Absence of ultrafast processes of repair of single-strand breaks in mammalian DNA.

INT . J. RADIAT . BIOL .,

1975,

VOL .

27,

NO .

2, 121-133

Absence of ultrafast processes of repair of single-strand breaks in mammalian DNA B . PALCIC f and L. D . SKARSGARD British Columbia Cancer Institute and University of British Columbia, 2656 Heather Street, Vancouver, B .C ., Canada

Int J Radiat Biol Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.

(Received 7 October

1974 ;

accepted 23 December

1974)

Town, Smith and Kaplan (1972) reported that the yield of DNA single-strand breaks (SSB) in E . coli is largely independent of the presence of molecular oxygen during irradiation . They suggested that the oxygen enhancement ratio (o .e .r) normally observed is due to the presence of an ultrafast repair mechanism acting (in bacterial cells) mainly on anoxically-produced breaks . To determine whether similar mechanisms exist in mammalian cells, we carried out comparable experiments on two cell-lines, one from Chinese hamster, the other from mouse. Both heat inactivation and chemical inhibition were used to eliminate the supposed enzymic ultrafast repair . Although heat treatment inactivated all the enzymatic processes assayed, it did not alter the o .e .r. for SSB production, which remained about 3 .0 . The presence of sodium cyanide, hydroxyurea, iodoacetic acid, EDTA and quinacrine all failed to alter significantly the o .e .r. Isolated nuclei also demonstrated the full o .e .r. For these cell-lines at least, ultrafast repair does not seem to exist . Isolated Adenovirus 2, which presumably lacks enzymic activity, demonstrated an o .e .r . of 3 . 6 for SSB production . From these results and others it seems unlikely that the so-called ultrafast enzymic repair is a general phenomenon accounting for the o .e .r . in a wide range of biological systems. Rather, the o.e .r . for SSB seems to result from differences in the direct physico-chemical effects of radiation under aerobic and anoxic conditions in most organisms.

1. Introduction The DNA molecule is presumably one of the most important targets of radiation damage in the living cell . Many studies of radiation damage have been undertaken with various organisms exposed to ionizing radiation, and several types of DNA damage have been assayed . The production of DNA single-strand breaks has been, perhaps, the most extensively studied . Generally, the method of alkaline sucrose gradients (McGrath and Williams 1966) is used for determination of single-strand breaks . It has been reported that the yield of DNA single-strand breaks in mammalian cells is increased if molecular oxygen is present during the time of irradiation (Palcic and Skarsgard 1972 a, b, Dugle, Chapman, Gillespie, Borsa, Webb, Meeker and Reuvers 1972) . These observations were similar to those for bacterial cells (Dean, Ormerod, Serianni and Alexander 1969, Lehnert and Moroson 1971, Johansen, Gurvin and Rupp 1971) . Town, Smith and Kaplan (1972) also found an oxygen-effect on the yield of single-strand breaks in E. coli cells . However, they proposed that bacterial cells possess an ultrafast repair system which operates mainly on anoxic breaks, rejoining them before they can be assayed and they suggest that this accounts f Research Fellow of the National Cancer Institute of Canada. R.B .

21


122

B. Palcic and L. D . Skarsgard

for the observed oxygen enhancement ratio (o .e .r) . This ultrafast repair is apparently completed within a few minutes after irradiation and it has been suggested that it is an enzymatic process . They showed, for example, that when bacterial cells were heated for 5-10 min at 52°C, the ultrafast repair process was inhibited, presumably due to denaturation of the repair enzymes at this high temperature, and the yield of single-strand breaks was the same, whether the heat-treated bacterial cells were irradiated under anoxic or aerobic conditions . Cold shock at 0°C gave a similar effect . Treatment of E. coli with several chemicals known to be either general SH enzyme inhibitors, such as N-ethylmaleimide (NEM) and iodoacetic acid (IA), or inhibitors of DNA repair, such as ethylenediaminetetraacetic acid (EDTA), sodium cyanide (NaCN) and quinacrine also, in some cases, increased the number of observable anoxic breaks ; the same effect was observed with hydroxyurea, which is known to sensitize bacterial cells . From their results, Town et al . concluded that at least the heat treatment of E . coli (52°C, 0°C) could inhibit the ultrafast repair, and they were thus led to postulate that in E . coli cells the initial yield of single-strand breaks is largely independent of the presence of oxygen . Under anoxic irradiation, however, some of the breaks are rapidly rejoined by the ultrafast repair system, giving an apparent 02 effect, they suggested . The question arises whether such a repair system exists in mammalian cells . If it does, it might account for the observed oxygen effect on the yield of DNA single-strand breaks (more specifically, alkali labile bonds) in irradiated mammalian cells, an effect which has usually been ascribed to a difference in the number of initial lesions produced under aerobic and anoxic conditions . In this paper we present data which argue against the presence of an ultrafast repair system in mammalian cells ; rather, the results indicate that for both anoxic and aerobic irradiation, most (if not all) of the DNA breaks are caused by a direct physico-chemical action of ionizing radiation . 2 . Materials and methods

2.1 . Cells Chinese hamster cells of the CH2B 2 line (Agnew and Skarsgard 1972) were grown as monolayers in plastic culture flasks (Falcon Plastics), in minimal essential medium .(MEM, F-16, Gibco) supplemented with 10 per cent undialysed foetal calf serum (FCS, Gibco), NaHCO3 (2 . 2 mg/ml) and 90 units/ml of a penicillin-streptomycin mixture (Microbiological Associates) . The cells were sub-cultured at 2-day intervals by detaching them from the plastic surface using 0 . 1 per cent trypsin (Bacto-Trypsin, Difco) . The doubling-time was 12-14 hours . Mouse fibroblast cells of the L-60 line were maintained as previously described (Palcic and Skarsgard 1972 a) . Adenovirus 2 with its DNA uniformly labelled with 14C thymidine was kindly supplied by Dr . A. Rainbow, McMaster University . 2.2.

Radioactive labelling

Most experiments required cells with their DNA uniformly labelled by a radioactive precursor . For these purposes 1 . 2 x 106 cells were seeded into plastic culture flasks (250 ml, Falcon Plastics) containing 20 ml of growth-


Absence o f ultrafast repair in mammalian DNA

123

medium and allowed to grow, attached to the surface, for one day (37°C, 95 per cent air, 5 per cent C02) . Thereafter, the medium was replaced with 50 ml of fresh medium containing tritiated thymidine (3 HTdR, > 50 Ci/mM, Amersham-Searle) at an activity of 0 .25 µCi/ml . The culture was then incubated at 37°C for one more day. The labelling was terminated by an additional 1-hour incubation in medium that was not radioactive . Labelled cells were collected by trypsinization with 0 . 1 per cent trypsin, washed twice with growth-medium, and finally resuspended in physiological buffered saline (PBS) from which Mg++ and Ca++ ions were omitted . This labelling procedure yielded rates of approximately 0 . 5 counts per min per cell, a level which does not introduce enough single-strand breaks to alter significantly the control DNA sedimentation profile (Palcic 1972) . 2.3 . Irradiation procedure Cell suspensions were loaded into several special glass irradiation vessels (Parker, Skarsgard and Emmerson 1969), which were placed in a 0°C water-bath for the duration of the experiment . Oxygen or nitrogen (containing less than 5 parts/million 02) was then passed over the suspension for 1 hour before and during irradiation . The gas flow-rate was - 1 litre/min and the suspensions were agitated with a magnetic stirrer . The cell suspensions were irradiated at a dose-rate of 330 rads/min using a 137 Cs unit, or at a dose-rate of 480 rads/min using a therapeutic 60 Co unit . 2.4. Alkaline sucrose gradients Linear 5 to 20 per cent alkaline sucrose gradients were prepared in 17 ml cellulose nitrate tubes (Beckman) using an automatic gradient former (Isco, Model 570) . Gradient solutions contained 0 . 3 M NaOH, 0.01 per cent SDS (sodium dodecyl sulphate) and 0 .001 M ethylenediaminetetraacetate (EDTA) and appropriate concentrations of sucrose . A 0 . 5 ml layer of lysing solution containing 0 .5 M NaOH, 0 . 2 per cent SDS and 0 . 01 M EDTA was placed on the top of each gradient . All solutions were made with double-distilled water and were passed through a membrane filter having a 0 .22 µm pore size . An aliquot of cells (1-2 x 10 4 cells in 0.02 ml) was carefully added to the lysing solution on the top of the gradient using a precooled 50 pi microsyringe . The cells were lysed for 6 or 12 hours at room temperature (-22-C). Centrifugations were performed at 20°C using the SW 27 .1 rotor in a Beckman L-65 preparative centrifuge . The angular speed and the time of centrifugation were varied depending on the experiment and are thus indicated in each figure representing a sedimentation profile . After centrifugation, 25 fractions of 0 .75 ml were collected from each gradient using an Isco Model D fraction collector . After adding 0. 2 ml of 4 M HCl and 5 ml of scintillation cocktail (Aquasol, New England Nuclear) to each fraction, the count rates were measured in a Beckman liquid scintillation counter . The measured radioactivity was plotted for each fraction as a percentage of the total counts . The weight average molecular weight and number average molecular weight were calculated by the method which we have described in detail (Palcic and Skarsgard 1972 a) . Calibration of the gradient technique (17 ml tubes, SW 27 .1 Beckman rotor) was accomplished by measuring the

124

B . Palcic and L . D . Skarsgard

sedimentation properties of T4 and T7 phage and Adenovirus 2 DNA . Specifically, the values of 0 in the expression ~dt


Si =

(1)

were determined for each of the three types of DNA . In this expression, Si is the sedimentation constant of the particular DNA molecule in water at 20°C, di is the distance from the middle of the fraction to the point where the molecule began sedimenting, w is the angular velocity in revolutions/min and t is the time of sedimentation . The constant relating these quantities, P, was found to have an average value of 6. 51 x 10 10 svedberg x (r .p .m .) 2 x hours x cm1 . 3.

Results

3 .1 . Heat treatment Cell suspensions (1 x 10 6 cells/ml) were heat-inactivated by incubating cells at a prescribed temperature (52°C, 65°C or 75°C) for 10 or 15 min. Aliquots of 10 ml were placed in 20 ml test-tubes, which were gently shaken in a heated water-bath during the heat treatment . At the end of the incubation period, the cells were quickly cooled to 0°C, at which temperature they were generally kept throughout the remainder of the experiment until they were lysed on the top of an alkaline sucrose gradient . Heat treatment alone produced DNA single-strand breaks . In figure 1, typical results are shown for unirradiated cells treated for 10 min at 65°C or for an equivalent time at room temperature . These cells, too, were first cooled to 0°C and then immediately lysed . The DNA of heat-treated cells was of much smaller size ; in fact, heat treatment produced approximately the same number of DNA breaks as when untreated cells were irradiated to a dose of 4 . 5 krads in the presence of oxygen . Incubation of cells at any of the elevated temperatures (52°C, 65°C or 75°C) produced various degrees of DNA breakage ; thus, in each experiment we also measured the number of breaks produced due to the heat treatment alone . A typical sedimentation profile from heat-treated

20

Z HH 0 U

Q F

15

10

H H Z

U 5

w CL

0 5

10 FRACTION

15 20 NUMBER

25

DIRECTION OF SEDIMENTATION -->

Figure 1 . Sedimentation profiles of unirradiated heat-treated cells. CH2B2 cells were either heat-treated at 65°C for 10 min ( .) or kept at room temperature, 22°C, for 10 min (o) . They were then quickly cooled to 0 ° C . After this they were lysed for 6 hours at 22°C in a lysing solution on the top of an alkaline sucrose gradient . Gradients were then spun at w=8000 r .p .m . for 32 . 7 hours at 20°C in a Beckman preparative ultracentrifuge (SW 27 .1 rotor, 17 ml tubes) .


125

and irradiated cells is shown in figure 2 . In this case, cells were heated for 15 min at 75°C and then irradiated to a dose of 9 krads under an anoxic atmosphere (N 2 ) or 3 krads under an aerobic atmosphere (0 2) . In both cases, DNA sedimented to approximately the same position, indicating equivalent size distributions for the single-stranded DNA molecules . Consequently, these profiles suggest that the o .e .r. for DNA SSB in heat-inactivated cells is approximately 3, the ratio of the N 2 /02 doses .


20 I-

Z

o, s U J

I

Q I-

10 O

U

o

•+

~ 5

IL

O 5 10 15 FRACTION

20

25

NUMBER

-DIRECTION OF SEDIMENTATION

Figure 2 . Sedimentation profiles of irradiated heat-treated cells. CH2B, cells were heat-treated at 75 °C for 15 min and then quickly cooled to 0 ° C . Cell suspensions were then gassed for 1 hour before and during irradiation with N, (0) or O, ( •) . Immediately after irradiation (9 krads in N, and 3 krads in O,) the cells were lysed in a lysing layer at the top of an alkaline sucrose gradient for 6 hours at 22 ° C. Gradients were then centrifuged at w=20000 r .p .m. for 14 . 5 hours at 20 °C in a Beckman preparative ultracentrifuge (SW 27.1 rotor, 17 ml tubes) .

The net contribution to strand breakage made by radiation in heat-treated cells is described by the relation nirr -

Mw' 0 Mw(heat+irr)

- Mw,o

( 2)

Mw(heat)

where Mw ,o is the weight average molecular weight of DNA from control cells (no irradiation, no heat treatment) and Mw(heat) , Mw(heat+irr) are the weight average molecular weights of DNA from heat-treated and heat-treated, irradiated cells, respectively . nirr is then the number of breaks per molecule produced by irradiation alone . Molecular weights were calculated from profiles similar to those shown in figures 1 and 2 according to mathematical procedures which are described elsewhere (Palcic and Skarsgard 1972 a) . It should be noted that (2) is valid only for molecular size distributions which are random (Charlesby 1954) . The pooled data from all heat treatment experiments are presented in figure 3 . In this figure, the experimental points represent heat-treated cells and the solid lines are the best fits to these data ; the dashed lines represent the best fits to the results obtained with unheated cells (Palcic and Skarsgard 1972 a), for which the actual experimental points are not shown in figure 3 . The close agreement between the heat-treated and non-heat-treated data indicates quite clearly that heat treatment has not altered the o .e.r. value, which remains approximately 3 .

126


2


N2

5

10 DOSE (krads)

15

Figure 3 . The oxygen effect on single-strand break production in heat-treated cells . CH2B2 cells were held for 10 or 15 min at 52°C ( •, o), 65°C ( ., o) or 75°C (A, o) and immediately cooled to 0 ° C . They were then irradiated under anoxic (N 2) or aerobic (02) conditions, lysed on the top of an alkaline sucrose gradient for 6 hours at room temperature and centrifuged in an SW 27 .1 Beckman rotor (17 ml tubes) . Weight average molecular weights were calculated from sedimentation profiles such as those shown in figure 2 and the net contribution of radiation-produced breaks was computed (heat treatment without radiation also caused some single-strand breaks) . Radiation-produced breaks are plotted as a function of dose . Dashed lines are those from cells which were not heat-treated (points not plotted) .

A few similar experiments were performed with mouse L-60 cells and here too, heat treatment did not affect the o .e .r . values for radiation-induced SSB . As a further check on the effectiveness of our heat treatment, assays were performed for several enzymatic processes in normal and in heat-treated cells . The table shows results of observations of DNA, protein and RNA synthesis . It can be seen that all macromolecular synthesis is effectively stopped in CH2B 2

Macromolecule

Precursor

TCA precipitable counts in 106 cells (counts per minute) 23°C

65°C

75°C

84016

31

23

DNA

( 3H-TdR)

Protein

( 3H L-leucine)

3369

35

31

( 3H uridine)

71528

23

14

RNA

CH2B 2 cells were grown as monolayers, collected by trypsinization and washed twice in growth medium . Then they were incubated for 10 min at 23 ° C, 65°C or 75 °C . Immediately thereafter, cells were assayed for DNA, protein or RNA synthesis by incubating cells for 30 min at 37 ° C in 1 ml of growth medium in the presence of 20 µCi/ml 3 H-TdR (- 50 Ci/mM 3H thymidine, Amersham Searle), 20 µCi/ml L-leucine (30 Ci/mM, International Chemical and Nuclear Corp .) or 20 µCi/ml 3H uridine (28 Ci/mM, Schwartz-Mann) . After incubation, the cells were quickly cooled to 0°C, washed twice in 10 ml of cold growth medium and lysed in 1 ml of 1 per cent SDS for 1 hour at room temperature . TCA precipitable counts in lysates were determined in a Beckman scintillation counter . The counts tabulated include background, which averages - 20 c .p .m . Macromolecular synthesis in normal and heat-treated cells .

Absence of ultrafast repair in mammalian DNA

127


cells by treatment at 65°C or 75°C for 10 min . In cells which had been held at 23°C, however, active macromolecular synthesis was observed . The capacity of heat-treated cells to rejoin single-strand breaks was also examined. It was found that heat-treated cells lack the capacity to rejoin single-strand breaks, as is illustrated in figure 4 . Here, Chinese hamster cells were incubated for 10 min at 52°C, then irradiated under anoxia with a dose of 2 krads . Immediately after irradiation they were resuspended in growth medium and incubated for 60 min at 37°C . Such incubation would result in a nearly complete restoration of broken DNA molecules in unheated cells ; yet, as is demonstrated in figure 4, no rejoining was observed in heat-treated cells . It was also observed that single-strand breaks induced by heat treatment alone could not be repaired. Similar experiments were carried out in which heattreated cells were irradiated in the presence of oxygen and then incubated for 60 min at 37°C ; again, no rejoining was found . Incubation at 37°C after heat treatment of the cells did not result in any significant further degradation of DNA molecules, indicating that DNA breaks due to heat treatment must have occurred during the period at the elevated temperature .

20 U) F z

oU

15

J

a o

10

U

5

F F Z

~

w 00

5

10 FRACTION

o

15

20

25

NUMBER


>

Figure 4 . Post-irradiation incubation of heat-treated cells . CH2Ba cells were heattreated at 52°C for 10 min, then quickly cooled to 0 ° C . They were irradiated under anoxia with a dose of 2 krads at 0 ° C . Immediately after irradiation they were either lysed (o) or further incubated at 37 °C for 1 hour and then lysed (o) for 12 hours at the top of an alkaline sucrose gradient . Gradients were centrifuged in a Beckman preparative ultracentrifuge (SW 27 .1 rotor, 17 ml tubes) at w = 20 000 r .p .m . for 6 hours . 3 .2 . Chemical treatment

Chinese hamster cells were treated with the same drugs and drug concentrations as were used in the experiments performed by Town et al. (1972) with E . coli cells . Labelled cells were prepared as described in §2 .2 and finally resuspended in PBS . The appropriate drug was added to the desired final concentration and the cells were incubated for 1 hour at 37°C, followed by an additional hour at 0 ° C while 0 2 or N2 was passing over the stirred cell suspension . Cells with the drug present were kept at 0°C during irradiation (with the respective gas still flowing) and until the cells were lysed . The results are presented in figure 5 .

B. Palcic and L. D . Skarsgard


128

With the exception of EDTA treatment, all drugs produced some DNA breaks even without irradiation . Here again, the net contribution of radiationinduced breaks was determined by using an expression analogous to (2), and the results are represented by the experimental points . For comparison, the production of single-strand breaks in cells not treated with drugs is shown as the dashed line, as in figure 3 (experimental points not shown) . The treatment of cells with 1 mM sodium cyanide, 10 mM hydroxyurea, 5 mM iodoacetic acid and 20 mM EDTA had no significant effect on the yield of DNA single-strand breaks under either aerobic (02 ) or anoxic (N 2 ) conditions . Treatment of cells with 0 . 2 mM quinacrine increased the sensitivity of DNA to radiation ; however, this was true under both aerobic and anoxic conditions, so that the o .e .r . remained approximately the same . The only treatment which selectively affected the yield of breaks under anoxic conditions was that where cells were exposed to 0. 5 mM NEM (N-ethylmaleimide) .

°

1 mM NaCN 20 mM EDTA

15

.0 5 mM IA ACID

•

10

~ . 10 mM H . UREA

0

0 .2 mM QUINACRINE

O

0 .5 mM NEM

5

O

5

N2

10

DOSE (kradc)

Figure 5 . The oxygen effect on the production of single-strand breaks in chemicallytreated cells . CH2P 2 cells in PBS were treated with the chemicals indicated in the figure (1 hour, 37 ° C), then irradiated under anoxic (N 2) or aerobic (O s) conditions at 0°C . Immediately after irradiation, cells were lysed at the top of an alkaline sucrose gradient for 12 hours at room temperature and then the gradients were centrifuged in a Beckman preparative ultracentrifuge (SW 27 .1, 17 ml tubes) . Weight average molecular weights were calculated from the sedimentation profiles and the net contribution of radiation-induced breaks was computed . Dashed lines are those from untreated cells (points not plotted) .

The increased yield of SSB under anoxic conditions in the presence of NEM cannot be taken as evidence of ultrafast repair because of this drug's known properties as an anoxic radiosensitizer . This fact has already been mentioned by Town et al. (1972) . In fact, it has been shown by Dugle et al. (1972) as well as in our own laboratory (Palcic, Agnew and Skarsgard 1974) that most anoxic radiosensitizers selectively enhance the yield of SSB under anoxic irradiation, with no effect on aerobic cells . In both laboratories, these results have been interpreted as indicating that these sensitizers act in a fashion similar to that of oxygen, namely, by enhancing the yield of initial lesions by direct physicochemical interaction rather than by interference with some mechanism of enzymic repair .


129


3 .3 . Adenovirus 2 It is a general property of isolated virus that, by itself, it lacks enzymatic activity. Only when it is introduced into the host cell does it manifest enzymic function . We therefore decided to examine the yield of SSB in Adenovirus 2 under both aerobic and anoxic conditions, since it seemed that if there were an oxygen effect for SSB production in virus, it would strongly support the idea that this is a general phenomenon and not something peculiar to mammalian cells . Isolated and purified virus was irradiated under conditions identical to those of the mammalian cell experiments . Concentrated inactivated viral suspensions in CsCI were diluted 1 : 200 with PBS . Aliquots of 10 ml were loaded into the irradiation vessels and kept under sterile conditions at 0°C throughout the experiment . 0 2 or N2 was flowed over stirred suspensions for 1 hour before and during irradiation . A typical result is shown in figure 6 . A dose of 27. 6 krads was delivered under aerobic (0 2 ) and anoxic conditions (N2 ) . Viral DNA irradiated in the presence of oxygen displayed much more damage than that irradiated with the same dose under anoxia . Pooled data from all such experiments are presented in figure 7 . The ratio of slopes of the lines representing the best fit through the experimental points in oxygen and in nitrogen gives an o .e .r . value of 3 . 6.

F- UNIRRADIATED VIRUS

I

•

o

0 5

P

!• 10

FRACTION

\

15

h

20

25

NUMBER


Figure 6 . Sedimentation profiles of irradiated Adenovirus 2 . Adenovirus 2 was irradiated in buffered saline under conditions identical to mammalian cell experiments . A dose of 27 . 6 krads was delivered under anoxic (o) or aerobic (9) conditions . Immediately after irradiation the virus was lysed on the top of an alkaline sucrose gradient (1 hour, room temperature) and then centrifuged at w = 27 000 for 13 . 5 hours in a Beckman SW 27 .1 rotor (17 ml tubes) .

4. Discussion 4 .1 . Heat treatment The results presented in §3 .1 show that exposure of mammalian cells for 10-15 min to elevated temperatures (52 ° -75 ° C) does not affect the yield of DNA single-strand breaks produced by radiation under either anoxic or aerobic conditions . The o .e .r . for SSB is about the same in heat-treated and normal cells, approximately 3 . 0 . Yet, in the same cells, this heat-treatment was shown to block effectively all the enzymic processes examined including DNA, RNA and protein synthesis . Also, the rejoining of DNA SSB, a phenomenon that


130


has been found to operate in all mammalian cell-types examined and is presumed to be enzymic in nature, is also completely inhibited by heat treatment . Although a few cellular enzymes undoubtedly retain some activity even after this heat treatment, it is clear that all enzymic processes should be substantially inhibited . Consequently, the heat treatment results provide strong evidence that little, if any, enzymatic modification of DNA breaks occurs in normal mammalian cells kept at 0°C during and after irradiation . In earlier work, we showed that in normal cells (not heat-treated), incubation at 0°C for periods as long as 5 hours after irradiation produced no detectable changes in the DNA size distribution (Palcic and Skarsgard 1972 b) .

W J

U W

15

J

J Q

>

10

0= U) Y Q w CIO

5

O ce w m Z

10

20

30

40

50

60

DOSE (V.rads)

Figure 7 . The oxygen effect on the production of single-strand breaks in Adenovirus 2 . Adenovirus 2 was irradiated in buffered saline under aerobic ( .) or anoxic (o) conditions . The molecular weight of single-stranded DNA was determined from sedimentation profiles such as those shown in figure 6 . The number of breaks per single-strand molecule was calculated and is shown plotted against dose .

4.2 . Chemical treatment The results of the drug experiments are less conclusive than those of heat treatment for the reasons already described in §3 .2 . Also, it has not been established to what extent these chemicals can penetrate (passively or actively) into Chinese hamster cells, though with all of the agents except EDTA, some DNA breaks were observed due to the chemical treatment alone, indicating the presence of the drugs in the cell nucleus . In previously reported experiments (Palcic and Skarsgard 1972 b), we exposed Chinese hamster cells, mouse fibroblasts and human skin fibroblasts to 2,4-dinitrophenol (DNP) in vitro . The treatment of the cells with DNP (0 . 1 mM or 0 . 5 mM in PBS) was identical to the chemical treatments described in §3 .2 . DNP produced a severe depletion of intracellular ATP and this was accompanied by a large inhibition of all the energy-dependent enzymatic processes examined . It has also been shown that the rejoining of single-strand breaks is inhibited by DNP treatment (Moss, Dalrymple, Sanders, Wilkinson and Nash 1971, Palcic and Skarsgard 1972 b), and these findings accord with those of Matsudaira, Furuno and Otsuka (1970), who showed that in Ehrlich ascites-tumour cells ATP is required for the rejoining of single-strand breaks .


131

Nevertheless, when the o .e .r . for break production was measured in DNP-treated cells, it was found to be the same as in untreated cells (Palcic and Skarsgard 1972 b) .


4.3 . The o .e.r. in isolated nuclei In another series of experiments using mouse L-60 cells, we examined the capacity of isolated nuclei to rejoin DNA single-strand breaks (Palcic 1972) . Two procedures were used for the isolation of nuclei (Kemper, Pratt and Aronow 1969, Kidwell and Mueller 1969). Though it has been reported that under certain circumstances isolated cell nuclei are able to rejoin some singlestrand breaks on the addition of a cytoplasmic supernatant fluid and ATP (Matsudaira and Furuno 1970), we could not demonstrate rejoining of breaks in cell nuclei under any conditions . Yet, when cell nuclei were prepared even without the addition of the supernatant fraction and exogenous ATP, and then irradiated under anoxic or aerobic conditions (following a procedure identical to that for cellular experiments), the observed o .e .r . was the same as for intact cells, approximately 3 . 0 . Thus, both the DNP and isolated nuclei experiments support the hypothesis that no enzymatic processes are responsible for the observed o .e .r ., though the unlikely possibility that some enzymatic repair system not dependent on energy may be operative cannot be excluded . 4.4. Adenovirus 2 The lack of enzymatic activity in this organism before infection of a host cell makes it a very useful test system with regard to the question of whether, as a general rule, the observed oxygen effect on single-strand breaks simply reflects enzymic repair under hypoxic conditions . The data presented in §3 .3 show unambiguously that under conditions of irradiation identical to those used for mammalian cells, the production of DNA single-strand breaks in Adenovirus 2 has a substantial o .e .r . (3 .6) comparable to that observed in mammalian cells . The first o .e .r. measurement for SSB production in bacterial virus suggested that oxygen did not affect the yield of SSB (Freifelder 1966) . However, later studies (Boyce and Tepper 1968, Van der Schans and Blok 1970, Johansen et al. 1971) reported that there was an oxygen effect for SSB production by radiation in bacterial virus . These reports, together with the present work using Adenovirus 2, argue strongly against the hypothesis that, in general, an ultrafast enzymatic modification of anoxic breaks is responsible for the observed oxygen enhancement ratios in various biological systems .

5.

Conclusion All the observations reported in this work support the idea that the initial yield of DNA single-strand breaks produced by ionizing radiation is greater in the presence of oxygen and that this increase is not due to some enzymatic modification of anoxic (or aerobic) DNA damage but rather that it represents an increase in the direct physico-chemical action of radiation under aerobic conditions .

132

B . Palcic and L . D . Skarsgard ACKNOWLEDGMENTS


The authors express their gratitude to Miss K . Brierley and Miss Isabel Poyntz who provided capable technical assistance . This work was supported by the National Cancer Institute of Canada, the National Research Council and the British Columbia Cancer Treatment and Research Foundation . Town et al. (1972) rapportent que la production des cassures de chaines uniques d'ADN Bans E . coli serait largement independante de la presence d'oxygene moleculaire pendant le temps d'irradiation . Its ont suggere 1'existence d'un mecanisme ultra-rapide de reparation agissant dans lea cellules bacteriennes, et specifique pour lea cassures produites dans Ns, serait 1'explication de 1'effet oxygen qu'on observe . Nous avons realise des experiences comparables avec des cellules provenant de hamster chinois et de souris pour determiner 1'existence des mecanismes similaires Bans lea cellules de mammiferes . L'inactivation par la chaleur, et l'inhibition par des moyens chimiques ont ete utilisees pour eliminer cette reparation enzymatique ultra-rapide supposee . Bien que le traitement par chaleur ait inactive tous lea processus enzymatiques essayes, it ne changait pas l'effet oxygen pour la production de cassures de chains uniques qui est restee a 3 . 0 environ . La presence de sodium cyanide, d'hydroxyuree, d'acide iodoacetique, d'EDTA et de quinacrine, n'a pas reussi non plus a changer l'effet oxygen significativement . Les noyaux isoles ont montre un effet oxygen entier. Au moins pour ces deux lignes de cellules, it ne semble pas exister de reparation ultra-rapide . L'adenovirus 2 isole, qui lui-meme manque d'activite enzymatique, a montre un effet oxygene de 3,6 pour la production de cassures . Selon ces resultats et d'autres, it parait improbable que la reparation enzymatique ultra-rapide soit un phenomene general, responsable de l'effet oxygene dans un grand nombre de systemes biologiques . Au contraire, it parait que 1'effet oxygene pour des cassures de chaines uniques resulte de differences dans lea effets physico-chimiques directs de la radiation sur la plupart des organismes sous conditions aerobiques et anoxiques . Town et al . (1972) berichteten Bass die Ausbeute an DNS-Einzelstrangbrilchen (SSB) in E. coli weitgehend unabhangig von der Gegenwart molekularen Sauerstoffes wahrend der Bestrahlung sei . Sie schlugen vor, dass fur das normalerweise beobachtete OER ultraschnelle Reparatur-Mechanismen (in bakteriellen Zellen) verantwortlich seien, welche hauptsachlich bei in N 2 produzierten strangbruchen wirken . Wir fuhrten vergleichbare Experimente mit zwei Zell-Arten aus, chinesischer Hamster and Maus, um das Vorhandensein ahnlicher Mechanismen in Sauger-Zellen zu untersuchen . Sowohl Warme-Inaktivierung als auch chernische Inhibierung wurden angewendet, urn diesen enzymatischen ultraschnellen Reparatur-Mechanismus zu eliminieren . Wahrend Warmebehandlung alle getesteten enzymatischen Prozesse inaktivierte, wurde das OER fur die SSB-Produktion nicht verandert . Es blieb ungefahr 3,0 . Ebenso hatte die Gegenwart von Natriumcyanid, Hydroxyharnstoff, Iodessigsaure, EDTA and Chinacrin keine wesentliche Veranderung des OER zur Folge . Auch isolierte Zellkerne wiesen ein unveranderte OER auf. Zumindest fur diese zwei Zell-Arten scheint der ultraschnelle Reparatur-Mechanismus nicht zu existieren . Der isolierte Adenovirus 2, welcher keine enzymatische Aktivitat aufweist, zeigte ein OER von 3,6 fur SSB-Produktion . Nach diesen and anderen Resultaten erscheint unwahrscheinlich, dass der sogenannte ultraschnelle enzymatische Reparatur-Mechanismus ein generelles Phanomen and fur das OER in einem weiten Bereich biologischer Systerne verantwortlich ist . Eher scheint das OER fur SSB aus Unterschieden der direkten physikalisch-chemischen Bestrahlungseffekte in den meisten Organismen unter 0 2 and N2 Bedingungen zu resultieren . REFERENCES

AGNEw, D . A ., and SKARSGARD, L . D ., 1972, Radiat. Res ., 51, 97 . BoYcE, R. P ., and TEPPER, M ., 1968, Virology, 34, 344 . CrARLESEY, A ., 1954, Proc . R . Soc . A, 224,120.


133

C . J ., ORMEROD, M . G ., SERIANNI, R . W ., and ALEXANDER, P ., 1969, Nature, Lond., 222, 1042 . DUGLE, D . L ., CHAPMAN, J . D ., GILLESPIE, C . J ., BORSA, J ., WEBB, R . G ., MEEKER, B. E., and REuVERS, A. P ., 1972, int . Y. Radiat . Biol., 22, 545 .


DEAN,

FREIFELDER, D ., 1966, Radiat . Res ., 29, 329 . JOHANSEN, I ., GuRVIN, I ., and Rupp, W. D ., 1971, Radiat . Res ., 48, 599 . KEMPER, B . W ., PRATT, W. B ., and ARONOw, L., 1969, Molec. Pharmacol., 5, 507 . KIDWELL, W. R., and MUELLER, G . C ., 1969, Biochem . biophys . Res . Commun., 36, 756 . LEHNERT, S ., and MoROSON, H ., 1971, Radiat . Res ., 45, 299 . MATSUDAIRA, H ., and FuRuNo, I ., 1970, Abstracts of the Fourth International Congress of Radiation Research, Evian, France, p . 140. MATSUDAIRA, H ., FURUNO, I ., and OTSUKA, H ., 1970, Int . Y . Radiat . Biol., 17, 339 . McGRATH, R. A., and WILLIAMS, R . W., 1966, Nature, Lond ., 212, 534 . Moss, A . J ., Jr ., DALRYMPLE, G . V., SANDERS, J. L ., WILKINSON, K . P ., and NASH, J . C ., 1971, Biophys. J., 11, 158 . PALcic, B ., 1972, Ph .D . Thesis, McMaster University, Hamilton, Ontario, Canada . PALCIC, B ., AGNEW, D . A., and SKARSGARD, L . D ., 1974, Radiat. Res., 59, 81 . PALCic, B ., and SKARSGARD, L . D ., 1972 a, Int. J. Radiat. Biol., 21, 417 ; 1972 b, Ibid., 21, 535 . PARKER, L., SKARSGARD, L . D ., and EMMERSON, P . T., 1969, Radiat . Res ., 38, 493 . SCHANS, G . P. VAN DER, and BLOK, J ., 1970, Int. -7. Radiat . Biol ., 17, 25 . TOWN, C . D ., SMITH, K . C ., and KAPLAN, H . S., 1972, Radiat. Res., 52, 99.

Fluorometric analysis of DNA unwinding to study strand breaks and repair in mammalian cells.

DNA strand breaks, repair, and survival in x-irradiated mammalian cells.

Strand breaks of mammalian mitochondrial DNA induced by carcinogens.

Error-Prone Repair of DNA Double-Strand Breaks.

Recombinational repair of radiation-induced double-strand breaks occurs in the absence of extensive resection.

DEK is required for homologous recombination repair of DNA breaks.

The origins of DNA breaks: a consequence of DNA damage, DNA repair, or apoptosis?

DNA excision repair in mammalian cell extracts.

RECQL4 Promotes DNA End Resection in Repair of DNA Double-Strand Breaks.

The repair of large DNA adducts in mammalian cells.

Chromatin modifications and DNA repair: beyond double-strand breaks.

Hierarchies of DNA repair in mammalian cells: biological consequences.

SMCHD1 accumulates at DNA damage sites and facilitates the repair of DNA double-strand breaks.

DNA polymerase delta-dependent repair of DNA single strand breaks containing 3'-end proximal lesions.

The Role of Long Non Coding RNAs in the Repair of DNA Double Strand Breaks.

Effect of differentiation on the repair of DNA single strand breaks in neuroblastoma cells.

Significance and measurement of DNA double strand breaks in mammalian cells.

On the initiation of mammalian RNA polymerase at single-strand breaks in DNA.

Letter: Modification of end-groups in DNA strand breaks of irradiated thymocytes during early repair.

Effect of heat on induction and repair of DNA strand breaks in X-irradiated CHO cells.

DNA single-strand breaks during repair of UV damage in human fibroblasts and abnormalities of repair in xeroderma pigmentosum.

Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells.

In vitro repair of double-strand breaks accompanied by recombination in bacteriophage T7 DNA.

The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage.