 20 light flashes had n o effect on cell survival. In log-cells the survival fraction of UV-irradiated cells without pre- illumination increased slightly after 1-flash PR (b-1). However, when the cells were preilluminated, a large increase of the survival fraction with 1-flash PR was observed (b-2).

Figure 2 shows the variations of NpREact as function of the number of flashes for preillumination in stat-cells (a) or log-cells (b) of strain XS 774-6A. The NpREact in cells of both growth phases was saturated after more than 10-flashes preillumination. In log-cells not preilluminated, the average values of A D p R E obtained in 14 independent experiments and the standard deviation was 0.115 k 0.034 J m-2 and increased to 0.460 k 0.061 J m-’ after pre- illumination with 20 light flashes. The NpREact

calculated using (Y ADpRE was 28 k 8 without preillumination and 110 k 15 after preillumination. In stat-cells, the average A D p R E obtained in 4 independent experiments and the standard deviation Was 0.376 f 0.034 J m-’ (NpREact = 90 f 8)

without preillumination and 0.494 f 0.036 J m-2 ( N P R E ~ ~ ~ = 119 f 9) after preillumination.

In order to determine whether preillumination may have any effect in a strain without active photolyase molecules a photoreactivationless mutant (phr 1) of strain XS 774-6A was isolated, and treated in the same way as the strain used in Fig. 1. The UV-survival curve remained unchanged in spite of preillumination and photoreactivation.

Variation of buffer at 40°C

Log-cells and stat-cells of strain XS 774-6A were resuspended in buffer for 30 or 60 min at 40°C before UV-irradiation (buffer holding) and then was determined from a A D p R E . Buffer holding for 60 min before UV-irradiation did not affect the survival curve. Preillumination by 20 flashes was performed before andlor after buffer holding.

Figure 3 shows NpREact vs time of buffer holding depending on preillumination. When log-cells were preilluminated neither before nor after buffer holding, only a small decrease of NpREact occurred during 60 min buffer holding. The remarkable increase of NpREacr evident after preillumination decreased after buffer holding for 30 and 60 min to about 55 and 40% respectively. In cells, however, preilluminated before and after buffer holding NpREact decreased after 60 rnin buffer holding only to about 75%. This is approximately the same per- centage of decrease in NpREact as obtained in stat-cells neither preilluminated before nor after buffer holding.

by resuspension of cells in

DISCUSSION

The experiments reported show that the fluence decrements A D p R E obtained by 1 PR flash become larger when the cells have been preilluminated before UV-irradiation.

For an explanation of the increase in A D p R E after preillumination three assumptions shall be discussed.

(1) There may exist two classes of P R E molecules, one of which becomes active after preillumination. The latter class shall be called “inactive” PRE molecules. These inactive P R E molecules may represent either an inactive condition (precursor) of the active P R E molecules or a second type of PRE molecules that never become active without pre- i l luminat ion. T h e results showing tha t pre- illumination does not produce active P R E molecules in a phr 1-mutant exclude the latter assumption.

(2) The number of active PRE molecules may be constant before and after preillumination but the number of photoreactivable complexes increases by preillumination, for example by an alteration of the accessability of DNA in the chromosomes. The validity of this assumption may be checked by the data presented in Fig. 1. As can be seen in Fig. 1

Factors modifying photolyase concentration I1 229

Figure 1. Survival curves in UV-irradiated yeast cells of stationary or logarithmic growth phase without PR (0) or after PR with 1 flash (0). (a-1) stat-cells without preillumination; (a-2) stat-cells after preillumination with 20 light flashes; (b-1) log-cells without preillumination; (b-2) log-cells after

preillumination with 20 light flashes.

150 I t

" 0 10 20-30 40 0 10 20 30 40

NUMBER OF LIGHT FLASHES

Figure 2. Variation in NPREact as function of the number of light flashes for preillumination. (a) stat-cells: (b) log-cells.

(b-l), the fluence decrement obtained from survival curves with and without 1 flash PR is constant, when the cells not preilluminated were irradiated with a fluence from 3.0-5.3 J m-2. This shows that maximal complex formation has occurred. If, however, in cells not preilluminated only a certain proportion of pyrimidine dimers is accessible to PRE molecules, this proportion certainly increases with increasing UV fluence resulting in an increasing formation of photoreactivable complexes and con- sequently in an increasing fluence decrement. The constant fluence decrement obtained therefore disproves this assumption.

(3) The increase in A D p R E may be caused by an induction of the synthesis of active PRE molecules by preillumination. This assumption cannot be excluded strictly but seems rather unlikely, because cells irradiated with UV immediately after preillumin- ation (20 flashes every 10 s) and then photo- reactivated with a single light flash after keeping them for 15 min in the dark for maximum complex formation (that means altogether for 25 min from the beginning of preillumination) showed the same increase of NpREact as cells kept at 30°C for 180 min after preillumination and then UV-irradiated.

Summing up, the assumption that there exist PRE molecules in yeast cells that may occur in an active condition as well as in an inactive condition becoming activated after preillumination seems to be the most likely. It is in accord with the interpretation of Harm and Rupert (1976) about an activation of inactive PRE molecules by preillumination.

From this point of view the reduction of N ~ R E ~ ~ ~ in log-cells, preilluminated only after buffer holding (Fig. 3) can be explained by a loss of the ability for activation by preillumination of inactive PRE molecules. Inactive activable molecules alter to not activable PRE molecules. The rate of this alteration is smaller in log-cells preilluminated before buffer

Figure 3. Variation of NPREsct with or without pre- illumination as function of time of buffer holding at 40°C before UV-irradiation. Log-cells were not preilluminated (O), preilluminated only after buffer holding (0) or preilluminated before and after buffer holding (A). Stat-cells were preilluminated neither before nor after buffer holding (U). Average values obtained in 2-4

independent experiments.

230 ATSUSHI FUKUI and WOLFGANG LASKOWSKI

holding than in log-cells not preilluminated before buffer holding (Fig. 3). This preillumination effect agrees with the thermal stability-effect of pre- illumination on PR activity of active PRE molecules reported by Harm and Rupert (1976).

In the previous paper the effect of continuous light-illumination on N p ~ E was investigated (Fukui, Laskowski, 1984). The N ~ R E ~ ~ ~ of stat-cells grown in the dark at 30°C was about 90. A n increase to about 119, is produced by flash preillumination as shown in the present paper. A preillumination of 24 h during buffer holding at 30°C lead to a further increase of NPKL;acr to about 141. The highest value of N ~ R E was obtained after incubation under continuous light- illumination (NpREact = 247). About half of the increase of NpREact in stat-cells after buffer holding in light may be due to an activation of inactive PRE molecules. Most of the increase of NpREilct in stat-cells grown in light, however, is contributed by an effect of illumination on cells progressing from logarithmic to stationary growth phase.

In the previous paper (Fukui and Laskowski, 1984) it has also been shown that &REact in stat-cells grown in the dark at 23, 30 or 37°C is 229, 92 and 31, respectively. This difference of NpREact in stat-cells grown a t various temperatures can hardly be explained solely by thermodynamic inactivation of active P R E molecules. At 37°C the low NpREact in stat-cells may be caused by fast thermodynamic alterations of inactive PRE molecules, assumed to be the precursors of active P R E molecules. The difference, however, between NpREact in stat-cells grown at 23 or 30°C, cannot be due to an alteration of inactive PRE molecules during cell multiplication, because the number of active plus inactive PRE molecules per cell, NpREact plus NpREinact, in log- cells after buffer holding at 30°C in the dark for 24 his

about the same as that before buffer holding, indicating that inactive P R E molecules are not altered to a not activable condition at 30°C (unpublished data). To explain these data an additional assumption is necessary, postulating that the number of inactive and/or active PRE molecules is determined by an enzyme controlled action depending on tempera ture . In spi te of the uncertainties mentioned, the determination of NpREinact in log- and stat-cells may be important for an investigation of the effects of temperature on N P R E ~ ~ ~ .

Acknowledgements-We wish to express out gratitude to Professor Dr. Matsudaira and Dr. Hieda for borrowing the flash device.

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