Mutation Research, 3° (1975) 43-54 ~i Elsevier Scientific Publishing Company, A m s t e r d a m - - P r i n t e d in The Netherlands

43

STUDIES ON T H E INDUCTION OF MITOTIC GENE CONVERSION BY U L T R A V I O L E T IRRADIATION. II. ACTION SPECTRA

TAKASHI ITO AND KATSUMI KOBAYASHI

Institute of Physics, College of General Education, University of Tokyo, Komaba, Tokyo I53 (Japan) (Received August I6th, 1974) (Revision received March I7th, 1975) (Accepted March 2ISt, 1975)

SUMMARY

Action spectra for the induction of intragenic mitotic recombination (gene conversion) at the trp5 locus by UV are presented for three cell stages (To, T9 and T~e) taken from synchronously growing cultures of Saccharomyces cerevisiae. The spectra over the range from 23o to 3oo nm were taken mostly in 5-nm steps. The peak of action spectra was significantly shifted, regardless of the stage, toward the longer wavelengths as compared with that of the absorption spectrum of DNA (258 nm) or even that of thymine (265 nm). In one extreme case (T~), the peak was shifted 17 nm from the absorption peak of DNA. Further, the spectrum changed its shape as the cell stage advanced from non-dividing (unbudded) (To) to a dividing phase (T~6). Furthermore, the induction cross section decreased by a large factor (about 4o), regardless of the wavelength, in going from To to T~8. From observations of the high photoreversibility of induced conversions, the major primary damage was thought to be pyrimidine dimers in the DNA. One plausible explanation, though not quite satisfactory from the quantitative viewpoint for these findings was that the increasing RNA during growth would screen the incident UV differentially with respect to the stage. If this explanation is correct, thymine dimers may still be considered, in spite of the shifts and deformations in the action spectra, as the major primary damage that triggers the long series of processes leading to gene conversion. Conventional methods for obtaining action spectra are discussed in comparison with the present method, which was based on sensitivity parameter a in the proposed dose (t)-frequency (f) relation, f -- (at)~ (~ is the multiplicity parameter).

INTRODUCTION

A broad maximum around 26o nm in UV action spectra for mutation induction and lethal action in general has been taken to mean that these effects are produced through damage in DNA ~-4. For non-reciprocal recombination, known as gene con-

44

T. l-r(), K. K O B A Y A S H I

version, no attempt to take the action spectrum has been made so far. The gelm conversion is not a simple process but, as many lines of work suggest, may involve interesting steps, namely breaks, recombination and, perhaps, partial replication in the limited segment of DNA strands, much the same as the repair processes of radiationinduced damage (see also refs. 5, 6). Besides, bringing the homologous chroinos()mes into physical closeness may be a basic condition to be provoked whatever the direct causes may be. Furthermore, the UV dose-frequency curve for gene conversion was not linear in Saccharomyces 7. Since we need an appropriate sensitivity (cross section) parameter in making the action spectrum, this should pose a problem. Fortunately, however, previous analysis 7 showed that the dose-frequency curve could be satisfactorily described by two parameters, an intrinsic sensitivity parameter or "cross section" a of the target, and hit number or "multiplicity" parameter a. Then, a would be the appropriate parameter with which action spectra may be constructed. In this study, as a continuation of the preceding work, we have attempted to obtain action spectra, using this sensitivity parameter, for the induction of gene conversion at various cell stages taken from synchronously growing cultures over the region of wavelengths from 23o to 3oo nm. Since, in nmch of the action spectra work for a suspected DNA damage, experimental data are badly lacking in the critical region from 254 to 28o nm (see, for example, ref. i), this region has now been covered with 5-nm steps. MATERIALS

AND METHODS

The yeast strain, the methods of culture, the synchronization and the detection of genetic changes (gene conversion) are as described in previous work 7. Briefly, a diploid Saccharornyces cerevisiae heteroallelic at trp5 locus, derived from D 4 of ZIMMERMAN.XAXD SCHWAIER~, was used throughout. The method of synchronization followed that of MITCHISO~TAND VINCEXT9. The following synchronized cells were used : To (unbudded small cells selected by the sedimentation method from the late logarithmic phase); T9 (9o-min culture of To) which corresponds to about the beginning of the DNA synthesizing stage1°; Tie (I6o-min culture of To) which corresponds to the stage when the nuclear division is finished (see Fig. I in the preceding paperT). Washed cells, suspended in distilled water (lO 7 cells/ml), were irradiated in a I-cm quartz cuvette by monochromatic UV irradiation from a large grating monochromator 1~. The incident intensity at the sample position ranged from I.O to IO. lO 3 erg/cm 2 sec (depending on the wavelength) with the same total band-width of 4.4 nm; actual irradiation times were adjusted to give an equal absolute energy for all wavelengths. (In view of the simplicity in calculations and consistency with part I in this series of papers, the doses have been expressed in sec. The absolute dose has been given when necessary.) In other experiments when more spectral purity was desirable as to the irradiating wavelength, the band-width was narrowed to 2.o nm, the intensity being uniformly reduced. The doses were chosen in the survival range higher than 6o%. The region from 245 to 28o nm was covered with 5-nm steps. The cells were stirred continuously with a small magnetic stirrer during irradiation. The photoreactivation of UV-treated cells was performed by illumination with two 2o-W daylight fluorescent lamps at constant temperature (31.5°). The illumination time was 9 ° min at a lamp-sample distance of IO cm. According to our previous experience, 9° min achieves maximal photo-

UV

A C T I O N S P E C T R A FOR G E N E C O N V E R S I O N

45

reactivation. Methods of assay for gene conversion were based o n ZIMMERMANN AND SCHWAIERs and have also been described previously 7. Action spectra were obtained by plotting the values of parameter a as a function of wavelength. The determination of a was based on the previously adopted formula f - (at)L in which ~. = 2.02 for To, a ~ 1 . 6 4 for T9 and a ~ 1.o 9 for TI~ (Ref. 7). Action spectra obtained by a conventional method in which the frequencyf at a fixed dose (an equal incident energy) is directly plotted against the wavelength are also presented for comparison. Since only a relative value of a is important in the present experiments no attempt to correct the incident dose for scattering has been made; scattering factors on the cell surface, at the air-quartz interface, and at the quartz-water interface were assumed to be equal for all wavelengths used. For absorption spectra measurements, calf thymus D N A (Type I) and soluble R N A from yeast were purchased from Sigma Chemical Co., and thymines (reagent grade) from Tokyo Kasei Co. All measurements were made in sodium phosphate buffer (0.067 M) adjusted to pH 6.8. RESULTS

Determination of parameter a and action spectra of To and To Based on the rationale described in previous workL parameter a was determined for each wavelength by measuring the induced frequency at the predetermined dose. The value of parameter a. used in the calculations was taken from previous work 7. In this way, the whole dose-frequency curve is not necessary so long as the selected experimental points fall within the range in which the proposed formula is applicable. Measurements of the induced frequency were first carried out for two cell stages To and Tg, presumably having quite different conformational states of DNA. Two differTABLE I INDUCED FREQUENCY OF GENE CONVERSION f ( - 1 0 4 ) AT TWELVE WAVELENGTHS IN THE U V REGION FOR THE TWO CELL STAGES T o AND T9, SELECTED DURING SYNCHRONOUS GROWTH Wave-

Induced frequencies

length

Stage T O

( n.O

7° sec b

o.8o 2.22 4.17 7-35

230 240 245 25 ° 254 260

. 15.o

265 270 275 28o 29 ° 30o

21.2 22. 4 21.8 18. 7 3.82 . .

.

at two doses a Stage T 9

Relative to 2 7 0 n m

iio sec b

Relative to 2 7 0 n m

7° sec b

o.o36 o.o991 o.186 0-328 . 0.670

3.03 5.23 9.1o 18-3

o.0779 o.134 o.234 0.47o

.

38.7

0.995

36.8e 38.6 e 39.6 e 42.8 11.34

o.946e I.OOe I .o2 e I.IO o.292

0.946 I.OO 0.973 0.835 o.171 .

.

.

Relative to 2 7 0 n m

.

4 .62 6.48 7.1o

. o.222 . °.4°2 0.563 o.617

8.68 11. 5 . . 7.87 5.20 0.59

0.755 I.OO . 0.684 o.452 o.o51

2.55 .

.

Iio sec b

Relative to 2 7 0 n m

. 3.73

o.142

9.48 lO.9 18.5

0.362 o.416 0.706

21. 4 26.2

o.817 I.OO

22.8 8.39 1.o9

0.870 0.320 o.o416

.

.

a S p o n t a n e o u s l e v e l s ( I . 6 9 . lO -4 f o r T o a n d 1 . 4 2 . lO -4 f o r Tg) h a v e b e e n s u b t r a c t e d . bThe dose of 7 ° sec is e q u i v a l e n t to 8.82. i o a erg/crn ~ for all w a v e l e n g t h s ; t h e dose of IiO sec is e q u i v a l e n t to 1 . 3 9 . lO 5 e r g / c m 2 for all w a v e l e n g t h s . e A p p a r e n t s a t u r a t i o n in t h e i n d u c t i o n of g e n e c o n v e r s i o n 7.

4b

"f. I T ( I ,

K.

KOI',AYASHI

TABLE iI V A L U E S O F P A R A M E T E R a " 1() 4 ( s e c 1) AT T 0 A N I ) A S S U M E D AS 2 . 0 2 F O R T 0 A N D ] . 0 4 F O R Z 9

T 9 CALCIJLATEI)

7"0

Wavelenth ¢nm)

23 ° 240 245 250 254 26o 265 27o

275 28o 29 ° 3 °o

f

FROM

(al) x,

\VI[F;Rt-; ,% IS

T.

Dose (sec)~ . . . . . . . 7° 2io

~)ve~'agi;-~'~rma/ized" at 270 n m

Dose (sec)a-70 IlO

1.31 2.I8 2.98 3.95

1.62 2.12 2.80 3.96

t.47 2.I5 2.89 3.96

o.214 o.313 0.420 0.576

5.63 6.69 6.88 6.78 6.28 2.85 -

5.74 5.6o la 5.76b 5.8Ib 6.o 4 3.12

5.69

o.827 (3.972 i.ooo 0.985 o.895 o.435

-o.91 . . 1.31 1.61 1.7 ° 1.93 2.29

6.16 2.99

.

1.8i 1.41 o.37

0.73 . 1.29 1.41 1.94 2.13 2.41

.

1.2I 1.2o o.35

,.tverag,

Normali:ed at 270 tTm

0.82 . 1.3 ° r.5i ~.82 2.o3 2.35

o.349 0.553 0.643 (3.774 o.864 [.ooo

2.Ol 2.31 o.36

o.855 o.557 o.153

aSee f o o t n o t e to T a b l e I for t h e m e a n i n g of dose. bThese v a l u e s h a v e been o m i t t e d in later calculations (see f o o t n o t e c to Table l).

10

I

I

I

I

E

I

I

I

5

i

1.0 0.5

2

N

0.2 x

0.5

0.1 0

~_

O.2

0.05 DNA

0.1

I I f 230 250

I

I 270

Wavelength

I

I 290

(rim)

I

0.02 310

i

230

I

J

250

f

r

270

Wavelength

[

I

290

absorption I

I

J

310

(nm)

Fig. i. Action s p e c t r a ( w a v e l e n g t h d e p e n d e n c e of p a r a m e t e r a of To(: ) a n d Tg(./ ) for t h e induction of gene conversion at t r p 5 . Fig. 2. A c o m b i n e d action s p e c t r u m (O) b a s e d on t h e a v e r a g e of a a t T Oa n d T~. T y p i c a l a b s o r p t i o n s p e c t r u m of D N A (--) (in o.o67 M p h o s p h a t e buffer, p H 6.8) is included for c o m p a r i s o n . B o t h s p e c t r a h a v e been n o r m a l i z e d to i a t t h e p e a k w a v e l e n g t h .

ent dose levels were selected in the survival range higher than 6o%

for each wave-

length. The data are listed in Table I for twelve wavelengths ranging from 23o to 3oo n m . T a b l e I I s h o w s t h e c a l c u l a t e d v a l u e s o f p a r a m e t e r a. A c t i o n s p e c t r a f o r To a n d T~ w e r e t h e n o b t a i n e d b y p l o t t i n g a a s a f u n c t i o n o f w a v e l e n g t h , a s s h o w n i n F i g . I. I n b o t h To a n d Tg, t h e m a x i m u m o c c u r r e d a t 2 7 0 n m . T h e t w o s p e c t r a r e s e m b l e d

47

U V ACTION SPECTRA FOR GENE CONVERSION

one another in shape although the absolute values of a differed by a factor of about 3 between the two. The ratios a,~/a2:o (where ~ denotes the wavelength) are listed in the last column of Table II. The close resemblance of two spectra allows us to combine these ratios for plotting a single representative action spectrum. As shown in Fig. 2, the combined spectrum is shifted b y 12 nm toward the longer wavelength as compared with the absorption spectrum of DNA. Action spectra based on a conventional method Even for the effect with non-linear responses it is customarily permissible to use comparisons of the induced effects at a fixed equivalent dose for obtaining the action spectrum. The action spectra based on this direct comparison of induced frequency, f~/f27o (where ~ denotes the wavelength), are shown in Fig. 3 (triangles) for A

~.

1.o

E E

0.5

-o E

.N E

~"

0.2

./

O

~

0.1 0.05

E

o

~

0.02

/ 1

230

I

I

250

I

I

270

I

I

290

I

I

I

310

Wovelength ( n m ) Fig. 3- Action s p e c t r a based on the direct comparison of frequency, f~/f=7o, at a fixed dose for T o and T o (a conventional method). A, To (7o-sec data); A, T9 (average of 7o-sec and i i o - s e c data). Action s p e c t r u m based on a (O) is reproduced from Fig. 2.

To and T,. Clearly these spectra are deformed from the spectrum (filled circles) based on parameter a. In a special case, where ~ is sufficiently close to I (linear dose response), this distortion should disappear. Photoreactivation of UV-induced gene conversion If UV action spectra for gene conversion are generally shifted significantly toward the longer wavelength in reference to the absorption of DNA, what would be the target in which primary damage occurs ? A test of photoreactivation by visible light is the most effective means for diagnosing in vivo the nature of the induced damage. The results for To and T9 are summarized in Table I I I along with the survival data. The UV dose used here is relatively low and comparable to those used in other experiments. Judging from the high photoreactivability both in To and Tg, the majority of the induced damage responsible for the effect in question must be pyrimidine dimers irrespective of the stage. Thymines, which contribute to most of the pyrimid-

4~

1". IT(), I,2. 1,2()t',\Y,\~;HI

TABLE

li I

PHOTORJ~;ACTIVATION OF If\" (27O Uln) INI)UCEI) KILLING AND GEN[-; CONVF, RSION Cell

,S'arvival ( ~!;~)

stage

No tr~atment

~jo scc (" I "a

9 o sec U 1" 4 y o rain V I ) ~

l~h, oto

(a)

(t~J

(e)

T0

IOO

(~4..4

94-7

S 5. i

]'9

I oo

S ~ .o

97.2

~5.3

( ; e n e conversion ./'re~ u e n c y

TO T~

] . , ~ 9 - I O -'5 3 . 8 1 . 1 o -5

(~!o) c

reactivabdity

Photo reactivability

3.21 . i o a 2 . 3 O . l O :~

l . I 4. i o 3 8.19.1o ~

(%) e

68.o 65. 5

a i . i 3 . l O a e r g / c n l 2. bVL, visible light. ephotoreactivability:

(e b) .'~: I O O / ( l o o b) for k i l l i n g ; ( b - e ) ;x: I O o / ( b a) for g e n e c o u v e r s i o n .

ine dimers, however, are known to have the absorption peak at 265 nm (see also Fig. 5).

More precise action spectra of To and TI~ If thymine dimers are the major primary damage responsible for the induction of gene conversion in To and Tg, w h y do not action spectra precisely overlap the absorption spectrum of thymine residues ? Our action spectra showed the peak at 27o nm whereas the absorption peak of thymine occurs at 265 nm as mentioned. (DNA has tile absorption peak at 258 nm.) Among several possibilities that might explain this discrepancy we tested the interference by shielding substances such as R N A against the penetration of incident UV. For this purpose we chose T16, because by this stage the cell should accumulate a large amount of R N A 1° as compared with To. Furthermore, the value of parameter a at T16 was 40 times less than that of To at 27 ° nm, an unexplained finding in the previous work 7. The experiments were carried out with a narrower bandwidth of 2.0 nm to achieve higher resolution. The action spectrum for To was also made with the same culture as a reference. By taking advantage of the known value of ~ for To and TI~ (ref. 7), we only measured the frequency at a single optimal TABLE

IV

V A L U E S OF P A R A M E T E R a AT T O A N D T16 C A L C U L A T E D F R O M f FOR Z 0 A N D I . O 9 FOR T l g Dose

used here was 7 ° sec (equivalent

--

(at) ~,

W'HERE ~ IS A S S U M E D AS 2.02

t o 8 . 8 2 . i o 4 e r g / c m ~) f o r T o a n d i 1o s e c ( e q u i v a l e n t

t o 1 . 3 9 . lO5

e r g / c m 2) f o r TI~.

Wavelength

TO

(nm)

f. zo 4

245 25 ° 254 26o 265 27o 275 280 29 °

3.02 5.64 9.25 7.39 25.6° 33.88 34.87 33.o3 8.46

T16 a . ±o 4

Normalized

(see 1)

at 270 n m

2.58 3.52 4.5 ° 6.I5 7.44 8.55 8.68 8.45 4.3 °

0.302 o.412 o.526 °.719 °-87° 1 .ooo I .Ol 5 0.988 0.503

a f. zo 4

4.38 7.17 7.95 9.64 1 ~. 14 14.74 17.26 17.21 5.95

a. zo G

Normalized

(sec -1)

at 2 7 5 n m

7.54 i 1.85 13.°3 15.55 17.76 22.96 26.53 26.46 9-99

0.284 0.447 o.491 o.586 o.669 o.865 I.OOO 0.997 0-377

a

UV

ACTION SPECTRA FOR GENE CONVERSION

49

dose as j u d g e d b y previous experience a n d c a l c u l a t e d t h e value of a for each wavelength. The results are listed in T a b l e IV a n d shown in Fig. 4 as a function of wavelength. Several f e a t u r e s were noted. (I) The 4 ° times difference b e t w e e n To a n d TI~ in a was g e n e r a l l y e x t e n d e d to t h e whole range of wavelengths. (2) The action s p e c t r u m of T16 was shifted f u r t h e r (17 nm) t o w a r d the longer w a v e l e n g t h as c o m p a r e d w i t h t h e a b s o r p t i o n s p e c t r u m of D N A . (3) A n o t a b l e d e f o r m a t i o n in t h e action s p e c t r u m of T16, as c o m p a r e d with t h a t of To, was seen in t h e range b e t w e e n 250 a n d 27o nm. I t should 20

10

I I I I I I I I I --

5

A

7u

2

v

1

,qr 0 x

0.5

t3

: