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INT . J . RADIAT . BIOL .,
1987,
VOL .
52, NO . 1, 107-113
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Possible occurrence of DNA double-strand breaks during
repair of u .v.-induced damage in yeast
M . FRANKENBERG-SCHWAGER, D . FRANKENBERG
and R . HARBICH
Institut fur Biophysikalische Strahlenforschung der Gesellschaft fur
Strahien-und Umweltforschung mbH, D-6000 Frankfurt 70, Paul-Ehrlich-Str .
20,
F.R . Germany
(Received 10 October 1986 ; revision received 15 December 1986 ;
accepted 23 December 1986)
The yeast mutant rad54-3, which is temperature conditional for dsb rejoining, is
sensitive to u . v. light when held at the restrictive temperature following exposure .
We propose that this is attributable to the enzymatic formation of dsb in DNA
containing u .v . lesions and a subsequent lack of dsb repair in this mutant .
1.
Introduction
In yeast three pathways for dark repair of u .v . damage to DNA have been
described (Brendel and Haynes 1973, Game and Cox 1973, Cox and Game 1974,
Haynes and Kunz 1981) : excision repair pathway controlled by the RAD3 epistasis
group, the error-prone repair pathway controlled by the RAD6 epistasis group, and
the recombinational repair pathway (Resnick 1975, Game 1983) controlled by the
RAD52 epistasis group . The excision repair pathway is the most important one for
the removal of u . v . -induced damage, whereas the recombinational repair pathway is
of minor importance (Game 1983) . For mutants blocked in all three repair pathways,
one or two thymine dimers per cell constitute a lethal hit (Cox and Game 1974) .
Mutants blocked only in the pathway controlled by the RAD52 epistasis group
are very X-ray-sensitive but, since they have a functional excision repair pathway,
they exhibit a relatively high resistance to u .v .-light (Game and Mortimer 1974) .
Most of these X-ray sensitive mutants are recombination-deficient (Saeki et al.
(1981)), see Game (1983)) and some have been shown to be defective in the repair of
radiation induced DNA double-strand breaks (dsb) (see Haynes and Kunz (1981)) .
For E . coil K-12 evidence has been presented that after exposure to u .v .-light dsb
appear in the DNA of these cells due to the enzymic repair of base damage (Bonura
and Smith 1975 a, Wang and Smith 1986 a, Sharma and Smith 1986) . In human
fibroblasts the formation of dsb during repair of u .v .-light induced damage was
reported (Bradley and Taylor 1981, Wang and Smith 1986 b) . We were interested in
the possibility that such enzymatically produced dsb also occur in yeast cells after
u.v . -irradiation . If dsb are formed in the course of repair of u .v .-induced base
damage, dsb repair deficient cells might be expected to be more sensitive to u .v .-light
than wild type cells . In order to test this, we have studied the survival response as a
function of u .v . fluence of yeast mutant strains which are temperature conditional for
dsb rejoining . From this type of approach, i .e . using the increased sensitivity of a
M . Frankenberg-Schwager et al .
108
mutant deficient in the repair of a specific lesion as an indicator of the presence of this
unrepaired lesion induced by a given agent (see Moustacchi et al . (1983)), the
involvement of dsb can be reasonably inferred without actually measuring these
breaks .
2.
Materials and methods
2 .1 . Yeast strains
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In these studies we used the following four diploid yeast strains :
1 . rad54-3 (g580) which was kindly provided by Dr J . Game . At 36 ° C the cells
are extremely X-ray sensitive and are also deficient in genetic recombination . In
contrast, at 23 ° C cells become radioresistant and genetic recombination is observed
(Game 1983) . We shall refer to this strain as rad54-3G . Its genotype is :
his 1-1 trp 2
rad54-3 ade 4 leu 2 Mata can 1T ura 3
+
ura
3
hom
3-10
his
l-7 trp 2
rad54-3 + leu 2 Matcc CANS
2 . rad54-3 (X754-3B6D) was kindly donated by Dr M . Budd . At 36 ° C this
mutant is unable to rejoin X-ray induced dsb resulting in its maximal sensitivity to
X-rays, whereas at 23 ° C dsb rejoining occurs and cells are radioresistant (Budd and
Mortimer 1982) . We shall designate this strain as rad54-3B to distinguish it from the
strain rad54-3G described above . Its genotype is:
a his l leu 2 + trp 5 + tup 7 rad54-3
a his 1 + ade trp 5 ura 3 tup 7 rad54-3
The RAD54-3 strains belong to the RAD52-controlled epistasis group .
3 . rad2-17rad52 (PR114) was kindly given to us by Dr M . A . Resnick . This
strain was chosen for its high sensitivity to u .v . light, conferred on it by the
homozygous rad2-17 gene . This strain is deficient both in the excision of u .v .induced dimers and in the rejoining of dsb (Resnick and Martin 1976) . Its genotype
is :
a rad2-17 rad52 tup leu 1-12 trp 5-48 ura 1 his ade
a rad2-17 rad52 tup
+
ura 1 his ade
+
4 . PR90 was also given to us by Dr M . A . Resnick and was included in this study
as a strain yielding the wild-type u .v .-response . Although heterogenous for rad2-17
in this strain all three u .v . repair pathways are functional (Resnick and Martin 1976) .
Its genotype is :
a
+
phr I tup arg 4-4 thr 1
+
+
ura 1 his ade
a rad2-17 phr I tup +
+ leu t-12 trp 5-48 ura 1 his ade
2.2 . Experimental conditions
Yeast cells were grown on YPD-agar (1 per cent yeast extract, 2 per cent peptone,
2 per cent glucose, 1 . 5 per cent agar) to stationary phase (the two PR-strains at 30 ° C,
rad54-3 strains at 36 ° C), harvested, washed twice in 67 mmol 1 -1 phosphate buffer,
pH 7 . 0, sonicated to separate clumped cells, and irradiated in ice-cold phosphate
buffer with u .v-light (254 nm) . U .v .-dosimetry was kindly performed by Dr M .
Brendel as described previously (Fath and Brendel 1975) . After irradiation cells were
Double strand breaks in yeast after u .v . -irradiation
109
diluted appropriately, and incubated on YPD-agar until macrocolonies could be
counted to assay for survivors . The PR-strains were incubated at 30°C, whereas the
two rad54-3 strains were incubated at 36°C (restrictive for dsb rejoining) as well as at
23 °C (permissive for dsb rejoining) .
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3.
Results
Survival curves were measured after u .v . exposure of the yeast strain PR90, two
rad54-3 mutant strains of different origin (rad54-3G and rad54-3B), and the rad217rad52 double mutant . Figure 1 shows a summary of the survival curves of these
various different yeast strains after u .v .-irradiation . As expected, rad2-17rad52 cells
-2 )
were very u .v .-sensitive (D 37 =0.86Jm
and cells of PR90 were highly u.v .resistant (D 37 =70.5Jm -2 ), exhibiting wild type response . Shouldered survival
curves of both rad54-3 strains were observed at the temperature restrictive (36°C)
and permissive (23 °C) for dsb rejoining (figure 2) . When dsb rejoining was
prevented (36°C) rad54-3 cells exhibit a remarkable resistance to u .v .-light as
figure 3) . The D 37 value
compared to the very u .v .-sensitive strain rad2-17rad52 (see m-2,
for strain rad54-3G was 11 . 5 J m -2 and for rad54-3B 20. 8 J
whereas it was only
0. 86 J m-2 for the mutant radl2-17rad52 (see the table) . Cells of strain rad54-3G
were found to be more u .v .-sensitive than those of the mutant rad54-3B when
incubated at 36°C (figure 2) . When dsb rejoining was permitted after u .v .-irradiation
by incubation at 23°C the u.v .-resistance of rad54-3 cells increased and the two
_ 2,
rad54-3 strains showed identical survival curves with a D 37 value of 59 .5 J m
close to that of the strain PR90 (D 37 =70. 5 J m -2 ) giving wild-type response . Based
on the data of Waters and Moustacchi (1975) that u .v . light induces on average 163
pyrimidine dimers per 10 7 nucleotides per (J m -2 ) and taking 1 . 8 x 10 10 g mol-1 as
0.1 -
rad2rad52
005 -
Figure 1 . Summary of the survival curves of the four different strains studied . The
experimental data points are shown for the strain PR90 exhibiting wild-type response
(0) ; for the sake of clarity those for the other strains are shown in figure 2 and 3 . The
curves presented were fitted by eye through the mean values of the experimental points .
M. Frankenberg-Schwager et al .
110
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50
UV-fluence
J •m 2
100
Figure 2 . Survival curves of strains rad54-3G (open symbols) and rad54-3B (closed
symbols) at the temperature permissive (23°C, circles) and restrictive (36°C, triangles)
for dsb rejoining .
UV-fluence
J-M -2
0
0.5
1.0
1.5
2.0
i
05
S
0.2
0.1 -
0105 .
Figure 3 . The survival curve of strain rad2-17rad52 (V) . For comparison, the survival
curves of rad54-3B (
) and rad54-3G (--) at 36 ° C are also shown .
the molar mass of DNA of diploid yeast (Lauer et al . 1977), we calculated the
number of dimers per diploid yeast cell induced by the u .v . fluence D 37 yielding a
surviving fraction of 0. 37 for the strains used (see the table) .
4.
Discussion
The survival curves presented in figures 1 and 3 show that, relative to strain rad217rad52, cells of rad54-3 at 36°C are more u .v .-resistant, presumably because they
are able to repair thymine dimers at this temperature (table 1) . However, comparing
the rad54-3 survival curves obtained at 36°C with those at 23 ° C (figure 2) it is clear
Double strand breaks in yeast after u .v .-irradiation
111
The number of thymine dimers per diploid cell induced by the u .v .-fluence D 37 , yielding a
surviving fraction of s=0 . 37 .
Strain
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rad2-17rad52
D37(J
m
0. 86
_ 2)
Thymine dimers
induced/cell
771
rad54-3G, 36 ° C
rad54-3B, 36°C
11 . 5
20. 8
10 310
18647
rad54-3G, 23 ° C
rad54-3B, 23°C
59. 5
53 342
PR90
70. 5
63 203
that u .v .-resistance is higher at 23' - C, indicating that a temperature-dependent repair
process is involved . In view of the finding that rad54-3 cells are deficient in the
rejoining of dsb at 36 ° C but not in 23 ° C (Budd and Mortimer 1982) the above results
may shed some light on the nature of the molecular lesion involved in u .v .-induced
killing of rad54-3 cells at 36 ° C . We therefore propose that the differences between
the u .v . survival curves of the rad54-3 strains at 36°C and 23 ° C may be caused by the
formation of dsb which arise in the course of repair of u .v .-induced base damage .
For E. coli K-12 quantitative evidence was presented that dsb which are formed
enzymatically after u .v .-irradiation (Bonura and Smith 1975 a, Wang and Smith
1986 a, Sharma and Smith 1986) are responsible for u .v .-induced killing (Bonura
and Smith 1975 b) . These authors have suggested that dsb may arise during excision
repair of u .v .-induced dimers when (a) excision in one strand proceeds, past an
incision break on the opposite strand or, alternatively, past a dimer which is
subsequently excised and/or (b) enzymatically excised gaps on opposing strands
overlap . The average patch size in u .v .-irradiated E . coli has been determined to be
30 nucleotides (Setlow and Carrier 1964) . However, . Cooper and Hanawalt (1972)
found that repair patches vary in size ; some patches are at least as large as 1500
nucleotides whereas most patches seem to comprise only a few nucleotides .
A similar situation may apply to human cells where the formation of dsb after
u .v .-irradiation has been reported to occur in the DNA of excision-proficient lung
fibroblasts, whereas in excision-deficient human skin fibroblasts (XPA) no such
effect was found (Bradley and Taylor 1981) .
For yeast, Resnick and Martin (1977) have determined an average patch size of
5-10 nucleotides . No attempt has been made yet to study the possible heterogeneity
of patch size distribution in this organism . In view of these findings the probability of
formation of enzymatically induced dsb in u .v .-irradiated yeast is expected to be low .
However, the mutant rad54-3 which at 36°C is unable to rejoin dsb provides a very
sensitive system to reveal dsb . Since in yeast approximately one unrepaired dsb per
cell is observed per lethal event (Ho and Mortimer 1975, Resnick and Martin 1976,
Frankenberg et al . 1984, 1986), only one enzymatically induced dsb per genome is
expected to enhance killing of rad54-3 cells at 36°C but not at 23 ° C .
Thus we interpret our results as evidence for the formation of enzymatically
induced dsb after u.v . -irradiation in yeast cells . If this interpretation is correct, the
u .v . -sensitivity of mutants belonging to the RAD52 epistasis group may be due to
their inability to repair enzymatically induced dsb rather than to their lack of repair
of dimers . Also, the minor importance of the RAD52-controlled pathway for u .v .
112
M . Frankenberg-Schwager et al .
repair may be explained by the low probability of formation of enzymatically
induced dsb .
Although this interpretation is the most reasonable one, it may also be possible
that the observed effect is due to other temperature-dependent repair processes
involving recombination (Game 1983) such as long-patch excision repair and postreplication repair, as described for bacteria . Recently, evidence for the post-
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replicative formation of dsb has been presented for u .v .-irradiated human (SV40)transformed fibroblasts (Wang and Smith 1986b) and E . coli (Wang and Smith
1983) . These workers suggest that a dsb may occur when a single-strand break is
introduced into the parental DNA strand opposite the unrepaired DNA daughterstrand gap .
In conclusion we show here that the yeast mutant rad54-3, which is temperature
conditional for dsb rejoining, is sensitive to u .v . light when held at the restrictive
temperature following exposure . We propose that this is attributable to the
formation of dsb in DNA containing u .v . lesions and a subsequent lack of dsb repair
in the rad54-3 mutants . These enzymatically formed dsb may arise by overlapping
excision patches or by a postreplicative repair process .
Acknowledgment
We are indebted to Professor Dr M . Brendel for providing the u .v .-irradiation
facility and for performing the dosimetry and to Dr P . E . Bryant for critically reading
the manuscript .
References
BONURA, T ., and SMITH, K . C ., 1975 a, Enzymatic production of deoxyribonucleic acid
double-strand breaks after ultraviolet irradiation of Escherichia coli . Journal of
Bacteriology, 121, 511-517 .
BONURA, T ., and SMITH, K . C ., 1975 b, Quantitative evidence for enzymatically-induced
DNA double-strand breaks as lethal lesions in u .v .-irradiated pol + and polAl strains of
E. coli K-12 . Photochemistry and Photobiology, 22, 243-248 .
BRADLEY, M . 0 ., and TAYLOR, V . I ., 1981, DNA double-strand breaks induced in normal
human cells during the repair of ultraviolet light damage . Proceedings of the National
Academy of Sciences, USA, 78, 3619-3623 .
BRENDEL, M ., and HAYNES, R . H ., 1973, Interactions among genes controlling sensitivity to
radiation and alkylation in yeast . Molecular and General Genetics, 125, 197-216 .
BUDD, M ., and MORTIMER, R . K., 1982, Repair of double-strand breaks in a temperature
conditional radiation-sensitive mutant of Saccharomyces cerevisiae . Mutation Research,
103,19-24 .
COOPER, P . K ., and HANAWALT, P . C ., 1972, Heterogeneity of patch size in repair replicated
DNA of Escherichia coli . Journal of Molecular Biology, 67, 1-10 .
Cox, B ., and GAME, J ., 1974, Repair systems in Saccharomyces cerevisiae . Mutation Research,
26, 257-264.
FATH, W . W ., and BRENDEL, M ., 1975, Improved assay of thymine dimers . Zeitschrift fur
Naturforschung, 30c, 804-810 .
FRANKENBERG, D ., FRANKENBERG-SCHWAGER, M ., and HARBICH, R ., 1984, Interpretation of
the shape of survival curves in terms of induction and repair/misrepair of DNA doublestrand breaks . British Journal of Cancer, 49, Suppl . VI, 233-238 .
FRANKENBERG, D ., GOODHEAD, D . T ., FRANKENBERG-SCHWAGER, M ., HARBICH, R ., BANCE,
D . A ., and WILKINSON, R . E ., 1986, Effectiveness of 1 . 5 keV aluminium K and 0. 3 keV
carbon K characteristic X-rays at inducing DNA double-strand breaks in yeast cells .
International Journal of Radiation Biology, 50, 727-741 .
Double strand breaks in yeast after u . v .-irradiation
C ., 1983, Radiation-sensitive mutants and repair in yeast . Yeast Genetics,
Fundamental and Applied Aspects, edited by J . F. T . Spencer, D . M . Spencer and A . R .
W . Smith (New York, Berling, Heidelberg, Tokyo : Springer Verlag), pp . 109-137 .
GAME, J . C ., and Cox, B ., 1973, Synergistic interactions between rad mutants in yeast .
Mutation Research, 20, 35-44 .
GAME, J . C ., and MORTIMER, R . K ., 1974, A genetic study of X-ray sensitive mutants in yeast .
Mutation Research, 24, 281-293 .
HAYNES, R . H ., and KUNZ, B . A ., 1981, DNA repair and mutagenesis in yeast . The Molecular
Biology of the - Yeast Saccharomyces, Life Cycle and Inheritance, edited by J . N .
Strathern, E . W . Jones and J . R . Broach (Cold Spring Harbor, New York : Cold Spring
l larbor Laboratory), pp . 371 414 .
Ho, K ., and MORTIMER, R . K ., 1975, X-ray dominant lethality and chromosome breakage and
repair in a radiosensitive strain of yeast . Molecular Mechanisms for Repair of DNA,
edited by P . C . Hanawalt and R . B . Setlow (New York : Plenum Press), pp . 545-547 .
LAUER, G . D ., ROBERTS, T ., and KLOTZ, L . C ., 1977, Determination of the nuclear DNA
content of Saccharomyces cerevisiae and implications for the organisation of DNA in
yeast chromosomes . Journal of Molecular Biology, 114, 507-526 .
MOUSTACCHI, E ., FAVAUDON, V ., and BISAGNI, E ., 1983, Likelihood of the new anti-tumoral
drug 10-(y-diethylaminopropylamino)-6-methyl-5H-pyrido (3, 4 : 4, 5)pyrrolo(2, 3g)isoquinolin (BD-40), a pyridopyrroloisoquinoline derivative, to induce DNA strand
breaks in vivo and its non-mutagenicity in yeast . Cancer Research, 43, 3700-3706 .
RESNICK, M . A., 1975, The repair of double-strand breaks in chromosomal DNA of yeast .
Molecular Mechanisms for Repair of DNA, Part B, edited by P . C . Hanawalt and R . B .
Setlow (New York : Plenum Press), pp . 549-556 .
RESNICK, M . A ., and MARTIN, P ., 1976, Repair of double-strand breaks in the nuclear DNA of
Saccharomyces cerevisiae and its genetic control . Molecular and General Genetics, 143,
119-129 .
RESNICK, M . A ., and MARTIN, P ., 1977, Nuclear DNA synthesis in yeast and the effect of
irradiation . International Journal of Radiation Biology, 31, 365-375 .
SAEKI, T ., MACHIDA, I ., and NAKAI, S ., 1981, Genetic control of diploid recovery after
gamma-irradiation in the yeast Saccharomyces cerevisiae . Mutation Research, 73,
251-265 .
SETLOW, R . B ., and CARRIER . W . L ., 1964, The disappearance of thymine dimers from DNA :
An error-correcting mechanism . Proceedings of the National Academy of Sciences, 51,
226 .
SHARMA, R . C ., and SMITH, K . C ., 1986, Repair of DNA double-strand breaks in u .v .irradiated E. coli uvrBrecF cells is inhibited by rich growth medium . Mutation Research,
DNA Repair Reports, 166, 23-28 .
WANG, T. V ., and SMITH, K . C., 1983, Mechanisms for recF-dependent and recB-dependent
pathways of postreplication repair in u .v.-irradiated Escherchia coli uvrB . Journal of
Bacteriology, 156, 1093-1098 .
WANG, T . V ., and SMITH, K . C ., 1986 a, Postreplication formation and repair of DNA doublestrand breaks in u .v .-irradiated Escherichia coli uvrB cells . Mutation Research, DNA
Repair Reports, 165, 39-44 .
WANG, T . V ., and SMITH, K . C ., 1986 b, Postreplication repair in ultraviolet-irradiated
human fibroblasts : Formation and repair of DNA double-strand breaks . Carcinogenesis,
7, 389-392 .
WATERS, R ., and MOUSTACCHI, E ., 1975, Dose dependence of the excision of ultra-violetinduced pyrimidine dimers from nuclear deoxyribonucleic acids of haploid and diploid
Saccharomyces cerevisiae . Journal of Bacteriology, 121, 901-906 .
GAME, J .
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