REPAIR O F UV-INDUCED PYRIMIDINE DIMERS IN

DROSOPHILA MELANOGASTER CELLS I N VITRO*

JAMES E. TROSKOt AND KATHY WILDER

Department of Human Development, Michigan State Uniuersity,
East Lansing, Michigan 48823
Manuscript received August 24, 1972
Revised copy received October 25, 1972
Transmitted by DAVIDSCHLESSINGER

ABSTRACT

Tissue culture cells of Drosophila melanogaster were given various doses of

ultraviolet light. The results indicate that Drosophila cells do have a dark-
repair excision mechanism which is not sensitive to caffeine. Pyrimidine dimers
were destroyed by photoreactivating illumination in these cells and this
destruction probably represents monomerization of the pyrimidine dimers.

HE detailed molecular basis for mutagenesis in either prokaryotes or eukary-

Totes has not been delineated. However, experimental progress has implicated
the non-repair of DNA lesions or the type of enzymatic mechanism involved in
DNA repair with the mutation process ( WITKIN1969; BRIDGES1969).
Drosophila melanoguster continues to be one of the most convenient and well-
studied eukaryotic organisms in mutagen testing. Therefore, with recent avail-
ability of Drosophila cells in tissue culture (SCHNEIDER 1971), it is now tech-
nically feasible to characterize some DNA repair mechanisms.
Several investigations have examined ultraviolet radiation effects on killing
and mutagenesis (DEMEREC et al. 1942; GOLDMAN and SETLOW1956; PRUD-
HOMMEAU 1972), as well as the effect of photoreactivation on UV-induced muta-
genesis and phenocopy induction in Drosophila (ALTENBURG and ALTENBURG
1957; PERLITSCH and KELNER1953; BROWNING and ALTENBURG 1962). Several
unsuccessful attempts have been made to detect the photoreactivation enzyme in
Drosophila (MUHAMMED and TROSKO 1967; COOK1970). This report includes
the demonstration of direct photoreactivation and the dark repair-excision of UV-
induced pyrimidine dimers in Drosophila cells grown in vitro.

MATERIALS A N D METHODS

Tissue Culture: Tissue culture cells of Drosophila melanogaster derived from embryos by
DR. I. SCHNEIDER (line e ) , obtained from DR. GEORGE MIKLOS(University of California, San
Diego), were used for all the following experiments. The cells were grown at 25C in Schneider's
* This investigation was supported b y U.S. A.E.C. Contract AT-(ll-1)-1704, by Public Health Service Research
Grant No. CA 13W-01 from the National Cancer Institute and by N.I.II. Research Career Development Award 1 KM
CA 24085-01to Dn. TROSKO.
{- On leave o f absence at the MrArdle Iiiboratory for Cancer Kesearch. Univerqity of Wisconsin, hIadison. M'iscunsin
53706.

Genetics 73: 297-302 February, 1973

298 J. E. TROSKO A N D K. WILDER
medium-revised (Grand Island Biological), supplemented with Difco Bacto-peptone (500 mg/l00
ml) and with 15% calf serum. Cells were plated in 60 mm Falcon plastic petri dishes for
experiments.
Zrradiafion Procedures: All the UV irradiations were performed with a germicidal lamp
(G 25T8, delivering primarily 254 mp light), mounted in a Microvoid transfer hood. The incident
dose rate to the surface of the 1/2 ml Drosophila Ringers solution containing the cells in a 60 mm
petri plate was 25 ergs/mmZ/sec (dose rate determined by a YS1 Model 65 radiometer, Yellow
Springs Instruments, Yellow Springs, Ohio). After UV irradiation, the Ringers solution was
decanted and 3 ml of Schneiders media were placed on the cells to be tested for dark repair. The
cells were placed in a dark incubator for twenty-four hours Photereactivaitng light was delivered
by three General Electric black-light lamps (GE-Fl5T8-BLB; 320-450 mp, predominantly 360
mp) and one standard 15 watt daylight fluorescent bulb. Lamps were positioned to give a flux of
4800 ergs/mmz/minute through 1 cm of plate glass. Cells that were photoreactivated after UV
irradiation were placed in 1.5 ml of the Schneiders medium for the radiation exposure.
Pyrimidine Dimer Assay: To analyze pyrimidine dimers in these Drosophila cells, the follow-
ing procedures were used. Drosophila cells were inoculated into 60 mm Falcon plastic petri dishes
(1 X IO6 cells/plate) . After one days growth, fresh medium with tritiated thymidine (10 pc/ml;
53.4 Ci/mM, New England Nuclear) was added. After two days incubation in the radioactive
medium, non-radioactive medium was placed on the cells for 4 hours. Prior to irradiation, the
medium was decanted from the plates, the edge of the monolayer of cells was scraped with a
rubber policemanto remove cells that would be shadowed by the edge of the petri dish and the
cells were carefully washed twice with Drosophila Ringers solution. The cells were irradiated in
0.5 ml of the Ringers solution. Immediately after the irradiation, the cells were either harvested
or re-incubated in the dark in medium or photoreactivated. After the respective radiation or post-
irradiation treatment, cells were fixed in cold 5% trichloroacetic acid and the insoluble residue
was analyzed for pyrimidine dimers by two -dimensional chromatography after hydrolysis in
formic acid (CARRIER and SETLOW 1971).

RESULTS A N D DISCUSSION

Results of the experiments, designed to examine the ability of Drosophila cells,

in vitro, to dark repair UV-induced pyrimidine dimers, are shown in Table 1.
Pyrimidine dimers are produced linearly within the dose range given to these
cells. These data are consistent with the interpretation that these cells were able
to excise pyrimidine dimers from their DNA. Because of the technical difficulties
of measuring the excised pyrimidine dimers in the tissue culture medium after
the twenty-four-hour dark incubation, we did not check for the appearance of
pyrimidine dimers in medium as they were disappearing from the TCA-insoluble
fraction of the cells. At relatively low doses, approximately 40% of the pyrimi-
dine dimers were removed, whereas at the high dose point, 90% of the pyrimidine
dimers are still associated with the TCA-insoluble material. However, the data
indicate that a constant number of pyrimidine dimers is excised in 24 hours.
This observation has been observed before in bacterial and human cells ( SETLOW
1964; CLEAVER and TROSKO 1970; SETLOW et a2. 1969).
Caffeine, given as a posttreatment to UV-irradiated bacteria, synergistically
increases the UV-induced killing and mutation frequencies (WITKIN1958; LIEB
1951) by inhibiting the excision of pyrimidine dimers (LUMB,SIDEROPOULOS
and SHANKEL 1968; SETLOW and CARRIER 1968; SIDEROPOULOS and SHANKEL
1968). The results in Table 2 indicate that the excision of UV-induced pyrimi-
 R E P A I R O F U V - I N D U C E D D A M A G E I N DROSOPHILA 299
TABLE 1
r2
Dark repair ( D R ) of ultrauiolet light-induced pyrimidine dimers ( X T ) * in
Drosophila melanogaster cells grown in uitro
0 hours ~ 4: hi-DHf
Cuunts i n XT: X T (7;)
- Counts in xi' x'r ( % )
I

Experinient U\' treatment Counts in T T Counts in T T

A. None 0/474,000 0.00
15 secs 680/93 1,000 0.07 110/415,000 0.03
20 sec 660/612,000 0.1 1 350/602,000 0.06
30 sec 1,4~0/1,015,000 0.14 350/552,000 0.06
45 sec .......... ... 450/267,000 0.17
60 sec 2,250/787,000 0.28 290/110,000 0.26
B. 15sec 260/241,000 0.1 1 140/198,000 0.07
20 sec 890/647,000 0.14 180/215,000 0.09
30 sec 780/320,000 0.21. 710/352,000 0.20
60 sec 1,230/312,000 0.39 560/162,000 0.35
r2 A A
' X T refers to both TT and U T dimers, since chromatography procedures used here do not
separate them from each other.
t DR refers to post radiation dark repair incuhation.
$ IO-minute counts were made of each sample. A background count of 30 cpm was subtracted
from each sample.
Cells were irradiated at 254 mp. The dose rate for experiment A was not determined accur-
ately. The incident dose rate in experiment B was 25 ergs/mmz/sec.

dine dimers in Drosophila cells is not inhibited by caffeine posttreatment. The

same rewlt is noted in human cells ( RECANet al. 1968).
Figure 1 illustrates the results of photoreactivation treatment on UV-irradiated
Drosophila cells. The fact that these cells did not excise a significant fraction of
pyrimidine dimers at the high dose range in twenty-four hours indicates that the
pyrimidine dimers were destroyed rather than excised. Photoreactivating enzyme
activity has been demonstrated in the arthropod, Anagasta kiihniella (Flower

TABLE 2
Lack of an effect of cafjeine on the excision of UV-induced pyrimidine dimers
Drosophila melanogaster cells in uitro'

Experiment Trea mien t

C. UV alone 560/427,000 0.13

U V f 24 dark incubation 37 0/5 13,000 0.07
UV +1 x lo-,<M Caffeine-
dark incubation 320/421,000 0.07
D. UV alone 1,080/937,000 0.12
+
U V 24 dark incubation 340/400,000 0.08
+
U V 1 x I @ * M Caffeine-
dark incubation 180/267,000 0.07
-~~ ~ ~

* Cells were irradiated with 254 mp UV light. Total incident dose was 500 ergs/"' at a dose
rate of 25 ergs/mni"sec. A background count of 28 cpm was subtracted from each sample, 10-
minute counts were made on each sample.
300 J. E. TROSKO A N D K . W I L D E R

0.50

0.40

0.30

0.20

4
ae
0.10

0.08

0.06

0.04
I I I I I I I I I I I

10 20 30 40 50 60

MINUTES PHOTOREACTIVATION
FIGURE 1 .-Light-dependent dimer reversal in vitro in Drolrophila melanogaster cells which
received 1500 ergs/mm2 of 254 mp UV. Ordinate: percent of total thymine as thymine-or uracil-
thymine dimer (XT). Abscissa: time in photoreactivatinglight at 25C.

moth) (COOKand MCGRATH1967). Together with the latter observation and

with observations of biological photoreactivation in Drosophila, we infer that the
destruction of pyrimidine dimers is due to the monomerization of the dimers.
These results indicate that Drosophila melanogaster has at least the excision
and photoreactivation repair mechanisms. It remains to be shown whether
recombination-repair (RUPP and H O W A R D - F L A N D E R S 1968) or postreplication
repair (LEHRMANN 1972; BUHLet al. 1972) mechanisms are found in Drosophila
cells. The excision repair pattern in Drosophila is similar to that which has been
reported in human cells (REGAN,TROSKO and CARRIER 1968) while the presence
of the photoreactivation repair mechanism makes Drosophila cells similar to the
cells of marsupial mammals (COOK 1970) and different from human cells
(CLEAVER 1966; TROSKO 1970). These observations should be of some value either
in studies of comparative mutagenesis (assuming the repair of non-repair of
damaged DNA is somehow related to mutagenesis) or of the mechanism of UV-
induced mutagenesis in eukaryotes.
 R E P A I R O F U V - I N D U C E D DAMAGE IN D R O S O P H I L A 301
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