Radiation-induced chromosome damage in X-ray-sensitive mutants (xrs) of the Chinese hamster ovary cell line

255

Mutation Research, 166 (1986) 255-263 DNA Repair Reports, Elsevier MTR 06180

Radiation-induced chromosome damage in X-ray-sensitive mutants (xrs) of the Chinese hamster ovary cell line L . M . K e m p i a n d P.A. J e g g o 2,. I Department of Zoology, University College London, Gower Street, London WCIE 6BT, and 2 National Institute for Medical Research, The Ridgeway, Mill Hill, London N W 7 1AA (Great Britain) (Received 11 March 1986) (Revision received 4 June 1986) (Accepted 12 June 1986)

Summary The frequency of both spontaneous and X-ray- (95 rad) induced cytogenetical aberrations has been determined for 2 X-ray-sensitive strains (xrs-6 and xrs-7) of the Chinese hamster ovary cell line, and their wild-type parent (CHO-K1). Increased levels of spontaneous aberrations were not a general feature of the x r s strains, although xrs-7 did show a 2-fold increase in chromatid gaps. Unsynchronised populations of xrs ceils, estimated to have been irradiated in late S and G 2, showed a 3-5-fold increase in chromatid gaps, breaks and exchanges compared to CHO-K1. The irradiation of synchronised populations of xrs-7 and CHO-K1 in G 1 demonstrated a 3-5-fold increase in chromosome breaks, gaps and exchanges in xrs-7. In addition xrs-7 displayed a large increase in chromatid-type aberrations, particularly triradials. These X-ray-sensitive strains have previously been shown to have a defect in double-strand break rejoining (Kemp et al., 1984), and an increased number of double-strand breaks (DBSs) remain in their DNA after irradiation compared to wild-type cells. The increased number of DSBs remaining in these strains 20 min after irradiation, correlates well with the increase in chromosome breaks.

6-X-ray-sensitive strains ( x r s ) of the Chinese hamster ovary cell line (CHO-K1) have been previously described (Jeggo and Kemp, 1983), and all lie in the same genetic complementation group (Jeggo, 1985a). The x r s strains also show increased sensitivity to bleomycin (Jeggo and Kemp, 1983), ,/-rays, hydrogen peroxide (unpublished observations) and ot particles (Thacker and Stretch, 1985), in addition to showing a more variable and less marked response to certain alkylating agents and UV light (Jeggo and Kemp, 1983). These x r s strains are particularly interesting since they all show a defect in the rejoining of

* To whom correspondence should be addressed.

DNA double-strand breaks (DSBs) (Kemp et al., 1984), and they represent the first mammalian cell mutants with this defect to be described. In contrast they appear to show a normal capacity to rejoin X-ray-induced single-strand breaks (SSBs). Strains of this type, with a defect in DNA repair, will prove valuable in helping to understand mechanisms of DNA repair in mammalian cells, and radiation-sensitive mutants in particular may help to elucidate the effects of radiation damage in the cell. One major biological effect of radiation damage is the production of chromosome aberrations. The human syndrome ataxia telangiectasia (AT) and the mouse cell line M10, which are both sensitive to radiation as judged by cell survival studies, also show an elevated level of sponta-

0167-8817/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

256

neous and radiation-induced chromosome aberrations (Taylor et al., 1976; Taylor, 1978; Takahashi et al., 1982). It was therefore interesting to investigate the spontaneous aberration frequency in these xrs strains as well as examining the yield of aberrations following irradiation. Furthermore, these xrs strains provide a unique opportunity to investigate the role of DSBs and the mechanism of DSB rejoining in the production of chromosome aberrations. This is particularly important since there is increasing evidence supporting the idea that a DSB, whether induced directly or indirectly, is the lesion responsible for chromosome aberrations induced by ionising radiation (Natarajan and Obe, 1978; Natarajan et al., 1980; Bryant, 1984). In this report, we examine the cytogenetical response of two of these X-ray-sensitive strains, and discuss the results in terms of the knowo defect in DSB rejoining.

incorporation of [3H]thymidine into TCA-precipitable counts. Synchronised cells were seeded at a density of 1.5 × 104 cells/35-mm dish. At 2 hourly intervals duplicate cultures were pulse labelled for 30 min with 5/~Ci/ml [3H]thymidine (46 Ci/mM, Amersham International). TCA-precipitable counts were measured by filtering the cell lysate through glass microfibre filters and counting the filters in liquid scintillant.

X-Irradiation and metaphase preparation. Unsynchronised populations. Exponentially growing cells were irradiated with 95 rad of X-rays (Newton Victor X-ray machine operated at 120 kVp, 5.5 mA, at a dose rate of 48 rad/min) and arrested in metaphase by the addition of 0.1 mg/ml colcemid (Gibco-Biocult (UK) Ltd.), 2 h prior to harvesting. Cells in metaphase were harvested by mitotic detachment and chromosome preparations made by hypotonic treatment (0.56% potassium chloride), methanol-acetic acid fixation

Materials and methods

Cell culture conditions Cells were routinely grown in Eagle's minimal essential medium (Gibco F15) supplemented with 10% (v/v) foetal calf serum (heat inactivated at 56°C for 60 rain), penicillin-streptomycin (0.06 m g / m l and 0.1 mg/ml, respectively), glutamine (0.25 mg/ml) and pyruvate (0.11 mg/ml). Cells were maintained in 25-cm2 tissue culture flasks (Nunc) at 37°C in a 5% CO 2 humidified incubator. Synchronisation of cultures for G 1 irradiation Cells were seeded into 80-cm2 flasks (Nunc) at a density of approx. 4 × 106 and left overnight. Mitotic shake-off was performed as described by Zwanenburg (1983). Briefly, a preliminary shakeoff at 100 rpm for 3 min was used to remove loose or dead cells. Fresh medium was added and at 30-rain intervals mitotic cells were detached by shaking at 100 rpm for 1 min and then stored on ice in ice-cold PBS. Following 3 "shake-offs" the cells were pelleted, counted and plated as required. This was taken as zero time for each experiment. The synchrony and cell cycle progression of the mitotic population was checked by measuring the

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Fig. 1. Cytogenetical aberrations in X-irradiated cells. (A) Unsynchronised population of xrs-6, sampled 4 h after receiving a dose of 95 rad X-rays. (B) Synchronised population of xrs-7 irradiated in G 1 and sampled at the following mitosis. N.B.: Magnification differs between prints, a, chromatid gap; b, chromatid break; c, chromatid intrachange; d, chromatid interchange; e, chromatid interchange (quadriradial); f, triradial; g, dicentric; h, centric ring.

257

(3:1) and air drying. Slides were stained in 4% Giemsa. Synchronised populations. Mitotic cells were seeded in 25-cm2 flasks at a density of 105 cells/flask, incubated for 30 rain and irradiated with 95 rad X-rays. 3 h prior to harvesting, 0.1 mg colcemid was added. Metaphase preparations were then obtained at 17-24 h and 19-26 h post irradiation for CHO-K1 and xrs-7 respectively. Analysis of chromosome aberrations Aberrations were classified into 2 categories, chromatid type and chromosome type. Each category was further subdivided into gaps, breaks and various forms of exchange figures (see Fig. 1 and Table 2 for examples). All aberrations with a 3-armed structure (i.e. T- and Y-shaped figures) were classified as triradials. The significance of the difference in the numbers of chromosome aberrations was tested by assuming these numbers follow a Poisson distribution and comparing them using a normal theory approximation. Results

Analysis of chromosomes Chromosome analysis was made using 3 cell strains, the wild-type parent, CHO-K1 and 2 Xray-sensitive strains, xrs-6 and xrs-7. All 3 strains had a modal chromosome number of 23. Chromosome aberrations were examined under 3 conditions; those arising spontaneously, those arising from unsynchronised populations and those arising from synchronised populations after irradiation in G 1. Spontaneous chromosome aberrations Spontaneous chromosome aberrations were

scored from 300 metaphase preparations of each strain and the results are shown in Table 1. Although the dicentrics were classified as chromosome-type aberrations, they are probably of the derived type, arising from chromatid exchanges following cell division (Savage, 1976). Chromatid gaps were increased 2-fold in xrs-7 compared to CHO-K1, but this was not observed in the xrs-6 strain. No other class of aberrations was significantly increased in the xrs strains. Thus an increased level of spontaneous chromosomal aberrations does not appear to be a major feature of the xrs strains. Chromosome aberrations in unsynchronised populations Exponentially growing cultures of CHO-K1, xrs-6 and xrs-7 were irradiated with 95 rad X-rays and sampled at 2, 4, 6 and 24 h, post irradiation, with colcemid (0.1 mg/ml) present for the final 2 h. 150 metaphases were analysed at each sample time and the results shown in Table 2. Wild-type cells sampled at 2, 4, and 6 h were probably irradiated in G2, early G2/late S and S phase respectively, but this relationship may not hold for the xrs cells which may experience a longer mitotic delay. Wild-type cells irradiated at 24 h were most probably undergoing their second post-irradiation mitosis (cell cycle time of CHO-K1 is 14 h), but this also may not be the case for the xrs strains. Chromatid-type aberrations. The most striking difference between CHO-K1 and the xrs mutants is the increased level of chromatid-type damage (Table 2). The production of breaks, gaps and exchanges with time is shown graphically in Fig. 2. Chromatid breaks were significantly increased in the xrs mutants at all sample times. At 4 h, the

TABLE 1 SPONTANEOUS CHROMOSOMAL ABERRATIONS Number of cells observed

Chromosome-type aberrations

Chromatid-type aberrations

Dicentrics

Gaps

Breaks

Gaps

Triradials

Interchanges

CHO-K1

300

2

0

1

21

1

1

xrs-6 xrs-7

300 300

4 0

0 3

5 2

22 47 *

1 1

0 0

Cell strain

* Significantly different from CHO-K1 at the 1% level ( p < 0.01).

258 TABLE 2 CHROMOSOMAL ABERRATIONS AT EACH FIXATION TIME IN CHO-K1, xrs-6 and xrs-7 CELLS INDUCED BY X-RAYS (95 tad) Cell strain

Hours Number Cellswith Chromosome-type aberrations of cells aberrations B/F Gaps d. c. dc. a observed (%) rain rings

CHO-K1

2 4 6 24

150 150 150 150

67 74 40 18

xrs-6

2 4 6 24

150 150 150 150

97 95 67 34

xrs-7

2 4 6 24

150 150 150 150

99 99 72 37

1 2 0 0 11 *** 12 ** 1 0 5 0 0 3

Chromatid-type aberrations B/F

Gaps

Tri

Inter

Intra

19 73 23

164 129 35

2 3 4

6 6 8

2 2 2

1

12

0

0

0

7 1 2 1

1 0 1 0

1 0 0 1

16 12 3 13

5 0 0 0

8 3 2 2

0 0 1 2

5* 189 *** 504 *** 5 13 2 *** 311 *** 202"** 10 * 35 *** 1 115 *** 98 *** 7 17 22 12 ** 6 12 *** 6 *

3 1 4 4

20 * 4 3 2

4 1 0 0

1 0 0 3

19 3* 11 * 12

2 3 5 1

181"** 367"** 88"** 13"*

801"** 376"** 79"** 31"*

2 11" 11 5"

16 +* 18" 27"** 8 ++**

B/F, breaks and fragments; Gaps, achromatic lesions; d. rain, double minutes; c. ring, centric rings; dc., dicentrics; Tri, triradials; Inter, chromatid interchange; Intra, chromatid intrachange. + includes 1 complex aberration; + + includes 2 complex aberrations (see text). a For explanation of classification see Results. Levels of significance: Difference with CHO-K1 significant at * p < 0.05 level; ** p < 0.01 level; *** p < 0.001 level.

time of m a x i m u m b r e a k p r o d u c t i o n , xrs-6 a n d xrs-7 exhibited 4.2 a n d 5 times as m a n y breaks respectively as C H O - K 1 . However, the largest relative increase in breaks was seen at 2 h post i r r a d i a t i o n when the m u t a n t s show 10 times more breaks t h a n C H O - K 1 . C h r o m a t i d gaps were also significantly increased a n d at 2 h, w h e n the gap frequency was greatest, xrs-6 a n d xrs-7 displayed 3 a n d 4.8 times more gaps respectively t h a n C H O - K 1 . T h o u g h xrs-6 and xrs-7 show a n overall increase i n the level of breaks a n d gaps, these aberrations follow the same time course of a p p e a r a n c e as in C H O - K 1 (Fig. 2). Triradials a n d c h r o m a t i d exchanges are prod u c e d at a lower frequency t h a n breaks or gaps, b u t b o t h are increased in the xrs strains c o m p a r e d to C H O - K 1 (Table 2). W h e n all classes of chrom a t i d exchange (triradials, interchanges a n d intrachanges) are a d d e d together, the difference is highly significant, particularly at the 4- a n d 6-h sample times. The analysis of these data is complicated since the time of m a x i m a l exchange form a t i o n is 6 h in C H O - K 1 a n d xrs-7 a n d 4 h in

xrs-6 (Fig. 2). I n a d d i t i o n the n u m b e r of exchanges is small. Nevertheless the increase in c h r o m a t i d exchanges appear to be of the s a m e order of m a g n i t u d e as the increase in breaks a n d gaps, n a m e l y 3 - 4 - f o l d at the time of m a x i m a l production. Chromosome-type aberrations. These were observed in C H O - K 1 a n d xrs cells at b o t h early a n d late s a m p l i n g times (Table 2). I n T a b l e 2 one class of exchange a b e r r a t i o n has been classified as a dicentric, however the a c c o m p a n y i n g fragments, a n i n d i c a t o r of a true dicentric, were not scored a n d therefore these m a y represent i s o c h r o m a t i d / i s o c h r o m a t i d exchanges ( n o r m a l l y very rare aberrations) or t e r m i n a l c h r o m a t i d exchanges which c a n open out to resemble dicentrics. The a b n o r m a l l y high frequency of dicentrics seen at early s a m p l i n g times i n C H O - K 1 suggests that these m a y be incorrectly classified. Since isochromatidi s o c h r o m a t i d exchanges a n d t e r m i n a l exchanges are c h r o m a t i d - t y p e aberrations, in analysing the c h r o m o s o m e - t y p e aberrations the dicentrics have b e e n excluded. I n addition, c h r o m o s o m e breaks a n d isochromatid breaks are difficult to dis-

259 I

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Fig. 2. The frequency of X-ray-induced chromatid-type aberrations as a function of post-irradiation fixation time. Cells were irradiated with 95 rad X-rays and fixed at various times thereafter. The number of aberrations per cell was calculated from Table 2. Chromatid exchanges include triradials, interchanges and intrachanges. ©, CHO-K1; A xrs-6; n, xrs-7.

Irradiation o f cells in G~ S y n c h r o n i s e d p o p u l a t i o n s o f C H O - K 1 a n d xrs7 w e r e o b t a i n e d b y m i t o t i c s h a k e - o f f . 30 rain a f t e r m i t o t i c d e t a c h m e n t cells w e r e i r r a d i a t e d w i t h 95 r a d X - r a y s a n d 3 h p r i o r to h a r v e s t i n g c o l c e m i d w a s a d d e d . Cell c y c l e p r o g r e s s i o n w a s m o n i t o r e d in b o t h u n i r r a d i a t e d a n d i r r a d i a t e d cells b y

t i n g u i s h if t h e l a t t e r is o f the d o u b l y i n c o m p l e t e f o r m . D e s p i t e this d i f f i c u l t y in c l a s s i f i c a t i o n , t h e chromosome-type aberrations ( e x c l u d i n g dic e n t r i c s ) a p p e a r to s h o w a g e n e r a l s m a l l i n c r e a s e in t h e xrs cells c o m p a r e d to C H O - K 1 , b u t t h e d i f f e r e n c e is n o t as g r e a t o r as s i g n i f i c a n t as t h a t f o u n d for c h r o m a t i d - t y p e a b e r r a t i o n s .

TABLE 3 CHROMOSOME ABERRATIONS IN CHO-K1 AND xrs-7 CELLS IRRADIATED WITH 95 tad X-RAYS IN THE G 1 PHASE OF THE CELL CYCLE Cell Strain

Number of cells observed

Cells with aberrations (7o)

Chromosome-type aberrations B / F Gaps d. min c. rings

CHO-K1

150

13

5

1

0

0

7

4

xrs-7

150

56

26 *

3

2

11 *

26 *

23 *

dc.

Chromatid-type aberrations B / F Gaps Tri Inter 10 32 *

Intra

Complex

1

1

0

0

42 *

4

4

3

Abbreviations as described in Table. Complex, complex chromatid interchange involving 3 centromeres. * Difference with CHO-K1 significant at the 0.1% level (p < 0.001).

260

[3H]thymidine incorporation into D N A during a 30-min pulse given at 2 hourly intervals. 95 rad of X-rays given in G 1 delays the entry into S phase and depressed the level of D N A synthesis, to a greater extent in xrs-7 than in CHO-K1 (Fig. 3). A similar effect of enhanced radiosensitivity of D N A synthesis in xrs cells compared to wild-type cells, has been observed previously (Jeggo, 1985b). For CHO-K1, metaphases were collected from 17 to 24 h and for xrs-7, from 19 to 26 h post irradiation. No statistical differences were apparent between metaphases collected over the

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range of sample times, therefore the results from the different sample times were combined (Table 3). This suggests a constant radiosensitivity t h r o u g h o u t G 1 , but to substantiate this finding for all aberration categories a larger number of metaphases would need to be scored. G 1 irradiation resulted in 3-5 times more chromosome breaks, centric rings and dicentrics in xrs-7 than CHO-K1. In CHO-K1 chromatid-type aberrations were not significantly increased above the level found in unirradiated cells (Table 1), but in contrast chromatid breaks, gaps, exchanges and triradials were all seen at significantly increased levels in xrs-7 (Table 3). The most striking difference was the increase in triradials. While all other classes of aberration were increased 3-5-fold in xrs-7 compared to CHO-K1, triradials show an approximately 40-fold increase. It is unlikely that this increase in chromatid aberrations is due to G 2 cells contaminating the G 1 population, as chromatid aberrations were often found in cells which contained chromosome aberrations. The occurrence of both types of aberration within the same nucleus is very unusual, but is also seen in mouse M10 cells (Takahashi et al., 1982).

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Fig. 3. DNA synthesis in synchronised populations of irradiated (95 rad) and unirradiated cells. Synchronised populations of CHO-K1 and xrs-7 were obtained by mitotic detachment and seeded at a density of 1.5 × 104 cells/dish. At intervals [3 H]thymidine (5 # C i / m l ) was added to duplicate cultures for 30 min and TCA-precipitable counts were measured. A, unirradiated; ,,, 95 rad X-rays, 30 rain after mitotic detachment.

In this report, we have examined the cytogenetical response to X-rays of 2 X-ray-sensitive strains (xrs-6 and xrs-7), and the radio-resistant parent from which they were derived (CHO-K1). These x r s strains have been shown to have a defect in DSB rejoining and thus have an increased number of DSBs in their D N A after irradiation compared to control cells. We will attempt to evaluate the results on this basis. The xrs strains did not show a marked increase in the frequency of spontaneously arising chromosome aberrations, which suggests that a DSB is not a frequent lesion in unirradiated cells. The induction of aberrations by X-rays was investigated in unsynchronised and synchronised cultures. In the unsynchronised cultures, chromosome aberrations were examined at various times after irradiation, and although differences were apparent between the 3 different lines in the X-ray-induced perturbation of the cell cycle, samples from all 3 strains obtained at 2 and 4 h post

261 irradiation could be considered to have been in G 2 at the time of irradiation. X-Irradiation of cells (Table 2) resulted in a marked increase in chromatid-type aberrations, including breaks, gaps and exchanges in the two x r s strains compared to the parent strain. Using synchronised cultures of CHO-K1 and x r s - 7 aberrations were examined after irradiation in Ga phase and in this case an increase in chromosome aberrations (both deletion and exchange types) was observed in x r s - 7 as compared w i t h CHO-K1. Chromatid aberrations, particularly triradials were also found to be increased in x r s - 7 compared to CHO-K1. Chromatid damage of any sort is normally not observed in control cultures given this treatment. The origin of chromosome aberrations is still far from understood. Bender et al. (1974) proposed a model whereby DSBs, either directly induced or arising via the repair or replication of various forms of base damage and single-strand breaks, can give rise to deletions or exchanges. Furthermore there is accumulating evidence that DSBs may be the lesion responsible for chromosome aberrations induced by ionising radiation. Indirect evidence for this comes from the fact that high LET radiations are efficient in inducing DSBs as well as chromosome aberrations (Natarajan and Zwanenburg, 1982). More direct evidence has come from rendering irradiated cells permeable to Neurospora endonuclease, which converts X-ray-induced SSBs to DSBs. Under these conditions chromosome aberrations can be dramatically increased (Natarajan and Obe, 1978; Natarajan et al., 1980) and the production of DSBs and aberrations correlate (Natarajan and Zwanenburg, 1982). More recently this has been confirmed by the introduction of restriction enzymes into permeabilised cells (Bryant, 1984; Natarajan and Obe, 1984). In contrast to this, Preston (1982), based on studies of irradiated lymphocytes treated with cytosine arabinoside, has proposed that aberrations arise during the repair of induced DNA base damage and not directly from strand breaks. Since these x r s strains have been shown to have a defect in DSB rejoining (Kemp et al., 1984), they present a unique opportunity to investigate further whether DSBs are causal in the production of chromosome aberrations. Since chromosome and

chromatid exchanges do arise in these strains, this suggests that there must be at least two pathways of DSB rejoining, one utilising the x r s gene product and a second resulting in the formation of chromosome exchanges. Any DSBs not rejoined by either mechanism might be expected to appear as chromosome or chromatid breaks at mitosis. Gaps are also increased in these strains, which suggests that they too might be a cytogenetical manifestation of a DSB although other events may lead to gap formation. Furthermore, the 2-4-fold excess of DSBs remaining in the x r s strains 20 min after irradiation (Kemp et al., 1984) is of a similar magnitude to the increase in chromosome and chromatid breaks seen in these strains (approx. 5-fold). We propose that if a DSB induced by radiation in G 2 is not rejoined by the normal x r s repair mechanism it may appear cytogenetically as a break or interact with another radiation-induced DSB to produce an exchange aberration of the chromatid type. If a DSB is induced in G 1 and not rejoined this may replicate to produce 2 breaks at the same point in sister chromatids which provides the basis for chromosome-type damage (Savage, 1976). To explain the chromatid-type damage observed in x r s - 7 cells after irradiation in G1 we propose that a lesion, which primarily affects only 1 strand of the D N A double helix in Gx, is via the processes of replication and repair, converted to a DSB in G 2. The inability to rejoin these DSBs in the x r s strains will thus result in an increase in chromatid-type aberrations. There are various possibilities for this primary lesion in G~. One candidate is an SSB, and though the repair of this lesion is normally a fast process, some cells may replicate past this lesion before repair has occurred and produce a DSB break. In wild-type cells, this may have little consequence, since DSB will be rejoined prior to the onset of mitosis. Alternatively, a small number of SSBs may be repaired by the same mechanisms as those used for DSBs and may therefore remain unrejoined in the x r s strains and appear as DSBs following D N A synthesis. Indeed, there is evidence for two mechanisms of SSB rejoining in E. coli, one of which may involve recombination (Kapp and Smith, 1970; Town et al., 1973; Smith, 1978). No defect in SSB rejoining was observed in the x r s strains (Kemp et al., 1984), but the proce-

262 dures used would p r o b a b l y not detect a minor c o m p o n e n t of unrepaired breaks. A n o t h e r possibility is that radiation-induced base d a m a g e is, via the processes of repair and replication, converted to a DSB. Alternatively we should consider that the a b n o r m a l presence of chromatid d a m a g e in G 1 irradiated x r s - 7 cells, could result from an altered cell cycle such as premature initiation of D N A synthesis. The large increase in triradials after irradiation of xrs-7 in G 1 requires further c o m m e n t since this is a lesion observed infrequently in normal cells. We should mention that all 3-armed structures were scored as triradials even though a c c o m p a n y ing fragments were not always seen. Thus the triradials m a y represent a heterogeneous group consisting of the "classical triradials" (Savage, 1976), as well as other forms of chromatid exchange and possibly a type of aberration which is unique to x r s cells. The presence of chromatid and c h r o m o s o m e aberrations in the same nucleus is a characteristic feature of G 1 irradiated xrs-7 cells, although it is also observed in M10 cells (Takahashi et al., 1982). We propose that the lesions f r o m which these aberrations might arise (i.e. a DSB in one chromatid and 2 DSBs at the same point on sister chromatids) m a y also interact to produce triradials. Finally, it is interesting to c o m p a r e the cytogenetical response of these x r s strains to that obtained with other ionising radiation-sensitive strains. A T (Taylor et al., 1976; Taylor, 1978) and M10 (Takahashi et al., 1982) cells both show a pattern of radiation-induced cytogenetical d a m a g e similar to that observed in this study. Both have been shown to be proficient in SSB rejoining (Jaspers et al., 1982; Sato and H a m a - I n a b a , 1984), and though only A T cells have been examined for DSB repair, this process appears to be quantitatively normal ( L e h m a n and Stevens, 1977; Jaspers et al., 1982), although there are reports based on experiments with plasmid D N A that it m a y be qualitatively different (Cox et al., 1984). One explanation for the similar effects on the chromosomes of these two cell lines is that both m a y be defective in the rejoining of a small and as yet undetectable p r o p o r t i o n of SSBs and DSBs (Taylor et al., 1976). A n o t h e r is that misrepair as well as failure to repair DSBs influences the cytogenetical

o u t c o m e (Cox et al., 1984). However, the fact that A T cells have a similar pattern of X-ray-induced c h r o m o s o m e aberrations to these x r s cells, even though they clearly do not have the same level of defect in DSB rejoining suggests that some caution should be put in interpreting these data solely on the basis of a defect in DSB rejoining.

Acknowledgement We would like to acknowledge the interest and support of Dr. R. Holliday in this work. We would also like to thank Dr. J. Savage and Dr. P. Bryant for their useful comments.

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