- Email: [email protected]
Single strand interruptions in DNA and the effects of caffeine in Chinese hamster cells irradiated with ultraviolet light
BIOCHEMICAL
Vol. 36, No. 2, 1969
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SINGLE STRAND INTERRUPTIONS IN DNA AND THE EFFECTS OF CAFFEINE IN CHINESE HAMSTER CELLS IRRADIATED WITH ULTRAVIOLET
LIGHT*
J. E. Cleaver and G. H. Thomas Laboratory of Radiobiology University of California Medical Center San Francisco, California 94122USA Received May 22, 1969 DNA synthesized in bacteria after irradiation with ultraviolet
(UV) light
contains numerous interruptions in the polynucleotide chains (Rupp and HowardFlanders, 1968). These interruptions may be due to UV photoproducts on the opposite strand and are subsequently closed by a process distinct from semiconservative replication of DNA (Rupp and Howard-Flanders,
1968). These bacterial
experiments have now been repeated with Chinese hamster cells to determine whether a similar process exists in mammalian cells. Materials and Methods Chinese hamster cells (V79) were grown in plastic petri dishes using Eagle’s minimum essential medium with 15%fetal calf serum, at 3’7’C under a water saturated atmosphere of 5% CO2 in air.
For irradiation the cultures were
rinsed in buffer, exposed to UV light (2537 8) at 14 erg/mm2/sec at room temperature and then returned to 37’C. After irradiation,
cultures were labeled
with H3-thymidine (H3TdR 10 pCi/ml 12 Ci/mmole, New England Nuclear Corp.) for 30 min, rinsed in physiological saline, and scraped from the petri dishes. Approximately 5 x lo4 cells were immediately placed into 0.5 ml of 0.5 M sodium hydroxide on the top of a 5 to 20%linear alkaline sucrose gradient.
In some
experiments cultures were rinsed in buffer and grown for a further 5 hr in medium containing 5 pg/ml TdR and 5 pg/ml deoxycytidine, the dishes and placed on the top of the gradients.
before being scraped from The alkaline sucrose gradients
Work performed under the auspices of the U. S. Atomic Energy Commission
Vol. 36, No. 2, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were made in 30 ml tubes with 1.2 ml of 50%sucrose at the bottom and buffered throughout at pH 12.5 with 1mM EDTA and 0.9 M NaCl. After the cells had been placed on the top of the gradients, the tubes were stored at 4’C for 2 to 16 hr to ensure that the DNA was denatured and its strands fully separated (Davison, 1966). Gradients were then centrifuged at 25,000 rpm for 3 hr, after which 65 drop fractions were collected and the total acid insoluble radioactivity
determined in
each fraction using techniques previously described (Cleaver, 1969; Painter and Cleaver, 1969). Molecular weights were calculated with the formula of 14 Abelson and Thomas (1966) using C -labeled lambda phage DNA as a standard with a single strand molecular weight of 17 x lo6 (Rupp and Howard-Flanders, 1968). Results were expressed as a percentage of total radioactivity
in each
fraction, the latter being normalized to fractions of the total length of the gradient measured from the top. The average yield from these gradients was 81 f 9%. Results In the first experiment, cultures were irradiated with ‘70erg/mm2 and 700 erg/mm2, labeled for 30 min with H’TdR, and placed on the gradients immediately.
The radioactivity profiles showed a sharp peak near the bottom
of the gradients and a broad peak in the upper parts (Fig. 1). The proportion of activity in the bottom of the gradients and the median molecular weights in the upper parts both decreased with increasing UV dose. If we assume that the peaks near the bottom represent DNA molecules rather than a rapidly sedimenting gel (Lett aal. , 1967), then these molecules would have a molecular weight of 10’ or more. Sedimentation for only 1 hr, however, also produced a peak of activity near the bottom of the gradients and this fraction may therefore be a gel, and its position in the gradient may not be representative of the molecular weight of the DNA molecules therein.
The rapidly sedimenting gel does not, however,
contain a random sample of the newly synthesized H3-labeled DNA; as the molecular weight of the latter decreases, the low molecular weight molecules are
204
Vol. 36, No. 2, 1969
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fraction Figure
1.
top
Sedimentation profile in alkaline sucrose gradients of acid insoluble H3 activity from Chinese hamster cells irradiated with UV light, labeled for 30 min with H3TdR, and placed on the gradients immediately. Control A; 70 erg/mm2 0; 700 erg/mm2 IJ.
released preferentially the molecules decreased
from
from the gel (Fig.
weights
of
which form the broad peaks in the upper parts of the gradients
with increasing
(70 erg/mm2)
1). The median molecular
UV dose from 7.1 x lo7 (control)
and 0.09 x lo7 (700 erg/mm2).
This decrease
to 2.9 x lo7 is not due merely
to a slow synthetic rate subsequentto irradiation because when control cells were labeled for 30 min at 28’C, the median molecular weight was 7.0 x 107, the same as at 37’C.
This temperature reduces the total H’TdR incorporation
during 30 min labeling to the same level as irradiation with 130 erg/mm2 (Cleaver, 1967), but has no effect on the relative distribution of radioactivity the gradients.
in
These results suggest that DNA synthesized after irradiation has
a lower single strand molecular weight than in unirradiated cells and that the number of strands attached to high molecular weight fractions decreases as a 205
Vol.36,
No. 2, 1969
BIOCHEMICAL
AND WPHYSlCAL
RESEARCH COMMUNICATIONS
function of dose. When cultures labeled for 30 min were subsequently grown in nonradioactive medium for 5 hr, there was an increase in the molecular weight of the H3-labeled DNA (Fig. 2). This increase in molecular weight with time occured in both control and irradiated cultures, and appeared to be essentially complete after 2 hr of growth. When irradiated cultures were labeled and then grown in nonradioactive medium containing 2 x 10m3Mcaffeine, the increase in molecular weight did not proceed to completion (Fig. 2). Caffeine at this concentration did not prevent the increase in molecular weight in control cells.
I.0
0.5
Distance
Figure 2.
from
0
top
Sedimentation profile in alkaline sucrose gradients of acid insoluble H3 activity from Chinese hamster cells irradiated with UV light, labeled for 30 min with H3TdR, and grown for 5 hr in nonradioactive medium before being placed on the gradients. 100 erg/mm2 placed on gradient at end of labeling 0; 100 er /mm2 grown for 5 hr 0; 100 erg/mm2 grown for 5 hr in 2 x 10’ I M caffeine 0.
Discussion The newly synthesized DNA strands in unirradiated Chinese hamster cells !abeled for 30 min form two classes with different molecular weights: one may be a gel, and the other has a median of ‘7x 107. The formez May represent 206
Vol. 36, No. 2, 1969
BIOCHEMICAL
AND BlOPtiYSlCAL
RESEARCH COMMUNICATIONS
replicons which completed replication in the presence of H’TdR and the latter partially replicated replicons (Painter, Jermany and Rasmussen, 1966). Random breakage of newly synthesized DNA might also have contributed to the observed range of molecular weights although care had been taken to minimize shear. The results suggest that the size of the single strands synthesized after irradiation decreases as a function of dose as observed in irradiated Escherichia p&
(Rupp and Howard-Flanders,
1968). There is a close similarity between
the median molecular weights of DNA strands in irradiated Chinese hamster cells and E. coli; after 70 erg/mm2 the median molecular weight of the former was 2.9 x 10’ and after 60 erg/mm2 that of the latter was 1.7 to 2.5 x 10’ (Rupp and Howard-Flanders,
1968). In E. coli the molecular weight corresponds
to DNA strands with interruptions at approximately the same frequency as that of thymine dimers in the parental strands. A similar correlation may therefore be invoked for the interruptions in the Chinese hamster strands. In both control and irradiated cells the molecular weight of the strands increased with time, but the mechanisms in the two situations are probably different since caffeine only inhibited the increase in irradiated cells.
In
control cells, the increase may occur simply by extension of the length of newly synthesized strands. In irradiated cells the newly synthesized strands are extended by further replication, and the single strand interruptions must be linked together. Chinese hamster cells, therefore, may have a repair process similar to that controlled by ret mutations in bacteria (Rupp and Howard-Flanders,
1968) which enable cells to replicate their DNA despite
the presence of damage on the parental strands. Caffeine appears to exert its effect by inhibiting completion of the linkage of single strands. These observations are consistent with those of Rauth (1967) who observed that caffeine killed mouse L cells during the first S phase after irradiation with W light.
Rauth suggested that caffeine inhibited
a DNA repair mechanism, but it has now been shown that caffeine has no effect 207
Vol. 36, No. 2, 1969
on the excision-repair
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mechanism (Cleaver, 1969). Rather, the results of this
paper indicate that caffeine interferes specifically with a step associated with semiconservative DNA replication in irradiated cells (Cleaver, 1969). This may therefore be the mechanism whereby caffeine kills cells which would otherwise have survived irradiation with UV light (Rauth, 1967).
Abelson, J., Thomas, C.A. , J. Mol. Biol. 2, 262 (1966). Cleaver, J. E., Rad. Res. 30, 795 (1967). Cleaver, J. E., Rad. Res. 37, 334 (1969). Davison, P. F., J. Mol. Six 22-, 97 (1966). Lett, J. T., Caldwell, I. , Dean, C. J., Alexander, P. , Nature 214, 790 (1967). Painter, R.B., CIeaver, J.E., Rad. Res. E, 451 (1969). Painter, R. B., Jermany, D.A., Rasmussen, R. E., J. Mol. Biol. z, 47 (1966). Rauth, A.M., Rad. Res. g, 121 (1967). Rupp, W.D., Howard-Flanders, P., J. Mol. Biol. 3 291 (1968).
208
Vol. 36, No. 2, 1969
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SINGLE STRAND INTERRUPTIONS IN DNA AND THE EFFECTS OF CAFFEINE IN CHINESE HAMSTER CELLS IRRADIATED WITH ULTRAVIOLET
LIGHT*
J. E. Cleaver and G. H. Thomas Laboratory of Radiobiology University of California Medical Center San Francisco, California 94122USA Received May 22, 1969 DNA synthesized in bacteria after irradiation with ultraviolet
(UV) light
contains numerous interruptions in the polynucleotide chains (Rupp and HowardFlanders, 1968). These interruptions may be due to UV photoproducts on the opposite strand and are subsequently closed by a process distinct from semiconservative replication of DNA (Rupp and Howard-Flanders,
1968). These bacterial
experiments have now been repeated with Chinese hamster cells to determine whether a similar process exists in mammalian cells. Materials and Methods Chinese hamster cells (V79) were grown in plastic petri dishes using Eagle’s minimum essential medium with 15%fetal calf serum, at 3’7’C under a water saturated atmosphere of 5% CO2 in air.
For irradiation the cultures were
rinsed in buffer, exposed to UV light (2537 8) at 14 erg/mm2/sec at room temperature and then returned to 37’C. After irradiation,
cultures were labeled
with H3-thymidine (H3TdR 10 pCi/ml 12 Ci/mmole, New England Nuclear Corp.) for 30 min, rinsed in physiological saline, and scraped from the petri dishes. Approximately 5 x lo4 cells were immediately placed into 0.5 ml of 0.5 M sodium hydroxide on the top of a 5 to 20%linear alkaline sucrose gradient.
In some
experiments cultures were rinsed in buffer and grown for a further 5 hr in medium containing 5 pg/ml TdR and 5 pg/ml deoxycytidine, the dishes and placed on the top of the gradients.
before being scraped from The alkaline sucrose gradients
Work performed under the auspices of the U. S. Atomic Energy Commission
Vol. 36, No. 2, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were made in 30 ml tubes with 1.2 ml of 50%sucrose at the bottom and buffered throughout at pH 12.5 with 1mM EDTA and 0.9 M NaCl. After the cells had been placed on the top of the gradients, the tubes were stored at 4’C for 2 to 16 hr to ensure that the DNA was denatured and its strands fully separated (Davison, 1966). Gradients were then centrifuged at 25,000 rpm for 3 hr, after which 65 drop fractions were collected and the total acid insoluble radioactivity
determined in
each fraction using techniques previously described (Cleaver, 1969; Painter and Cleaver, 1969). Molecular weights were calculated with the formula of 14 Abelson and Thomas (1966) using C -labeled lambda phage DNA as a standard with a single strand molecular weight of 17 x lo6 (Rupp and Howard-Flanders, 1968). Results were expressed as a percentage of total radioactivity
in each
fraction, the latter being normalized to fractions of the total length of the gradient measured from the top. The average yield from these gradients was 81 f 9%. Results In the first experiment, cultures were irradiated with ‘70erg/mm2 and 700 erg/mm2, labeled for 30 min with H’TdR, and placed on the gradients immediately.
The radioactivity profiles showed a sharp peak near the bottom
of the gradients and a broad peak in the upper parts (Fig. 1). The proportion of activity in the bottom of the gradients and the median molecular weights in the upper parts both decreased with increasing UV dose. If we assume that the peaks near the bottom represent DNA molecules rather than a rapidly sedimenting gel (Lett aal. , 1967), then these molecules would have a molecular weight of 10’ or more. Sedimentation for only 1 hr, however, also produced a peak of activity near the bottom of the gradients and this fraction may therefore be a gel, and its position in the gradient may not be representative of the molecular weight of the DNA molecules therein.
The rapidly sedimenting gel does not, however,
contain a random sample of the newly synthesized H3-labeled DNA; as the molecular weight of the latter decreases, the low molecular weight molecules are
204
Vol. 36, No. 2, 1969
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fraction Figure
1.
top
Sedimentation profile in alkaline sucrose gradients of acid insoluble H3 activity from Chinese hamster cells irradiated with UV light, labeled for 30 min with H3TdR, and placed on the gradients immediately. Control A; 70 erg/mm2 0; 700 erg/mm2 IJ.
released preferentially the molecules decreased
from
from the gel (Fig.
weights
of
which form the broad peaks in the upper parts of the gradients
with increasing
(70 erg/mm2)
1). The median molecular
UV dose from 7.1 x lo7 (control)
and 0.09 x lo7 (700 erg/mm2).
This decrease
to 2.9 x lo7 is not due merely
to a slow synthetic rate subsequentto irradiation because when control cells were labeled for 30 min at 28’C, the median molecular weight was 7.0 x 107, the same as at 37’C.
This temperature reduces the total H’TdR incorporation
during 30 min labeling to the same level as irradiation with 130 erg/mm2 (Cleaver, 1967), but has no effect on the relative distribution of radioactivity the gradients.
in
These results suggest that DNA synthesized after irradiation has
a lower single strand molecular weight than in unirradiated cells and that the number of strands attached to high molecular weight fractions decreases as a 205
Vol.36,
No. 2, 1969
BIOCHEMICAL
AND WPHYSlCAL
RESEARCH COMMUNICATIONS
function of dose. When cultures labeled for 30 min were subsequently grown in nonradioactive medium for 5 hr, there was an increase in the molecular weight of the H3-labeled DNA (Fig. 2). This increase in molecular weight with time occured in both control and irradiated cultures, and appeared to be essentially complete after 2 hr of growth. When irradiated cultures were labeled and then grown in nonradioactive medium containing 2 x 10m3Mcaffeine, the increase in molecular weight did not proceed to completion (Fig. 2). Caffeine at this concentration did not prevent the increase in molecular weight in control cells.
I.0
0.5
Distance
Figure 2.
from
0
top
Sedimentation profile in alkaline sucrose gradients of acid insoluble H3 activity from Chinese hamster cells irradiated with UV light, labeled for 30 min with H3TdR, and grown for 5 hr in nonradioactive medium before being placed on the gradients. 100 erg/mm2 placed on gradient at end of labeling 0; 100 er /mm2 grown for 5 hr 0; 100 erg/mm2 grown for 5 hr in 2 x 10’ I M caffeine 0.
Discussion The newly synthesized DNA strands in unirradiated Chinese hamster cells !abeled for 30 min form two classes with different molecular weights: one may be a gel, and the other has a median of ‘7x 107. The formez May represent 206
Vol. 36, No. 2, 1969
BIOCHEMICAL
AND BlOPtiYSlCAL
RESEARCH COMMUNICATIONS
replicons which completed replication in the presence of H’TdR and the latter partially replicated replicons (Painter, Jermany and Rasmussen, 1966). Random breakage of newly synthesized DNA might also have contributed to the observed range of molecular weights although care had been taken to minimize shear. The results suggest that the size of the single strands synthesized after irradiation decreases as a function of dose as observed in irradiated Escherichia p&
(Rupp and Howard-Flanders,
1968). There is a close similarity between
the median molecular weights of DNA strands in irradiated Chinese hamster cells and E. coli; after 70 erg/mm2 the median molecular weight of the former was 2.9 x 10’ and after 60 erg/mm2 that of the latter was 1.7 to 2.5 x 10’ (Rupp and Howard-Flanders,
1968). In E. coli the molecular weight corresponds
to DNA strands with interruptions at approximately the same frequency as that of thymine dimers in the parental strands. A similar correlation may therefore be invoked for the interruptions in the Chinese hamster strands. In both control and irradiated cells the molecular weight of the strands increased with time, but the mechanisms in the two situations are probably different since caffeine only inhibited the increase in irradiated cells.
In
control cells, the increase may occur simply by extension of the length of newly synthesized strands. In irradiated cells the newly synthesized strands are extended by further replication, and the single strand interruptions must be linked together. Chinese hamster cells, therefore, may have a repair process similar to that controlled by ret mutations in bacteria (Rupp and Howard-Flanders,
1968) which enable cells to replicate their DNA despite
the presence of damage on the parental strands. Caffeine appears to exert its effect by inhibiting completion of the linkage of single strands. These observations are consistent with those of Rauth (1967) who observed that caffeine killed mouse L cells during the first S phase after irradiation with W light.
Rauth suggested that caffeine inhibited
a DNA repair mechanism, but it has now been shown that caffeine has no effect 207
Vol. 36, No. 2, 1969
on the excision-repair
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mechanism (Cleaver, 1969). Rather, the results of this
paper indicate that caffeine interferes specifically with a step associated with semiconservative DNA replication in irradiated cells (Cleaver, 1969). This may therefore be the mechanism whereby caffeine kills cells which would otherwise have survived irradiation with UV light (Rauth, 1967).
Abelson, J., Thomas, C.A. , J. Mol. Biol. 2, 262 (1966). Cleaver, J. E., Rad. Res. 30, 795 (1967). Cleaver, J. E., Rad. Res. 37, 334 (1969). Davison, P. F., J. Mol. Six 22-, 97 (1966). Lett, J. T., Caldwell, I. , Dean, C. J., Alexander, P. , Nature 214, 790 (1967). Painter, R.B., CIeaver, J.E., Rad. Res. E, 451 (1969). Painter, R. B., Jermany, D.A., Rasmussen, R. E., J. Mol. Biol. z, 47 (1966). Rauth, A.M., Rad. Res. g, 121 (1967). Rupp, W.D., Howard-Flanders, P., J. Mol. Biol. 3 291 (1968).
208