Photochemistry and Photobiology Vol. 39. No. 5 , pp. 613-617, 1984. Printed in Grcat Britain. All rightsreserved

0031-8655/84S03.00+0.00 Copyright 0 1984 Pergarnon PressLtd

MODIFYING FACTORS OF THE CELLULAR CONCENTRATION OF

PHOTOLYASE MOLECULES IN Saccharomyces cerevisiae CELLS-I. EFFECTS OF TEMPERATURE AND LIGHT

ATsusnI Fuwi and WOLFGANG LASICOWSKI* Institute of Biophysics, Free University Berlin, D-1000 Berlin 33, W. Germany

(Received 13 September 1983; accepted 13 December 1983)

Abstract-The effects of temperature and light on the cellular concentration of photoreactivating enzyme (PRE) molecules in haploid Saccharomyces cerevbiae cells were investigated. (1) Temperature effect: The number of active PRE molecules per cell (NpRE) in cells grown at 37°C was about 13% of that grown at 23°C. although the amount of proteins per cell remained the same. (2) Light effect: NpRE in cells grown in light was about 2.8 times larger than that grown in the dark. The value of NpRE in cells grown in the light decreased more rapidly during holding in buffer in the dark than in the light. The NpRE decrease during holding in buffer in the dark was more rapid in cells grown in the light than grown in the dark. A comparable decrease was observed after holding in buffer in the presence of cycloheximide. (3) In cells harboring a plasmid containing the gene PHRl, N ~ R E was larger in cells grown at 23 than at 30°C.

INTRODUCTION

Deoxyribodipyrimidine photolyase, or photo- reactivating enzyme (PRE)? molecules bind to UV-induced pyrimidine dimers in DNA forming PRE-pyrimidine dimer complexes. The pyrimidine dimers a re monomerized by illumination with n e a r - U V or shor t -wavelength vis ible l ight (photoreactivation light, PRL) (Rupert, 1964). In this process, PRL is the energy source for splitting pyrimidine dimers. In addition, it was shown in vitro, using PRE of yeast, that PRL affects directly photoreactivation (PR) activity of PRE molecules. A preillumination of PRE molecules in vitro causes an increase in PR activity as well as an increase in PRE stability t o thermal inactivation (Harm and Rupert, 1970,1976; Harm, 1979). This effect is considered to be due to direct photochemical alterations within the PRE molecules (Harm and Rupert, 1976). It indicates that PRL is a factor modifying the concen- tration of active PRE molecules. Besides, tem- perature has also been shown to be a factor modifying the activity of PRE molecules in vitro (Harm and Rupert, 1976).

vivo, whether temperature and light illumination during cell growth affect the cellular concentration of photolyase molecules in yeast. In the present work, the number of active PRE molecules per cell (NPRE) was determined in Saccharomyces cerevbiae cells grown at various temperatures by using PRL flashes.

To our knowledge, it has not been investigated i n ,

*To whom correspondence should be addressed. tAbbreviations: CHX, cycloheximide; AD, fluence

decrement; ADF, fluence decrement with 1 flash PR; ADpRE. saturated fluence decrement with 1 flash PR; NpRE, number of PRE molecules per cell; PR, photoreactivation; PRE, photoreactivating enzyme; PRL, photoreactivation light.

Cells transformed with a plasmid carrying the gene PHRl were also used. Besides effects of the temperature on NPRE, the effect of light on N ~ R E and t h e s tab i l i ty of PRE m o l e c u l e s w e r e a l s o investigated.

MATERIALS AND METHODS

Srrains. The experiments were performed with the haploid excision-repair defective strain X S 774-6A of Saccharomyces cerevisiae (a , radl-1, PHR1) (Nakai and Matsumoto, 1967) and the repair competent strain GRF18 (a, RAD, PHR1, his3, l e a ) . Cells of GRF18 have been transformed either with plasmid pJDB207 (Beggs, 1979) or with plasmid A8-3 that contains the gene PHRl otherwise identical with plasmid pJDB207 (Yasui and Chevallier, 1983).

Cell preparation. Cells were inoculated in liquid medium (pH 5 ) containing 0.5% Difco yeast extract, 0.3% Difco bactopeptone, 1% glucose and shaken during culture. Cells were incubated at 23°C for 4 or 5 days and at 30 and 37°C for 2 or 3 days to obtain cells in stationary growth phase. The percentage of single cells was between 82 and 92%. Harvested cells were washed and resuspended in 0.05 M KH2P04 buffer (pH 4.8).

Holding of cells in buffer. In order to investigate the stability of PRE molecules in vivo, cells were held in buffer at a concentration of 2-4 X lo6 cells per me before UV-irradiation (buffer holding, Harm 1976).

Light used for illumination. Continuous light during incubation or buffer holding was obtained from a fluorescence lamp [Philips TL 65 W/24 Weiss (B)]. The distance between the lamp and the cell suspension was about 30 cm. The fluence rate of tight was 1.1 x lo3 Ix at the surface, as determined by a photometer (80 XOpto-meter; United Detector Techn., USA).

UV-irradiation and PR with light flashes. For UV-irradiation, cells were resuspended in buffer to a cell concentration of 0.5-1 x 106per me. Ultraviolet-irradiation was performed with a low-pressure Hg lamp (Osram HNS12, FRG). The incident fluence rate was 0.19 J rn-' s-', as determined by a photometer (80 X Opto-meter). The procedures of flash-photoreactivation of UV- irradiated cells have been described by Yasui and Laskowski (1975) and were performed at room temperature (about 23°C).

613

614 ATSUSHI FUKUI and H 'OLFGANG LASKOWSKI

Survival. Treated cells were plated on solid agar medium. After 4 days of incubation at 30°C in the dark colonies were counted.

Method for determination of the number of active photoreactivating enzyme molecules per cell ( NPRE). From two UV-survival curves either without additional photo- reactivation (PR) or with one PR flash the fluence decrement by a single light flash (A DF) defined by Harm el al. (1971) was obtained. A DF is the difference between the UV fluence with photorepair actually applied and the smaller UV fluence, which would give an identical survival without PR. The NpRE was calculated from aADpRE (Harm et al., 1971), where AD~RE is a saturated ADF and a is the number of pyrimidine dimers produced by a unit fluence of UV in the DNA of a cell and determined to be 240 (pyrimidine dimer)/J m-') per haploid yeast cell (Unrau et al., 1973). In this paper, it is assumed for simplicity that photorepairable as well as non-photorepairable lesions are pyrimidine dimers, as Harm et al. (1971) proposed.

Determination of the amount of protein per cell. Harvested cells were washed and resuspended in buffer (0.025 M K2HP04, 0.025 M KH2P04, NaOH: pH 7) containing 1 mM ethylenediaminetetraacetate (EDTA). The cells in the suspension were broken with a homogenizer (Zellhomogenizator Mod. MSK, B. BraunIMelsungen). During this treatment the cell suspension was cooled with liquid carbon dioxide. Thereafter, the concentration of broken cells was microscopically determined with a hemocytometer. The suspension was centrifuged for 30 min at SO00 g and the supernatant was used to determine the amount of protein. All treatments were performed at about 4°C. The amount of protein in the solution was determined by using a method of Lowry et al. (1951). The amount of protein per cell was calculated by using the amount of protein in the solution and the concentration of broken cells.

RESULTS

(a) NpRE in cells grown at various temperatures

Cells of strain XS 774-6A were incubated in liquid medium in the dark at 23 or 30 or 37°C until stationary growth phase was achieved. In Fig. 1

I

30 0

200

100

x 16'2

0' ' I 10 23 30 37

temperatvre f°C)

Figure 2. Dependences of NpRE (0) and amounts of protein per cell ( A ) on temperature (23, 30 and 37°C)

during cultivation in the dark (XS 774-6A).

examples of UV-survival curves without additional PR and with one PR flash after UV-irradiation are shown. The values of NpRE in cells grown in the dark at various temperatures are plotted in Fig. 2 and shown in detail in Table 1. N ~ R E in cells grown in the dark at 37°C was 30.5 and 0.55 of NpRE at 30°C (92.1) and 0.13 of N ~ R E at 23°C (229).

The amounts of protein in cells grown at various t e m p e r a t u r e s (23, 30 a n d 37°C) were a l so determined. These amounts are plotted in Fig. 2 and shown in Table 1. As shown in Fig. 2 and Table 1, the decrease of N p R E in cells grown a t 37°C is independent of the amount of protein per cell. In Table 1 the number of PRE molecules per g of the

C 0

0 2 I 6 0 2 4 0 2 4

UV f l u e n c e ( J m - 2 1

Figure 1. Survival curves without PR (0) or after PR with 1 flash (0) of UV-irradiated cells (XS 774-6A) grown at 23 (a), 30 (b), or 37°C (c).

Factors modifying photolyase concentration 615

Table 1. NPRE and protein content in cells grown at various temperatures

Temperature ~ ~ ~ ~~

Number of

A DPRE (JW 0.95 f 0.22* 0.38 k 0.04 0.13 f 0.04 NPRE ( a ) 229 f 52 92.1 f 8.6 30.5 f 9.1

Amount of total protein (@ell) (b) 3.4 x 3.2 x 4.0 x

Number of active PRE molecules per g of total protein (alb) 6.7 x l O I 3 2.9 X 10” 0.76 X

experiments 5 16 7

*Standard deviation.

total protein content at each temperature is also shown. This value in cells grown at 37°C was 0.11 of the value in cells grown at 23°C.

(b) Effects of light on NPRE

In order to investigate an effect of light during cell-culture on NpRE, liquid medium inoculated with cells of strain XS 774-6A was incubated at 30°C in the dark or in light. As shown in Table 2, the average NpRE obtained from 6 independent experiments was 247 in cells grown in light and 89 in cells grown in the dark. This result shows that N ~ R E in cells grown in light is about 2.8 times larger than that obtained by cultivation in the dark.

(c) Stability of PRE molecules in cells grown in the dark or in light

The stability of PRE molecules in cells (XS 774-6A) incubated at 30°C in the dark or in the light was studied. Before UV-irradiation, cells were held at 30°C for 24 or 48 h in buffer (0.05 M KH2P04) in the dark or in light (buffer holding). The NpRE

obtained in cells after or before buffer holding is plotted in Fig. 3. The N p R E for cells grown in the dark showed a small decrease after 48 h buffer holding in the dark. When buffer holding was performed in light, N p R E became slightly larger than that before

Table 2. N,,, in cells grown in the dark or in light

NPRE

Experiment In the dark In light

1 2 3 4 5 6

average

82 150 73 150

100 214 82 291

105 319 91 287 89 + l l * 247 f 58

*Standard deviation.

buffer holding [Fig. 3 (a-l)]. In cells grown in light, N ~ R E decreased about half after buffer holding for 24 and 48 h in the dark [Fig. 3 (b-l)]. There was no decrease, however, in case of buffer holding in light after 48 h.

In order to prevent protein-synthesis during buffer holding, cycloheximide (CHX, 30 pglmt) was given

I (a-1) . (a-2) I ( b - 1 ) , ( b - 2 )

0 24 480 24 4 8 0 24 480 24 48

buf fer holding I h ) Figure 3. The NPRE after buffer holding in the dark or in the light in stationary cells (XS 774-6A) grown in the dark or in light. (a-1) Cells grown in the dark were held in buffer in the dark or in light. (a-2) Cycloheximide (CHX, 30 @me) was given to the suspension of (a-1). (b-1) Cells grown in the light were held in buffer in the dark or in the

light. (b-2) CHX was given to the suspension of (b-1).

to the cell suspension. In cells grown in the dark or in light NPRE obtained after buffer holding with CHX is depicted in Fig. 3 (a-2) and (b-2). The values of N p R E

obtained after buffer holding with CHX in cells grown in the dark [Fig. 3 (a-2)] did not differ much from the values in Fig. 3 (a-1). In cells grown in light, as shown in Fig. 3 (b-2), N p R E decreased by more than half after buffer holding in the dark, but PRE molecules in the cells were stable during buffer holding in the light. These results show that most of the decrease of N p R ~ in cells grown in light, by buffer holding in the dark, cannot be due to the synthesis of a protein, that may inactivate PRE molecules.

(d) N p R E in cells of strain GRF18 grown ar temperature 23 or 30°C

Cells of strain GRF18 have been transformed either with the plasmid pJDB207 or with plasmid

616 ATSUSHI FUKUI and WOLFGANG LASKOWSKI

A8-3 carrying the PHRl gene. Survival curves of both strains grown at 23°C or 30°C in the dark are shown in Fig. 4 (both strains do not grow at 37°C). In cells transformed with plasmid pJDB207, the UV-irradiated cells were photoreactivated with 5 flashes [Fig. 4 (a-1) and (a-2)]. The light flashes were repeated at intervals of more than 10 min, in order to obtain the maximum number of PRE-pyrimidine dimer complexes at illumination with each light flash. The fluence decrement AD after PR with 5 flashes, that is 5 x ADPRE, in cells grown at 23°C (a-1) was larger than in cells grown at 30°C (a-2). The NPRE calculated from c~ADPRE was 460 in cells grown at 23°C. In Fig. 4 (a-2), the increase of the survival fraction after 5 flashes of PRL was very small, indicating that NpRE is less than 140. In Fig. 4 (b-1) and (b-2), results with cells

transformed with the plasmid A8-3 and cultured at 23 and 30°C in the dark, respectively, are shown. The UV-irradiated cells were illuminated with one flash. In cells grown at 23°C (b-1), fluence decrement after one flash PRL, ADF, increased with UV incident fluence until the survival fraction of without becoming saturated, however, ADF in cells grown at 30°C (b-2), was almost saturated at the survival fraction of The curves show that GRF18 cells transformed with plasmid A8-3, containing gene PHR1, have a large amount of PRE molecules (about 5500) when grown at 30°C and that NpRE is even larger in cells grown at 23°C (over 13000). These results show that incubation temperature affects NpRE in GRF18 cells transformed with the plasmid containing a PHRl gene as well as in GRF18 cells containing the plasmid without gene PHRl (pJDB207).

DISCUSSION

The following results were obtained: (1) NpRE in cells of strain XS 774-6A is affected by temperature during cell growth. NpRE in cells grown at 37°C is about 0.13 of that in cells grown at 23"C, although the amount of protein per cell does not differ. (2) If the cells are cultivated in light, NpRE is about 2.8 times larger than N ~ R E of cells grown in the dark. (3) NpRE in cells grown in light decreases more rapidly during buffer holding in the dark than in the light. A comparable rapid decrease of NpRE was observed a f t e r buffer holding in t h e presence of cycloheximide. The decrease of NpRE in cells grown in light, by buffer holding in the dark, is rapid in comparison with the NpRE decrease in cells grown in the dark. (4) In GRF18 cells, transformed with a plasmid carrying the gene PHR1, NpRE was larger in cells cultured at 23 than at 30°C.

Harm and Rupert (1976) have reported that PRE illuminated with white light is more stable to thermal inactivation at 65°C than PRE not illuminated. Our in vivo results (3) can hardly be explained by thermal inactivation at 30°C and stabilizing of PRE by illumination, because NpRE in cells grown in light decreased by buffer holding in the dark more rapidly than that in cells grown in the dark. For an explanation of our results one may assume that cells have stable as well as unstable PRE molecules. It follows, that the percentage of unstable PRE molecules in cells grown in light is larger than in cells grown in the dark, and that most PRE molecules in cells grown in the dark at 30°C are stable. Our results have further shown, that the decrease of N ~ R E is smaller by buffer holding in light than in the dark,

0 100 2000 100 2000 100 zoo0 100 200

UV iluence ( J m - 2 )

Figure 4. Survival curves without PR (0) or after PR (0 1 flash or 5 flashes) in GRF18 cells transformed with plasmid pJDB207 (a-1) and (a-2) or with a plasmid A8-3 [(b-1) and (b-2)]. (a-1) And (b-1) cells

were incubated at 23°C. (a-2) And (b-2) cells were incubated at 30°C.

Factors modifying photolyase concentration 617

especially for cells cultured in light. This stabilizing effect of illumination on PRE molecules in cells cultured in the light agrees with the results of Harm and Rupert (1976).

It was shown for strains XS 774-6A and GRF18, used in the present work, that NpRE is affected by temperature and light applied during cultivation of the cells. Besides PRE stability, also the production of PRE molecules may be affected. This effect may be exerted by the influence of temperature and light on various factors (i.e. synthesis, activity of PRE molecules, mRNA etc.), which remain to be determined. Presently we only know that the total protein content per cell is not affected by temperature (Fig. 2 and Table 1) and therefore the amount of active PRE molecules per g of total protein changes in cells grown a t various temperature.

In this paper, NpRE was calculated assuming that photorepairable as well as non-photorepairable lesions are pyrimidine dimers. In contrast to this assumption one may consider the non-photo- repairable lesions not to be pyrimidine dimers. This means that all of the pyrimidine dimers are repaired after maximum PR of UV-irradiated cells, and therefore there is no effect of any pyrimidine dimer on the survival fraction after maximum PR. Taking this point of view, each NpRE in this paper becomes about 60% larger than calculated from CYAD~RE in strain X S 774-6A, as discussed in a previous paper

(Fukui el al., 1981). In strain GRF18, each NpRE becomes about 80% larger (unpublished data).

Acknowledgements-We thank Dr. Akira Yasui for supplying the strain GRF18 harboring the plasmids pJDB207 or A8-3 and for helpful discussions. We also thank Renate Ackermann for perfect technical assistance.

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