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Muta t ion Research, 51 ( 1 9 7 8 ) 4 3 5 - - 4 3 9 © E l sev i e r /Nor th -Ho l l and Biomedica l Press

Short Communication

LIGHT-FLASH ANALYSIS OF THE PHOTOENZYMIC REPAIR PROCESS IN YEAST CELLS

I. DETERMINATION OF THE NUMBER OF PHOTOREACTIVATING ENZYME MOLECULES

ATSUSHI FUKUI, KOTARO HIEDA and YORIAKI MATSUDAIRA

Biophysics Laboratory, Department of Physics, Rikkyo University, Nishi-Ikebukuro, Toshima-ku, Tokyo (Japan)

(Received 28 December 1977) (Revision received 17 March 1978) (Accepted 7 April 1978)

Introduction Photoreactivating enzymes (PRE) combine with UV-induced pyrimidine

dimers in DNA to form PRE--pyrimidine dimer complexes. Such a complex is photolysed by energy absorption of near-UV or short-wavelength visible light; in this process the enzyme is released, and the dimer is split. Since a single intense light flash photolyses all the complexes present, the number of com- plexed PRE molecules should be equal to the number of pyrimidine dimers monomerized by such a flash. This number can be determined from the photo- reactivating recovery on the survival fraction by the illumination of a single flash.

Harm et al. [3] studied, by flash photolyses, the photoenzymic repair of UV lesions in DNA of Escherichia coli cells, which are prokaryotes. Further, Nishioka and Harm [5] investigated the dependence of the number of PRE molecules on growth phases in E. coli Bs.l-160 and Bs-1, and observed that there is an excess PRE product ion in Bs.l-160 cells entering the stationary phase, but that the difference in growth phase does not affect the PRE content of B~.~ cells. Yasui and Laskowski [8] determined the number of PRE molecules per cell in 2 strains of yeast, MB 1030-1A and S 24-12c.

In the work described in this paper, the number of PRE molecules in a UV- sensitive strain of yeast cells, which are eukaryotes, was determined by using a single light flash, and the variation in the number of PRE molecules in various growth phases was estimated.

A bbreviation : PRE, photoreactivating enzyme.

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Materials and methods Experiments were carried out with haploid yeast Saccharomyces cerevisiae

XS 774-6A (~ tad 1-1) [4], which is excision-defective [6]. Cells were incu- bated at 30°C with shaking in complete medium (pH 5) containing 0.5% Difco yeast extract, 0.3% Difco bactopeptone, and 1% glucose. The cell culture was incubated for 48 h, then diluted 64 times with fresh medium and cultured under the same conditions. Cells were sampled in various phases of growth, washed twice with distilled water, and resuspended in distilled water at about 106 cells ml- ' . The concentration of cells was determined by a hemocytometer . The cell samples were irradiated at 254 nm by a low-pressure mercury lamp (Toshiba GL 10). After the UV irradiation, the cells were kept in the dark for about 15 min at room temperature (about 25°C) in distilled water so that all the PRE molecules could form complexes with the pyrimidine dimers present. A single light flash was produced by simultaneous discharge of 2 photographic strobos (National PE-563). The cell sample was 5--6 cm from the source, and there was a glass filter between source and sample. 90% of the light energy was emitted in 3 msec. It had been confirmed by a preliminary experiment that the illumination of flash light to non-UV-irradiated cells did not affect the survival.

The samples, with and wi thout the illumination of a single light flash, were plated on complete agar medium. After a 4-day incubation at 30°C in the dark the colonies were counted. UV-irradiated samples were treated in the dark or under a yellow fluorescent lamp (National FL 20 Y-F).

Results and discussions Fig. 1 shows that a single flash was intense enough to photolyse all the

PRE--pyrimidine dimer complexes present. The dark-survival curves and the survival curves photoreactivated by a single

light flash were obtained for UV-irradiated yeast cells, which were taken at

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Fig. I . Increase in the survival o f yeast cells resulting from photoreac t iva t ion by single flashes o f v a r i o u s strength. Stat ionary phase cells were irradiated b y U V of 52 (o) o f 65 (A) erg • m m - 2 , held in the dark for a b o u t 15 m i n at r o o m temperature (about 2 5 ° C ) and i l luminated by a s ingle flash. The abscissa s h o w s re la t ive intensit ies o f f lash l igh t obta ined from 2 units o f the photogzaphic s trobo which can emi t 2 differ- ent intensities; HI (guide number 56 ) and L O (guide n u m b e r 28) . For other exper iments the highest inten- s ity (HI • H I ) was used.

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Fig. 2 . Surv iva l cu rves o f yeas t ce l l s harves ted at various phases of g r o w t h . Cells w e r e incuba ted at 3 0 ° C in the c o m p l e t e m e d i u m for 6 h (1) , 2 0 h (2) a n d 4 8 h (3) , w a s h e d w i t h d i s t i l l ed w a t e r and irradiated by UV. W i t h (o) and w i t h o u t ( o ) p h o t o r e a c t i v a t i o n b y i l luminat ion o f a s ingle l i gh t f lash , U V - i r r a d i a t e d cells w e r e p l a t e d and i n c u b a t e d at 3 0 ° C in t h e d a r k f o r 4 days.

various phases of growth. Fig. 2 shows some examples of these survival curves. In the figure, the dose decrement, AD, defined by Harm et, al. [3], is approxi- mately constant below 1% survival, for every example. The dose decrement expresses the photoreactivat ion effect , and is the difference between the UV dose with photorepair actually applied and the smaller UV dose, which would give an identical survival wi thout photorepair. Since UV radiation at 1 e rg . mm -2 produces about 24 pyrimidine dimers per haploid yeast cell [7], the approximate number of complexes repaired by a single flash is 24 × AD, the same as the number of PRE molecules present in this experiment. For each of the various phases of growth, AD was obtained; and the number of PRE molecules present in a cell was calculated from the value of AD.

Fig. 3 illustrates the growth curve and the variation in the number of PRE molecules in a cell as functions of incubation time. In the stationary phase, namely, at 0 h and over 48 h of incubation time, the number of PRE molecules was estimated as about 180. In the logarithmic phase, a decrease in the number of PRE molecules per cell was observed; and, when t_:e cells divided twice at 6 h of incubation time, the number of PRE reached the minimal value, namely, about 50, which is about 1/3.5 of the number in the stationary phase. The figure of 50 molecules is likely to be an underestimate, since by necessity the logarithmic-phase cells contain on the average more DNA than do stationary- phase cells. In the cells containing a double content of DNA, the number of dimers produced by 1 erg • mm -2 must be twofold; hence the number of PRE molecules obtained should also be double. The average DNA con t en t of logarithmic-phase cells was less than twofold; thus the number of PRE mole- cules should be less than 100. This value is still less than that in the stationary phase.

A similar tendency was reported by Boling and Setlow [1] for the PRE activ- ity of cell extracts from several strains of yeast measured by cont inuous illumi- nation. The ratios of the PRE activity in logarithmic-phase cells to that in sta-

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Fig. 3. N u m b e r of PR E mo le c u l e s in a yeas t cell (o, A, o) a n d cell concen txa t ion in a cu l tu re (o) as a func- t ion of i n c u b a t i o n t i m e . Th re e d i f f e r en t s y m b o l s for n u m b e r o f PRE molecu le s r ep r e sen t i n d e p e n d e n t e x p e r i m e n t s . T he cell cu l tu re i n c u b a t e d fo r 48 h was d i lu ted 64 t imes w i t h f resh m e d i u m at t i m e 0 of t h e abscissa and i n c u b a t e d a t 30°C w i t h shaking. N u m b e r s of PRE molecu le s were d e t e r m i n e d f r o m survival curves w i t h and w i t h o u t p h o t o r e a c t i v a t i o n (see Fig. 2 a nd t ex t ) .

tionary-phase cells have been reported to be 0.12--0.02, as compared with 0.3 for this experiment.

An uncertainty in the number of PRE molecules determined by the dose decrement, AD, comes from the observation that a small fraction of the total UV lesions are not photorepairable [3]. It is at present no t known whether non-photorepairable lesions are pyrimidine dimers or different types of UV photoproducts. The non-photorepairable fraction was about 40% in the yeast strain used in the present work [2] , which was larger than that in E. coil (about 15%) [3]. However, for simplicity in our calculations, we have made the same assumption as for E. coli [3] that both photorepairable and non- photorepairable lesions are directs; the number of PRE molecules was cal- culated to be 24 × AD as described before. However, Yasui and Laskowski [8] calculated 272 as the number of PRE molecules on the assumptions that non- photorepairable lesions are not the dimers and that 32 pyrimidine dimers are produced in the DNA of a haploid cell by UV irradiation at 1 erg • mm -2. Based on their assumptions, the number of PRE molecules was calculated as 340 in our case.

The experiments reported here showed that the number of PRE molecules in a eukaryotic cell could be determined by photoreactivation with a single light flash as well as in a prokaryote. The results suggest that the rate constant for formation of P R E - - p y ~ i d i n e dimer complexes and the activation energy of this reaction can be obtained. If the variation in these quantities could be obtained against the incubation time, it would give some information for the start of a study of the changes in the situation and the environment around the chromosome DNA in vivo [2].

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References

1 Boling, M.E., and J.K. Setlow, Photoreact ivat ing enzyme in logarithmic-phase and Stationary-phase yeast cells, Biochim. Biophys. Acta, 145 (1967) 502--505.

2 Fukui , A., K. Hieda and Y. Matsudaira, Light flash analysis of the pho toenzymat ic repair process in yeast cells, II. Determinat ion of the rate constant for format ion of PRE--pyr imidine dimer complex and i ts act ivat ion energy, R ikkyo University Press, RUP 78-5 (1978).

3 Harm, W., C.S. Ruper t and H. Harm, The s tudy of pho toenzymat ic repair of UV lesions in DNA by flash photolysis , in: A.C. Giese (Ed.), Photophysiology, Vol. VI, Academic Press, New York, 1971, pp. 279--324.

4 Nakai, S., and S. Matsumoto, Two types of radiation-sensitive mu tan t in yeast, Mutat ion Res., 4 (1967) 129--136.

5 Nishioka, H., and W. Harm, Analysis of pho toenzymat i c repair of UV lesions in DNA by single flashes, IX. Excess product ion of photoreact ivat ing enzyme in E. coil Bs.1-160 under different growth condi- t ions, and its suppression by adenine, Muta t ion Res., 16 (1972) 121--131.

6 Unrau, P., R. Wheatcroft and B.S. Cox, The excision of pyrimidine dimers from DNA of ul t raviolet i rradiated yeast , MoL Gen. Genet., 113 (1971) 359--362.

7 Uv_rau, P., R. Wheatcroft . B. Cox and T. Olive, The format ion of pyrimidine dimers in the DNA of fungi and bacteria, Biochim. Biophys. Acta, 312 (1973) 626--632.

8 Yasui, A., and W. Laskowski , Determinat ion of the number of photoreact ivat ing enzyme molecules per haploid Saccharomyces cell, Int. J. Radiat . Biol., 28 (1975) 511--518.