Ultraviolet-induced pyrimidine dimers in tetrahymena

285

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95930

ULTRAVIOLET-INDUCED P Y R I M I D I N E DIMERS IN T E T R A H Y M E N A I. REMOVAL IN T H E DARK

G. L. W H I T S O N , A. A. F R A N C I S AND W. L. C A R R I E R

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. (U.S.A.) (Received F e b r u a r y i4th, 1968)

SUMMARY

Thymine-containing pyrimidine dimers have been isolated and identified chromatographically from DNA hydrolysates of ultraviolet-irradiated Tetrahymena. Removal of these photoproducts has been observed during post-irradiation growth of cells in the dark. After 47 h, about 83 % of the pyrimidine dimers disappear from the trichloroacetic acid-insoluble fraction. Further analysis of both the triehloroacetic acid-insoluble and trichloroacetic acid-soluble fractions indicated that photoproducts accumulating in the soluble fraction are eventually lost from the cytoplasm of irradiated cells.

INTRODUCTION

There has been an expanded interest in the effects of ultraviolet light on cellular systems since the finding of BEUKERS AND BERENDS1 that dimerization of thymine molecules occurs in ultraviolet-irradiated frozen solutions of thymine. I n vivo studies with bacterial cells have shown that thymine dimers inhibit DNA synthesis 2. The findings that a photoreactivating enzyme from yeast destroys pyrimidine dimers in DNA (refs. 3-5) and also that repair in the dark involves excision of pyrimidine dimers in cells 6,7 have made it fruitful to study the relationship of the production and fate of pyrimidine dimers to the lethal and mutagenic effects of ultraviolet irradiation on cells. Most of the available data on the production and fate of pyrimidine dimers have come from either in vitro studies or from reports on bacterial cells. Although thymine dimers have been reported in eukaryotes, namely fish s and mammalian cells 9-13, it is only recently that they have been looked for and identified in protozoan cells14-17. The present paper is a report on thymine-containing pyrimidine dimers in Tetrahymena and their removal during post-irradiation growth of the cells in the dark. Abbreviation: BBOT, 2,5-bis-2-(5-tert.-butylbenzoxazolyl)-thiophene.

Biochirn. Biophys. Acta, 161 (1968) 285-290

286

G. L. WHITSON, A. A. FRANCIS, W. L. CARRIER

MATERIALS AND METHODS

Growth and labeling o! cells Tetrahylnena pyriformis strain GL-C was grown on medium consisting of 1 % proteose peptone, o.I % liver extract (NBC conc. I:2O), and the following salts: NaC1 (0.2 % w/v), KH~PO 4 (o.i % w/v), Na2HPO 4.12 H20 (o.I % w/v), and MgCI~. 6 H~O (0.03 % w/v). Cells were grown in IO ml of this medium overnight (12-15 h) to a concentration of 25 000-30 ooo cells/ml with IO/~C/ml of C3Hlthymidine (specific activity 3.0 C/mmole) added to the medium and then transferred to the same amount of unlabeled medium for 2 h. Immediately before irradiation, cells were removed from the medium b y centrifugation, washed and suspended in IO ml of the salt solution used for preparing media.

Irradiation 2-ml samples of cells in the salt solution were placed in quartz cuvettes of I-cm path length and irradiated with monochromatic light from a Hilger quartz prism monochromoter with a high-pressure mercury arc source. The incident intensity was about 540 ergs/mm ~ per rain. The samples were stirred continuously during irradiation with a magnetic stirrer. Corrections were made for dosimetry according to MOROWITZ 18.

DNA isolation and chromatography DNA was isolated using a modification of the MARMUR technique 19. The cells were first lysed with sodium lauryl sulfate and the bulk of the protein was precipitated by chloroform-iso-amyl alcohol (24:1, by vol.). During lysis, carrier DNA (IOO/~g from calf thymus) was added to aid in the recovery of labeled DNA. The isolated DNA was then precipitated with ethanol at room temperature and hydrolyzed with 97 % formic acid for I h at 175 °. In some cases cells were treated with 5 % trichloroacetic acid. The soluble fraction was washed 3 times with ethyl ether to remove the trichloroacetic acid, then both insoluble and soluble fractions were air dried and hydrolyzed with IOO % trifluoroacetic acid for I h at 155 ° (ref. 20). The separation of pyrimidine dimers from thymine was accomplished by paper chromatography. Some samples were chromatographed on Whatman No. I paper using n-butanolacetic acid-water (8o:12:3o, by vol.) for the first dimension 21 and n-butanol-water (86:14, by vol.) for the second dimension ~2. Others were chromatographed in the first dimension with n-butanol-water (86 : 14, by vol.) and in the second dimension with saturated ammonium sulfate-I M sodium acetate-isopropyl alcohol (40 : 9 : I, by vol.) 21. In both cases, chromatography in the second dimension results in the removal of residual thymine due to streaking in the first dimension. To determine the distribution of label along the chromatograms, we cut them into i cm strips and eluted them with I ml of water; the eluate was counted in a scintillation counter (Packard Model 3oo2) using IO ml of dioxane-2,5-bis-2-(5-tert.-butylbenzoxazolyl)thiophene (BBOT)-naphthalene scintillator (BBOT, 12 g; naphthalene, 50o g; and dioxane (Eastman), 4 1). Biochim. Biophys. Acta, 161 (1968) 285-29o

ULTRAVIOLET INDUCED PYRIMINE DIMERS

I.

287

RESULTS AND DISCUSSION

Production o[ pyrimidine dimers Pyrimidine dimers have been produced in both cells and isolated DNA of Tetrahymena irradiated with 2652 ~, of ultraviolet light. The irradiation of cells with 5400 ergs/mm 2 resulted in the conversion of 0.5 % of the thymine to thymine-containing dimers. Lower doses produced proportionally fewer photoproducts, and irradiation of isolated DNA produced about 5 times more thymine-containing dimers than were in the DNA of irradiated, intact cells. Cytoplasmic shielding of the DNA in cells probably accounts for the difference. We identified photoproducts in hydrolysates of DNA from irradiated Tetrahymena on the basis of their chromatographic mobilities as described by SETLOW AND CARRIER 23, and the results of one experiment are shown in Fig. I. With n-buta~00-

I0-

LO

03

o~

O.Ol V--A--A

0.001

oi,

~.2

o13 RF 6.~

o15

do

o.~

Fig. I. The distribution of radioactivity along cb_romatograms of acid hydrolysates of Tetrahymena DNA labeled with ? H ] t h y m i n e . The percent label observed in both tile pyrimidine dimer regions and thymine of ultraviolet-irradiated (5400 ergs]mm 2, ~ = 2652 A) ( O - O ) and nonirradiated ( A - A ) cells are shown. ~ represents a cytosine-thymine dimer (see RESULTS); T'~ a thymine-thymine dimer; and T, thymine.

nol-acetic acid-water as the solvent, paper chromatograms of the acid hydrolysates of cells showed a major peak at an R F of 0.3 ° and a minor peak at 0.22. The major peak (thymine-thymine dimer) contained 4 times more label than the minor peak (uracilthymine dimer). It has been shown that the uracil-thymine dimer (~'T) results from the conversion of cytosine to uracil by deamination during acid hydrolysis ~. Further proof that these products were indeed thymine-containing dimers was obtained by the kinetics of their monomerization to thymine upon reirradiation with 2400 ~,. After Biochim. Biophys..4eta, 161 (1968) 285-29o

288

G. L. WHITSON, A. A. FRANCIS, W. L. CARRIER

4" IO* ergs/ram2 of 24oo h, we found that 69 ~o of the total label appeared as thymine and 31% of the total label remained as unaltered photoproducts. These values agree with those obtained by SETLOW AND CARRIER2~ for the photoreversal of thymine dimers formed from the irradiation of frozen thymine. The finding that the amount of label in ~ dimers in Tetrahymena was 4 times greater than the amount of label in 6~ dimers is similar to the results obtained /X by SETLOW AND CARRIER23 for H. in/luenza DNA, but in contrast to the ratio of TT to ~ (3.0) found in Escherichia coll. These findings are probably attributable at least in part to the base compositions of the respective DNA's, in which it has been shown that both H. in/luenza 23 and Tetrahymena DNA have high A + T contents (75 % A + T for Tetrahymena 24) and E. coli has a lower A + T content (5o %) (ref. 25).

Removal ol dimers in the dark Evidence for repair synthesis of DNA in Tetrahymena during growth of cells in the dark after ultraviolet irradiation has been reported by BRUNK AND HANAWALT26,27. They observed the incorporation of 32p and 5-bromodeoxyuridine into parental strands of DNA (first labeled with all) after ultraviolet irradiation of cells and concluded that the incorporation of 32p-label was into regions of the DNA where pyrimidine dimers had been excised. Our direct observations verify this. We compared the amount of label in the pyrimidine dimer regions of chromatograms prepared from hydrolysates of ultraviolet-irradiated cells with those obtained from hydrolysates of unirradiated cells grown for different periods of time in the dark. In such experiments, equal aliquots of the original irradiated sample (252o ergs/mm 2 at 2625 ~) were hydrolyzed at different times after irradiation. The results of a typical experiment are summarized in Table I. Note that two controls were used in this experiment; one was an unirradiated sample hydrolyzed at zero time and the other, an unirradiated sample hydrolyzed at the end of the experiment. The percentage of total counts observed in the TABLE I THE

DISTRIBUTION

OF RADIOACTIVITY

IN CELLS FOLLOWING

ULTRAVIOLET

IRRADIATION

The f i r s t c o l u m n i n d i c a t e s t h e t y p e of t r e a t m e n t g i v e n t o e a c h s a m p l e of cells fol l ow e d b y g r o w t h in t h e d a r k . U l t r a v i o l e t i r r a d i a t i o n = 252o e r g s / m m * a t 2652 •. The s e c o n d c o l u m n i n d i c a t e s t h e t o t a l c o u n t s / r a i n [ S H ] t h y m i n e a n d c o l u m n 3 t h e p e r c e n t l a b e l e d t h y m i n e . T h e no u l t r a v i o l e t s a m p l e s (I a n d 7) are b a s e d on a s m a l l e r s a m p l e size t h a n t h e u l t r a v i o l e t s a m p l e s . All o t h e r p e r c e n t s in t h e t a b l e are r e l a t i v e to t h e t o t a l c o u n t s ( c o u n t s / m i n [ 3 H ] t h y m i n e ) i n s a m p l e n u m b e r 2. C o l u m n 4 i n d i c a t e s t k e P e r c e n t r a d i o a c t i v i t y of [3H ~ t h y m i n e as p e r c e n t p y r i m i d i n e d i m e r s ( ( I : T + T T ) / T × IOO ~ ~O PYPY) m t h e t r l c h l o r o a c e t l c a c i d - i n s o l u b l e f r a c t i o n a n d c o l u m n 5 i n d i c a t e s t h e p e r c e n t PyPy in t h e s o l u b l e f r a c t i o n .

Sample

i 2 3 4 5 6 7

No u l t r a v i o l e t - - o h Ultraviolet-- o h Ultraviolet+8 h U l t r a v i o l e t + 24 h U l t r a v i o l e t + 31 h Ultraviolet+47 h No u l t r a v i o l e t + 4 7 h

Total E~H]thymine (countsimin)

jail]thymine (%)

Insoluble /" (% PYPY)

Soluble

282 343 364 31o 216 165 274

ioo IOO lO6 9o 63 48 98

o.oo 7 o. 197 o.134 o. 119 0.098 0.034 o.ooo

o.oo 5 o.oo4 o.063 o.022 O.Ol 7 O.Oli o.ooi

ooo 3° o 40o ioo 300 700 90o

Biochim. Biophys. Acta, 161 (1968) 285-29o

/" (% PYPY)

ULTRAVIOLET INDUCED PYRIMIDINE DIMERS I.

289

pyrimidine dimer regions of the controls (both the trichloroacetic acid-insoluble fractions) was negligible and is attributed to experimental error and chromatographic streaking. A comparison of the total counts in labeled thymine in the two controls indicates labeled D N A is conserved and is not broken down in unirradiated cells. This is in direct contrast with the irradiated cells in which we have observed a loss of labeled thymine from the DNA during growth in the dark after irradiation (see columns 2 and 3, Table 1~). At the time of 47 h of growth in the dark, nearly 52 ~o of the labeled thymine is lost from the DNA. Pyrimidine dimers are also lost from the trichloroacetic acid-insoluble fraction during post-irradiation growth of cells in the dark. The percentages of the total radioactivity of the trichloroacetic acid-insoluble fraction that is observed in cells are shown in column 4 of Table I. About 83 % of the thymine-containing photoproducts disappear from the insoluble fraction of cells grown in the dark for 47 h. The rate at which these photoproducts are lost, however, is more rapid than the loss of labeled thymine from DNA. While the loss of labeled thymine is difficult to explain, it has been shown in another strain of T e t r a h y m e n a b y SHEPARD28 that more than the normal complement of DNA is synthesized in cells after irradiation of 2000 ergs/mm 2 of ultraviolet light at 254 °/~, and that there is a loss of this newly synthesized as well as preiabeled DNA during successive cell generations. The changes in percent radioactivity of [3H]thymine as pyrimidine dimers in the trichloroacetic acid-soluble fraction during growth in the dark are shown in column 5, Table I. As expected, we observed no excised photoproducts ( P ~ y dimers) in the trichloroacetic acid-soluble fraction immediately after irradiation. We did observe, however, (8 h later) accumulation of photoproducts in the trichloroacetic acid-soluble fraction and then a decrease. We compared the loss in total percent thymine containing pyrimidine dimers from both the trichloroacetic acid-soluble and trichloroacetic acid-insoluble fractions. Note in Fig. 2 that during at least the first 8 h after

50

~

~,

\\ xx

o

0 0

'o

,b

~o

s'o

4b

s'o

GROWTH IN DARK ( h ) AFTER U.V.

Fig. 2. The percent total thymine-containing pyrimidine dimers observed in the tricbloroacetic

acid-insoluble fraction (/x_ ~), trichloroacetic acid-soluble fraction ( O - - O ) , and both fractions combined (F1 - - - IN) after post-irradiation growth of cells in the dark at varying periods of time. Ultraviolet (U.V.) irradiation: 2520 ergs/mm2 at 2652 A. A

ultraviolet irradiation there is no loss in total pyPy dimers from the cells. I t is apparent, however, that later there is a loss of PyPy dimers from the trichloroacetic acid-soluble fraction because photoproducts accumulating in the fraction are lost from the cytoplasm of irradiated cells. Biochim. Biophys. Mcta, 161 (1968) 285-29o

290

G . L . WHITSON, A. A. FRANCIS, W. L. CARRIER

Although we have no data on the quantitative correlation of ultraviolet-induced photoproducts in cells with delays in cell division (15-2o h delay at 3125 ergs/mm 2, 2652 A), we have results which indicate that by the time cell division is resumed most of the pyrimidine dimers have been removed. It has been reported that photoreactivating light reduces dark repair in Tetrahymena, and that this effect was probably due to direct cleavage of pyrimidine dimers in situ 2e,27. We previously reported biological reversal (photoreactivation) with short exposures to near-ultraviolet light which involved only the disappearance of a few dimers 17. Studies now in progress concerning photoreactivation of thymine-containing photoproducts indicate that long exposures to either near-ultraviolet light (3650 A), black light, or white light destroys thymine-containing pyrimidine dimers in situ. It remains, to be seen whether or not there is a quantitative correlation between biological reversal and the monomerization of pyrimidine dimers.

ACKNOWLEDGEMENTS

Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation. The authors express their sincere appreciation to Drs. R. B. SETLOW, R. F. KIMBALl. AND J. D. REGAN for helpful criticisms in the preparation of this manuscript.

REFERENCES i 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

R. BEUKERS AND W. BERENDS, Biochim. Biophys. Acta, 41 (196o) 55 o. R. B. SETLOW, P. A. SWENSON AND L. CARRIER, Science, 142 (1963) 1464. D. L. WULFF AND C. S. RUPERT, Biochem. Biophys. Res. Commun., 7 (1962) 237. J. K. SETLOW AND R. B. SETLOW, Nature, 197 (1963) 560. J. S. COOK, Photochem. Photobiol. 6 (1967) 97. R. B. SETLOW AND W. L. CARRIER, Proc. Natl. Acad. Sci. U.S., 51 (1964) 226. R. P. BOYCE AND P. HOWARD-FLANDERS, Proc. Natl. Acad. Sci. U.S., 51 (1964) 293. J. D. REGAN AND J. S. COOK, Proc. Natl. Acad. Sci. U.S., 58 (1967) 2274. J. E. TROSKO, E. H. Y. CHU AND W. L. CARRIER, Radiation Res., 24 (1964) 667. D. L. STEWARD AND R. M. HUMPHREY, Nature, 212 (1966) 298. M. KLIMEK AND M. VLASINOVA, Intern. J. Rad. Biol., i i (1966) 329. J. E. TROSKO AND M. R. KASSCHAU, Photochem. Photobiol. 6 (1966) 215. J. D. REGAN, J. E. TROSKO AND W. L. CARRIER, Biophys. J . , 8 (1968) 319. G. L. WHITSON, A. A. FRANCIS AND W. L. CARRIER, J. Protozool., 14 (1967) (Suppl.), 8. B. M. SUTHERLAND, W. L. CARRIER AND R. B. SETLOW, Science, 158 (1967) 1699. B. M. SUTHERLAND, W. L. CARRIER AND R. B. SETLOW, submitted to Biophys. J., 8 (1968) 490. G. L. WHITSON, A. A. FRANCIS, W. L. CARRIER AND K. L. GRANDI, J. Cell. Biol., 35 (1967) I42A. H. D. MOROWlTZ, Science, i i i (195 o) 229. J. MARMUR, J. Mol. Biol., 3 (1961) 208. S. K. DUTTA, A. S. JONES AND M. STACEY, J. Gen. Microbiol., 14 (1956) 16o. K. C. SMITH, Photochem. Photobiol., 2 (1963) 503 . A. WACKER, H. DELLWEG AND n . WEINBLUM, Naturwissenscha]ten 47 (196°) 477. R. B. SETLOW AND W. L. CARRIER, J. Mol. Biol., 17 (1966) 237. N. SUEOKA, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1141. J. E. M. MIDGLEY, Biochim. Biophys. Acta, 61 (1962) 513 . C. F. BRUNK AND P. C. HANAWALT, Biophys. Soc. Abstr., (1967) 82. C. F. BRUNK AND P. C. HANAWALT, Science, 158 (1967) 663. D. C. SHEPARD, Exptl. Cell Res., 38 (1965) 57 o.

Biochim. Biophys. Acta, 161 (1968) 285-29o