light-flash analysis of the photoenzymic repair process in yeast cells: ii. determination of the...

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Mutation Research, 81 (1981) 27--36 © Elsevier/North-Holland Biomedical Press

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

II. DETERMINATION OF THE RATE CONSTANT FOR FORMATION OF PHOTOREACTIVATING ENZYME--PYRIMIDINE DIMER COMPLEXES AND ITS ACTIVATION ENERGY TERM

ATSUSHI FUKUI, KOTARO HIEDA and YORIAKI MATSUDAIRA

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

(Received 14 January 1980) (Revision received 17 August 1980) (Accepted 23 September 1980)

Summary

As reported in the previous paper, the number of deoxyribodipyrimidine photolyase or photoreactivating enzyme (PRE) molecules per yeast cell was determined by the use of intense light flashes. In the present work, the reaction rate constant for the formation of PRE--substrate complexes, k l, and the activation energy term of h l were determined for yeast cells in vivo by the use of light flashes. At 30°C, hi equalled (6.5 + 1.1) X 10 -s (nuclear volume) • (molecule) -1 sec -1, which corresponded to 1.1 × 10 s 1 mole -~ sec -~, on the assumption that a nuclear volume is 3 X 10 -Is 1. h, showed positive temperature dependence as d.escribed by the Arrhenius expression with an activation energy of 11.8 -+ 1.6 kcal mole -~.

Deoxyribodipyrimidine photolyase or photoreactivating enzyme (PRE) repairs pyrimidine dimers in UV-irradiated DNA according to the following reaction scheme (Rupert , 1962)

kl k3 E + S~-~ ES-----~ E + P (1)

k~ light

where E is the PRE, S the pyrimidine dimer (substrate), ES the PRE--substrate complex, and P the repaired product .

The average numbers of PRE molecules per cell in Escherichia coli (Harm et al., 1968) and in yeast (Fukui et al., 1978; Yasui, 1976; Yasui and Laskowski, 1975) have been determined by a single light flash. By illumination with light

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flashes in rapid sequence, the reaction rate constants, kl and k2, and their activation energy terms have been determined for E. coli cells in vivo (Harm, 1970) and for the system in vitro of transforming DNA and a PRE molecule from yeast (Harm and Rupert, 1970). Recently, it was reported that PRE molecules from baker's yeast consists of 2 dissimilar sub-units, one about 85 000 dalton and the other about 60000 dalton (Werbin and Madden, 1977); whereas PRE molecules from E. coli seem to contain only one polypeptide chain of molecular weight about 35 200 (Snapka and Fuselier, 1977). It is of interest to analyze the photoenzymic repair process in yeast cells because they have nuclei and their PRE molecules are different from those of E. coli. In the work described in this paper, k~ and its activation energy term E A for yeast cells in vivo were determined by the method of light flashes in rapid sequence.

Materials and methods

Strain. Haploid yeast Saccharomyces cerevisiae XS 774-6A (~ radl-1 ), which is excision-defective, was used (Nakai and Matsumoto, 1967; Unrau et al., 1971).

Cell preparation. Cells were incubated at 30°C, while being shaken, for 2 days in complete medium (pH 5) containing 0.5% Difco yeast extract, 0.3% Difco bactopeptone and 1% glucose. The stationary-phase cells were washed twice and resuspended in 0.13 M phosphate buffer (pH 7) to a concentrat ion of 106 cells ml -I.

UV irradiation. UV irradiation was carried out with a low-pressure mercury lamp (Toshiba GL 10). The layers of samples were less than 3 mm thick. The incident dose rate was 0.13 J m -2 sec -I, as determined by a photometer (Inter- national Light Inc., IL 600).

Photoreactivation (PR). Flashes for photoreactivating light (PR light) were obtained from 2-photographic strobos (National PE-563). The single light flash was intense enough to photolyze all the ES complexes present (Fukui et al., 1978). Continuous PR light was obtained from 4 closely spaced fluorescent lamps (Toshiba FL 10D) at 3--5 cm distance. By illumination with this continuous light for 60 min at 30 ° C, UV-irradiated cells were photorepalred at maximal level (Fukui, 1978). To exclude any uncontrolled PR, UV-irradiated samples were manipulated in the dark or under a yellow fluorescent lamp (National FL 20 Y-F).

Plating. Treated samples were appropriately diluted and plated on a complete agar medium. After a 4-day incubation at 30°C in the dark, the colonies were counted.

Method o f determination o f kl. Under the condition that the pyrimidine dimers are repaired by PR light immediately after the formation of ES complexes, that is that the number of ES complexes present is negligibly small, the change in the concentration of pyrimidine dimers is represented by the equation

d[S] /dr = --k 1 [E] 0[S] (2a)

that is,

ln([Slt /[S]t=o) = --kl[E]0t (2b)

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where [S]t expresses the concentration of pyrimidine dimers at time t, [E]0 the concentration of PRE molecules when all PRE molecules are free, and t the time duration from the beginning of PR light illumination. For simplicity, [S]t, [S]t=0 and [E]0 will be expressed as the number of molecules per nuclear volume.

In the present experiments, UV-irradiated cells were kept in the dark in buffer at room temperature (25°C) for more than 15 min so that the number of ES complexes could arrive at the equilibrium level of scheme (1) in the dark. Then, the cells were illuminated with intense light flashes in rapid sequence at At intervals (4.2 or 5.5 sec) for PR light of the conditions as described before. Since all ES complexes were photolyzed by the first flash of the sequential flashes, all PRE molecules became free. During the dark interval, At, a small number of PRE molecules combined with substrates. These ES complexes were photolyzed by the second flash. Subsequently, the formation and photolysis of ES complexes were repeated. The number of photorepairable substrates remaining after light flashes of n times was denoted by IS] , . After the first flash (n = 1), [S] , decreased according to the next equation in which [S]t=o, [S]t , and t in Eqn. (2b) are replaced by [S]I, [S] , , and (n -- 1)At resp.

ln ( [S] , / [S] ~) = --kl[E]0(n -- 1) At (2c)

Although Eqn. (2c) is exact only if the number of free PRE molecules, [E], remains constant in the dark interval, Eqn. (2c) gives good approximate values under our experimental conditions (see Discussion).

The number of substrates repaired by PR light was calculated from a • AD, where AD is the dose decrement defined by Harm et al. (1971) and a is the number of pyrimidine dimers produced by a unit dose of UV in nuclear DNA and assumed to be 240 (l~yrimidine dimers)/(J m -2) per haploid cell (Unrau et al., 1973). The [S] , was obtained from [S]0 -- a • AD,, where [S]0 is the number of pyrimidine dimers before the first flash and AD, is the dose decrement after light flashes of n times. [S]0 was calculated from Eqn. (3) described in Results, Part A; and the number of PRE molecules in a nucleus, [E]0, was calculated from a • AD~ (see Results, Part B). Thus, k~ can be calculated from Eqn. (2c).

Results

(A ) Dose decrements with a light flash and photorepairable substrates Fig. 1 shows the survival curves with no PR (dark) and with PR (1 flash and

maximal PR). The dose decrement, AD, was the difference between the UV dose with PR actually applied and the smaller UV dose, which would give an identical survival with no PR (Harm et al., 1968}.

The relationships between the incident UV doses and the dose decrements of a single flash, AD1, shown in Fig. 2, showed that AD1 was constant above the incident dose of 3 J m -2. Thus, as reported in the previous paper (Fukui et al., 1978), a • AD1 at the constant level corresponds to the total number of PRE molecules in a nucleus.

In the present work, we assumed, as did Harm et al. (1971), that all the UV- induced lesions were pyrimidine dimers. A discussion on this assumption is

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i0 -I

~ 10 .2

m lO- 3

10 -4 2 4 6 8

UV DOSE ( J m -2)

?E 1"

/ /*

1//// / , / / / / / . / / maximum PR

/

,/ I flash

2 4 6 8

UV DOSE ( J m -2 )

Fig. 1. Surviva l cu rves o f U V - i r r a d i a t e d y e a s t cells w i t h a n d w i t h o u t PR . ~, PR b y a single l igh t f lash; ©, PR b y c o n t i n u o u s i l l u m i n a t i o n fo r 6 0 m i n ( m a x i m a l PR) ; e , w i t h o u t PR . A single l igh t f lash w a s u sed to i l l u m i n a t e the cells a f t e r t h e y h a d b e e n k e p t fo r over 15 ra in a t r o o m t e m p e r a t u r e ( 2 5 ° C ) in t he d a r k so t h a t t he n u m b e r o f ES c o m p l e x e s c o u l d r e a c h the e q u i l i b r i u m level .

Fig . 2. D e p e n d e n c e o f the dose d e c r e m e n t s o n the i n c i d e n t dose o b t a i n e d f r o m Fig. 1. D, a s ingle l igh t f lash , o , m a x i m a l PR . The dose d e c r e m e n t w i t h a single l igh t f lash w a s c o n s t a n t a b o v e a U V dose o f 3 J m -2 . The d o t t e d l ine c o r r e s p o n d s to t he case in w h i c h the dose d e c r e m e n t s e q u a l l e d the i n c i d e n t UV doses .

given in the Discussion section. The dot ted line in Fig. 2, in which the dose decrement is equal to the incident dose, corresponds to the case in which all the UV-induced lesions could be photorepaired. From the slopes of this dot ted line and of the curve for maximal PR by continuous light, the ratio of photorepairable lesions to total UV-induced lesions was calculated. The average ratio over 8 Expts. was 0.633 + 0.041. Consequently the number of photorepairable substrates induced by UV, [S]0, was assumed to be calculable by the equation

IS]0 = 0.63 • ~ • Do (3)

where Do is the incident dose.

(B) Determination of kl Fig. 3 shows an example of the relation between the survival and number of

light flashes, n, where the incident dose is 6.5 J m -2, At 4.2 sec, and the temperature 30 ° C. Because this incident dose was larger than 3 J m -2 (see Section A), [E]0 can be calculated by a • AD1.

The number of ES complexes formed during the dark interval after the first flash, ~ • (LiD 2 -- AD1), was estimated from ~ • (AD3 -- AD1)/2, because AD 2 was not obtained because of experimental difficulty. The average of this number over 8 Expts. corresponded to 17.3% of the total number of PRE molecules. The value is so small that it is possible to use Eqn. (2c) for the determination of k, (see Discussion).

Fig. 4 shows ln([S]n/[S] 1) obtained from the data in Fig. 3. Eqn. (2c) shows that the values of ln( [S] , / [S] 1) decreased linearly with n. However, in this fig- ure, the values of ln( [S] , / [S] 1) above n = 7 deviated markedly from the initial slope. Accordingly, to obtain k,[E]0 by using Eqn. (2c), the ratio [S]3/[S]1 was used. 8 independent Expts. were performed. For each Expt., kl[E]0 and

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xl0 -3

2O

> 5

2 / /

0 i 1

.--O-"

!

20 40 60 (sec) • . , , . % . ., , .

3 5 7 9 ii 13 15 17 19

NUMBER OF LIGHT FLASHES (n)

F i g . 3 . Survival o f UV- irradiated cel ls i l l u m i n a t e d b y l ight f lashes in rapid s e q u e n c e . Cells w e r e irradiated w i t h U V at 6 . 5 J m - 2 , k e p t in the dark for m o r e t h a n 15 rain at 2 5 ° C , and i l l u m i n a t e d b y l ight f lashes at 3 0 ° C . T h e interval b e t w e e n l ight f lashes ( A t ) was 4 .2 sec . N u m b e r s on the u p p e r s ide o f the abscissa are the t i m e s ( s ec ) c a l c u l a t e d f r o m (n - - 1 ) A t in E q n . ( 2 c ) .

[E]o were obta ined, and h~ was calculated from them. The average values and standard deviat ions o f k l [E]0 , [El0, and kl were, resp., (1 .23 -+ 0 .50 ) × 10 -2 sec -1, 186 + 59 (PRE molecules ) • (nuclear vo lume) -1, and (6 .5 + 1 .1) × 10 -s (nuclear vo lume) • (PRE molecules ) -1 sec -1. The value o f hi corresponded to 1.1 × 10 -s liter mole -~ sec -1 on the assumpt ion that the nuclear vo lume in sta-

0 .9

~ 0 . 8

0 .7

0 .6

\k \ \\

\kk\ k N ~

N \

20 40 60 (sec)

i i I i I , i

; ; g ; 11 13 l~ ;7 NUMBEN OF LIGHT FLASHES (n)

Fig . 4 . Decrease in subs trates b y l ight f lashes in rapid s e q u e n c e . T h e rat ios , [ S ] n / [ S ] 1, w e r e ca l cu la t ed f r o m the data o f Fig . 3 and p l o t t e d o n a l o g a r i t h m i c scale as a f u n c t i o n o f the n u m b e r o f l ight f lashes (n) a n d t i m e (n - - 1 ) At . T h e va lue at t h e th ird f lash (n = 3) w a s u s e d for t h e d e t e r m i n a t i o n o f k 1 . F o r refer- e n c e t h e init ial s l ope o f the sol id l ine is i n d i c a t e d b y a d o t t e d l ine .

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xlO -3 20

i0

8

6

~t ~ ~' '5 ~' I t '

o io 20 3o

(n-l)At (sec)

NI/~ER OF FLASHES at 30°C

~ h at 16°c

Fig. 5. Surviva ls o f U V - i ~ a d i a t e d cells i l l u m i n a t e d by l igh t f lashes in rap id s equence at 16 or 30°C . Cells

were i r r ad ia ted w i t h 5.9 J m -2. The p r o c e d u r e s a f t e r U V - i r r a d i a t i o n were the s a m e as for Fig. 3, e x c e p t

t h a t the in te rva l b e t w e e n l igh t f lashes (At ) was 5.5 sec at 16°C. t ' is the t i m e w h e n a survival at 16°C

equals the survival a t 3 0 ° C a f t e r the th i rd f lash (n = 3). D i f f e r e n t abscissa scales are used fo r the n u m b e r

of f lashes at 30 and 16°C, and for the t i m e (n - - 1) t (sec).

tionary phase cells is 3 × 10 -is liter or 3 pm ~, which was estimated from elec- tron micrographs (Matile et al., 1969).

(C) Temperature dependence o f te 1 UV-irradiated cells were illuminated by light flashes in rapid sequence at

various temperatures between 9 and 30 ° C. The intervals of light flashes were 5.5 sec at 9--20°C and 4.2 sec at around 30°C. A typical Expt. is shown in Fig.

xl0 -5

02 ~ o ~..~

Q v 1

30 20 i0 (°C) I ,L i i J

3.3 O 3 .'40 3 [ 50

I/T (°K-l)

Fig. 6. T e m p e r a t u r e d e p e n d e n c e of ra te c o n s t a n t k 1 for E S - c o m p l e x f o r m a t i o n in the f o r m o f A r r h e n i u s p lo t s . Each k i n d o f s y m b o l r ep r e sen t s resu l t s o f an i n d e p e n d e n t E x p t . T h e s t ra igh t l ine was d r a w n f r o m

the average k 1 at 3 0 ° C and the average E A o f 8 E x p t s .

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5. The kl at 30°C was obtained from Eqn. (2c) as described in Materials and Methods. The kl at 16°C was obtained from kl at 30°C multiplied by t/t', where t was the duration between the first and third flashes (8.4 sec) at 30°C and t' was the time when a survival at 16°C equalled the survival at t as shown in Fig. 5. The values of k l obtained from 8 independent Expts. are shown in Fig. 6 in the form o f Arrhenius plots, where the values of k~ were plotted logarithmically versus the reciprocal o f absolute temperatures (T). The results o f each Expt. showed the clear tendency that ln(kl) decreased with 1/T. The activation energy EA for each Expt. was calculated from the Arrhenius Eqn.

EA(cal mole -1) = - -RT[ln(kl) -- ln(A)] (4)

where R is the gas constant (1.99 cal mole -1 K -1) and A is the frequency factor. Average EA and the standard deviation were 11.8 -+ 1.6 kcal mole -1. The frequency factor A that was calculated from the average k~ and EA was 4 × 1013 liter mole -1 sec -1.

Discussion

The results obtained in the present work are summarized in Table 1, in com- parison with those for yeast cells investigated by Yasui (1976), for E. coli (Harm, 1970), and for the system in vitro of yeast PRE and Haemophilus influenzae transforming DNA (Harm and Rupert , 1970).

The value of kl liter mole -1 sec -~ of yeast cells was about 400 times smaller than that of the system in vitro with yeast PRE.. Many factors can affect the value of kl; two of them will be discussed here: viscosity of solvent and com- peting effect of undamaged DNA. The viscosity of the system in vivo may be higher than in vitro, as discussed by Harm et al. (1971). Higher viscosity causes lower k l. With regard to the effect of undamaged DNA, Cook and Proctor (1974) found that the ES-complex formation was inhibited by the presence of an excessive amount of unirradiated DNA especially at concentrations above 10 pg/ml. The concentration of DNA of haploid yeast cells in vivo was estimated as 6 X 103 #g/ml from the molecular weight ( - 1 X 101° dalton) of DNA per haploid yeast cell (Hartwell, 1970) and from the assumption that a nuclear volume was 3 ~m 3. On the other hand, the concentration of 0.167 mg/ ml was used for the system in vitro (Harm and Rupert , 1970). Hence, the com- peting effect between pyrimidine dimers and the undamaged port ion of DNA for PRE molecules may be stronger for yeast cells than for the system in vitro, that is, k l may also be lower for yeast cells in vivo than for the system in vitro. Furthermore, the difference between the higher structures of DNA in chromosomes in vivo and in solution in vitro may be another possible factor causing the difference in k l.

In eukaryotes, the PR process of chromosomal DNA lesions which actually occurred should be restricted within a nucleus during such a short period as in the present experiments using light flashes. The nuclear PRE molecules may not go out from the nucleus and the extranuclear PRE molecules may not con- tribute to the observed process. To compare the PR process in yeast with that in E. coli, we used a nuclear volume to determine k~ in a unit of liter mole -~ sec -~ for yeast instead o f the cell volume used for E. coli (Harm et al., 1971).

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T A B L E 1

C O M P A R I S O N O F T H E R E S U L T S A M O N G Y E A S T C E L L S , E. coli C E L L S , A N D S Y S T E M IN V I T R O

Yeas t cells

P re sen t

E. coli cells S y s t e m in

by H a r m v i t ro by

Yasul ( 1 9 7 0 ) H a r m and ( 1 9 7 6 ) c R u p e r t

( 1 9 7 0 ) d

k l [ E ] 0 , sec -1 a ( 1 . 2 3 + 0 . 5 0 ) X 1 0 - 2 1 , 5 X 10 -2 f 5 . 2 × 1 0 - 2 f 1 . 0 X 1 0 - 1 f

k 1, ( v o l u m e ) • (mo lecu le s ) -1 sec-1 a (6.5 -+ 1,1) X 10 -S 4 .2 X 10 - s e 2.6 X 10 -3 e - -

k l , l i t e r m o l e - 1 sec -1 a 1 . 1 X 10 s f 7 . 4 X 1 0 S f 1 . 6 X 1 0 6 f 4 . 0 X 1 0 7 e

E A , kca l m o l e -1 11.8 + 1.6 5 .33 11 9.3

A , l i te r m o l e -1 sec -1 3.8 X 1013 - - 1.2 X 1014 1.6 X 1014

N u m b e r o f P R E m o l e c u l e s per cell 186 +- 56 350 20 - -

C o n c e n t r a t i o n o f P R E molecu les , m o l e l i ter -1 1 X 10 -7 f 2 X 10 -5 3 X 10 -8 f 2 .5 X 10 -9

V o l u m e used fo r ca lcu la t ion , l i t e r b 3 × 10 -15 3 X 10 -16 1 X 10 -1S _

a k l [ E ] 0 and h 1 were the va lues a t 30°C. b Nuc lea r vo l tune o f y e a s t and cell v o l u m e of E. coli were assturned for c o m p u t i n g in c o n v e n t i o n a l un i t s o f

k 1, A, and c o n c e n t r a t i o n of PRE,

c The s t ra in was MB 1 0 3 0 - 1 A ( h a p l o i d ) . ~ was a s s u m e d to be 320 p y r i m i d i n e d i m e r s per h a p l o i d yeas t .

/~ 1 [E] 0 was o b t a i n e d by the use of c o n t i n u o u s PR l ight .

d P R E f r o m y e a s t and Haemoph i lu s in f luenzae t r a n s f o r r a i n g D N A . e O b t a i n e d f r o m the va lues in the i r papers .

f Ca lcu la ted f r o m the a p p r o p r i a t e va lues l i s ted in th i s table .

The value of kl liter mole -1 sec -1 for yeast cells in vivo was about 10 times smaller than that for E. coli cells in vivo. The PRE of yeast cells was different from the PRE of E. coli cells with respect to the molecular weight and the com- position of sub-units (Snapka and Fuselier, 1977; Werbin and Madden, 1977). The difference in k, may be partially caused by the differences in PRE molecules for both organisms. However, the dependence of k~ on the chemical and physical situations of the solvent and DNA was great as indicated by the difference in k i between the system in vivo and of yeast PRE in vitro. Accord- ingly, it cannot be concluded to what extent the difference in the kl values corresponds to the differences in PRE molecules.

Eqn. (2c) indicates that l n ( [ S ] J [ S ] l ) should decrease linearly with n. In Fig. 4, the experimental points above 7 flashes deviated from the initial slope. In the cases of E. coli (Harm, 1970) and the system in vitro (Harm and Rupert , 1970) the same tendencies were observed, although the break points of the value of [ S ] , / [ S ] , were somewhat different from the present results. Cook and Worthy (1972) measured, in vitro, the time course of photoreactivation by yeast PRE of thymine-- thymine dimers in acetophenone-photosensit ized E. coli DNA, and observed the break after 80% photorepair. The break after only 20% photorepair in vivo (Fig. 4) may be another consequence of chromosome structure in the intact nucleus.

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For simplicity, it was assumed in the present calculation that both the photorepairable and non-photorepairable lesions are pyrimidine dimers, as Harm et al. (1971) assumed. But some of the non-photorepairable lesions may not be pyrimidine dimers (Johnson et al., 1973); and in the extreme case that none is, the dose decrement with maximal PR, ADmax, corresponds to the total number of pyrimidine dimers induced by UV. The number of ES complexes can be calculated from a • AD1 × (D0/ADmax), where Do/Dmax is equal to 1/0.63 (Re- sults, Part A), and the number of PRE molecules, [E]0, becomes 59% larger than by the previous assumption. But kl[E]0 and E A do not change in this case (see Eqns. 2b, 4). Hence k~ is 37% smaller than the values in Table 1.

The value of k~ was obtained with the use of light flashes by a method sim- ilar to that of Harm (1970). For the exact estimation of kl from Eqn. (2c), the number of ES complexes must be negligibly small after the first flash. But, in the present Expts., 17.3% of all the PRE molecules formed ES complexes at the end of the dark interval after the first flash. The rate of ES-complex formation, d[ES]/dt , decreases if the number of free PRE molecules decreases. If this decrease is considered under a reasonable approximation that the dissoci- ation of ES complexes is negligible, the rate of ES-complex formation, d[ES]/ dt, equals kl([E]0 -- [ES])([S]~ -- [ES]) in the dark after the first flash, where t is the time after the first flash. This expression is a substitute for Eqn. (2a). After integration of the former equation under the initial condition [ES] = 0 at time zero, k~ is represented by the equation

1 1 -- [ES]/[S]~ kl = In

t([S]l -- [E]0) 1 -- [ES]/[E]0

From this Eqn., k~ was calculated by the use of [ES] obtained from a • (AD3 - AD1)/2 and t = 4.2 sec. The average k~ and the standard deviation over 8 Expts. were (6.7 -+ 1.3) × 10 -s (nuclear volume) • (PRE molecules) -1 sec -1, and the average value was only 3% larger than that in Table 1. This difference is small as compared with the standard deviation in k l. The foregoing discussion shows that the apparently simple equation, Eqn. (2c), can be used for the calculation of k 1 for these Expts.

Based on the k~ value obtained, less than 0.1 dimers could be repaired per cell in a single flash, whose duration was 3 m sec (Fukui et al., 1978). There- fore, the duration of the flash was short enough.

In Table 1, the activation energy E A obtained in this work is large as compared with that of the system in vitro, and is almost equal to that of E. coli. However, the activation energy EA obtained here was about twice larger than E h in the paper of Yasui (1976), who obtained it by the use of continuous PR light. This difference between the present results and those of Yasui may have been caused by the differences in yeast strains and by the experimental methods.

The dark-survival curve (Fig. 1) showed a small shoulder, and the one lethal hit dose, that is a dose giving 37% survival, was about 1.8 J m -2, which corresponded to 330 pyrimidine dimers. These two observations showed that dark- repair mechanism(s) repaired dimers in the cells. However, the dimers were repaired in the dark mainly during incubation on plates but not during holding in buffer from UV irradiation to plating, because, during holding in buffer in

36

the dark, survival was only slightly decreased (Fukui, 1978). Moreover, ratios of survivals after PR by one flash to dark survivals were constant during incubation in buffer up to 6 h (Fukui, 1978). Therefore, it was concluded that inter- action of dark-repair mechanism(s} with PR had little role in our analysis.

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light-flash analysis of the photoenzymic repair process in yeast cells: ii. determination of the...

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