Blochtmwa et Btophystca Acta, 741 (1983) 341-347 341 Elsevter
BBA 91293
DNA REPAIR IN ULTRAVIOLET-IRRADIATED HeLa CELLS IS DISRUPTED BY APHIDICOLIN
THE INHIBITION OF REPAIR NEED NOT IMPLY THE ABSENCE OF REPAIR SYNTHESIS
ANDREW COLLINS
Cancer Research Campatgn Mammahan Cell DNA Repmr Group, Umverslty of Cambrtdge, Department of Zoology, Downing Street, CambrMge CB2 3EJ (U K)
(Received June 22nd, 1983)
Key words DNA repatr, Aphtdwohn, Ultravwlet trradtanon, DNA polymerase a, (HeLa cell)
Aphidicolin, a potent and specific inhibitor of eukaryotic DNA polymerase a, has been reported to inhibit repair DNA synthesis in ultraviolet-irradiated, normal human fibroblasts but not in HeLa cells. By the use of assays for repair other than the measurement of repair synthesis, it is shown here that repair in HeLa cells is in fact susceptible to aphidicolin. Severe inhibition of DNA repair, with failure of individual repair events to be completed, and a smaller number of lesions removed, can occur even though repair synthesis continues.
Introduction
There has been much debate about the roles of the different eukaryotic DNA polymerases in re- pair DNA synthesis. Early circumstantial evidence implicated polymerase fl [1,2], but it is now clear that polymerases a and fl both participate in re- pair, to extents whtch depend on the cell type and on the nature of the D N A damage inflicted. The key to this conclusion has been the use of inhibi- tots specific to each of these polymerases, in par- ticular aphidicolin, which inhibits polymerase a [3], and dideoxythymidine, or its triphosphate, which blocks polymerase fl much more effectively than a [4]. These inhibitors have been applied to in vitro systems of isolated nuclei or permeabilised cells, in which effects on repair DNA synthesis can be measured unambiguously [5-9]. In whole cells, inhibitors have been shown to cause an accumula- tion of DNA breaks representing repair sites at which enzymic incision has occurred but comple- tion and ligation of the repair patch have been prevented [10-13].
Experiments with aphidicolin have indicated a
major role for polymerase a in the repair of ultra- violet-damaged DNA in normal human diploid cells. Incubation of ultraviolet-irradiated cells with the drug leads to high levels of inhibited repair sites revealed as DNA breaks [10-12]; and in permeabihsed cells or cell lysates, repair DNA synthesis is severely depressed [5,6,8]. In non-di- viding whole cells, too, repair synthesis (measured as incorporation of [ ~ H]thymidine ([ 3 H]dThd) into DNA) is inhibited by aphidicolin [12,14,15].
But in the case of heteroploid HeLa cells, there are conflicting results. Although aphidicolin in- hibits repair DNA synthesis in cell lysates [7], it does not prevent [3H]dThd incorporation in non-S phase ultraviolet-irradiated cells, whether mea- sured by autoradiography [16] or by 5- bromodeoxyuridine labelhng and CsC1 density gradient sedimentation [17].
I set out to determine whether HeLa cells really differ from diploid human cells in their mode of repair of ultraviolet damage; or whether the ap- parent failure of aphidicolin to block DNA repair in these cells is an artifact of the methods of study employed. Since the results presented here point
strongly to the latter alternatxve, I have attempted to explain the paradox whereby repair is disrupted by polymerase inhibitors and yet repair DNA synthesis apparently continues.
Methods
Cell culture; chemicals Normal human diploid fibroblasts, BCL-D1
(from Gibco Europe), were grown in Eagle's minimal essential medium buffered with bi- carbonate and supplemented with 10% foetal calf serum (Glbco Europe). HeLa cells were grown m bicarbonate-buffered minimal essential medium with 5% serum (comprising 1 part foetal calf serum to 3 parts newborn calf serum, from Gibco Europe).
Hydroxyurea was obtained from Sigma Chemi- cal Co; aphidicohn was a gift from Dr A.H. Todd of ICI; and [methyl-3H]dThd (54 Ci /mmol) was from the Radlochemical Centre, Amersham Aphidicohn was dissolved in dimethylsulphoxide at 1-5 m g / m l and diluted with phosphate-buffered saline to make stock solutions at appropriate con- centrations.
Measurement of rephcatlve DNA synthesis Identical ahquots of either normal human or
HeLa cells (1 -2 .105) were inoculated in 35 mm Nunc dishes with 1 ml of appropriate growth medium and incubated overnight. Duplicate dishes received aphidicolin at different concentrations, or no inhibitor, and were then incubated for 30 mm with [3H]dThd (0.2/.tCi/ml) before lysing the cells in 1 ml of 0.5 M NaOH and precipitating mac- romolecules with 1 ml of 20% (w/v) tnchloroacettc acid. The precipitate was filtered on Whatman G F / C glass fibre filters, washed with 5% trlchlo- roacetic acid and ethanol, dried, and 3H incorpo- rated into DNA measured by scintillation count- rag.
Measurement of repair-related DNA break accumu- lation
The modification of the alkahne unwinding as- say for DNA breakage, using eight-chamber tissue culture shdes (Lab-Tek Division, Miles Laborato- ries), has been described [18]. Briefly, for normal human cells, each chamber of 4 slides received
2 -10 4 cells m 0.3 ml minimal essential medium (10% serum) with [3H]dThd at 0 2 ~tCl/ml, and the cells were incubated for 3 days to label DNA. HeLa cells were inoculated at 3 5 .10 4 per cham- ber, in minimal essential medium (5% serum), with 0.2 /~C1/ml [3H]dThd and 2- 10 v M unlabelled dThd, and incubated for 1 day The medium was then replaced with medium containing aphadlcohn at 2 or 10/xg/ml, with or without hydroxyurea at 1 - 10 2 M (one slide with each inhibitor treatment for each cell type) After 30 mm incubation, cells were irradiated, as described [18], with a range of ultraviolet doses from a germicidal lamp emitting at 254 nm. During further incubation with the same lnhlb~tors for 20 mln, excision repair was initiated. The slides were then rinsed with cold saline, and the cells in each chamber lysed with 50 /zl of alkaline sucrose solution (5% w / v sucrose/ 0.3 M NaOH/0 .5 M NaCI) for 15 mm at 4°C. After neutrahsation with 15/~1 2 M acetic acid, the DNA was transferred to Mdhpore HATF mtrocel- lulose fdters and digested with single-strand specific S1 nuclease [18]. The percent of DNA unwound in alkah and subsequently lost by S1 digestion reflects the frequency of DNA break>, and is calibrated by reference to the unwinding of DNA containing known numbers of breaks intro- duced by X irradiation
Measurement of ultraviolet damage and tt~ remot, al from DNA
HeLa cells were incubated for 2 days w~th [3H]dThd (0.2 ~Cl/ml) . The labelled cells were distributed into 6 35-ram Nunc dishes, at about 3 - 105 cells per dish, and after 3 h the medium was removed and the cells irradiated with ultraviolet (0.5, 1 or 2 Jm 2, two dishes at each dose) Medium was replaced, and aphidicohn (10 ~ g / m l ) added to one dish at each dose. The medium ( + aphidicohn) was changed twice during the next day. 26 h after irradiation, the medium was re- moved, the dishes transferred to ice, and the cells scraped off with a silicone rubber policeman m 1 ml of ice-cold low salt buffer (0.1 M NaCI/0.01 M EDTA/0.01 M Tns/0 .01 M mercaptoethanol/1 m g / m l bovine serum albumin, pH 8.0). Cells were pelleted in an Eppendorf Centrifuge 5414 (30 s). the pellets suspended in 0.5 ml low salt buffer and centrifuged again. The final pellets were sus-
pended in low salt buffer at a cell density of 4. 106/ml, and triplicate 5/xl aliquots (i.e., 2 .10 4
cells) added to 20/ t l of high salt lysis solution (2 M NaC1/0.01 M EDTA/0.002 M Tris/0.5% Tri- ton X-100, pH 8.0) in 1.5 ml polythene centrifuge tubes. This treatment produces nucleoids, i.e., pro- tein-depleted nuclei containing intact DNA, and it ensures maximum accessibility of cyclobutane pyrimidine dimers to ultraviolet endonuclease [19]. Lysis continued for 15 min on ice, in the dark. Then 0.45 ml of endonuclease buffer (0.01 M potassium phosphate/0.01 M mercaptoethanol/ 0.001 M EDTA, pH 7.5) was added, together with 25 /~1 of a crude ultraviolet endonuclease solution prepared from Mtcrococcus luteus essentmlly by the method of Carrier and Setlow [20] and equiva- lent to their fraction II. The enzyme and DNA were mixed by gently inverting the tubes, and digesUon proceeded for 1.5 h at 37°C. Tubes were placed on ice, 0.5 ml of ice-cold alkahne saline (0.11 M NaOH/0 .2 M NaC1/0.02 M EDTA) was added to each and rmxed by gently reverting the tubes, which were left on ice for 5 min to allow unwinding of DNA in the alkali. After neutralisa- Uon with 0.1 ml 1 M KH2PO 4, the samples were sonicated and the percentage of single-stranded DNA (reflecting the frequency of endonuclease-in- duced DNA breaks) was estimated by hydroxy- apatite chromatography.
A calibration curve was constructed with cell samples given doses of ultraviolet between 0 and 2 Jm -2 and assayed as described above for endo- nuclease-sensitive sites, immediately after irradia- tion. This perimtted results of experiments involv- ing repair to be expressed in terms of the ultra- violet dose equivalent to the residual d~mers detected as endonuclease sites.
R e s u l t s
Sensttwtty of rephcattve DNA synthests to mhtbttton by aphtchcohn
Proliferating cultures of HeLa cells and normal human fibroblasts were pulse-labelled with [DH]dThd in the presence of various concentra- tions of aphidicolin, to measure the inhibition of normal DNA synthesis by the drug. Fig. 1 shows that the two cell types are both susceptible to aphidicohn, with DNA synthesis in HeLa cells
343
60:
§s0
g4o
~ 3o ¢,1
15 .~ 20 'O .IZ I - N 10
7C
~O 0 t t t 001 01 1.0 10
Aphidtcottn concentrotton (IJg/ml)
Ftg 1 Inhibition of rephcattve DNA synthesis by aphldlcohn Aph]dlcohn, at the concentrattons shown, was added to duph- cate samples of proliferating HeLa cells (O) or normal human flbroblasts (zx), followed by [DH]dThd; after 30 mm lncubat]on, incorporation of 3H into DNA was measured. Data (means of duplicates) are expressed as the percentage of the incorporation in samples not incubated with aplu&cohn (Dtmethylsulpho- xtde at a concentration corresponding to that present with the apbadicohn had no stgnlficant effect on incorporation.)
being slightly more depressed at a given aphidico- lin concentration than DNA synthesis in normal fibroblasts. 50% inhibition is reached at a con- centration of about 0.02 /~g/ml. Clearly there is no evidence that an unusually aphidmolin-resistant form of DNA polymerase a is operating in HeLa cells.
Accumulatton of DNA breaks m ultraotolet-lrradm- ted cells with aphtdwohn
A sensitive test for the inhibition of repair DNA synthesis is the presence of elevated levels of breaks in preexisting DNA in ultraviolet-irradia- ted cells [21]. These breaks represent continuing enzymic incision at ultraviolet damage sites (prin- cipally cyclobutane pyrimidine dimers) without subsequent resynthesis and ligation. Normal pro- liferating human cells, as expected, accumulate breaks to the same level whether incubated with 2
o r 10 /zg a p h i d l c o l i n / m l ; h y d r o x y u r e a p resen t
w~th the a p h i d i c o h n has no s i gmf i can t ef fec t (Fig.
2a). H e L a cells show a d i f f e ren t pa t t e rn of re-
sponse (Fig. 2b). A c c u m u l a t i o n of b reaks wi th
a p h l d l c o l i n at 2 / x g / m l is low c o m p a r e d wi th the
level r e ached at 1 0 / x g / m l ; and at each a p h i d l c o h n
c o n c e n t r a t i o n , h y d r o x y u r e a has a p o t e n t m t i n g ef- fect. Thus , r epa i r in H e L a cei ls ~s re la t ive ly insen-
s i t ive to i n h i b i t i o n by aph id i co l in ; bu t u n d e r ap-
p r o p r i a t e cond i t i ons , the level o f b reaks d e t e c t e d
can reach the level seen wi th n o r m a l h u m a n
f ibroblas ts .
Effect of aphtdtcohn on removal of dlmers E v e n t h o u g h a p h i d i c o h n does no t p r e v e n t en-
z y m i c inc is ion , a n d p r e s u m a b l y excis ion, at D N A
TABLE I
REMOVAL OF CYCLOBUTANE PYRIMIDINE DIMERS (U LTRAVIOLET-EN DONUCLEASE-SENSITIVE SITES) IN THE PRESENCE AND ABSENCE OF APHIDICOLIN
In the absence of aphldlcohn, cells are likely to pass through one S phase during the 26 h incubation The presence of unbroken new DNA paired in the same duplex with DNA nicked by ultraviolet endonuclease at dlmer sites v.tll reduce the effectwe concentration of unwinding points, and the resid- ual dlmers in preexisting DNA will be underesumated b~ a factor of ~< 2 Applying this maximum correction gives the figures shown In square brackets still clearb¢ indicating greater removal of dlmers in the absence of inhibitor
Imtlal ultraviolet Jm 2 equivalent to dimers remaining dose (Jm -2 ) after 26 h
Without With aphldtcohn aphldlcohn
05 01102] 03 1 0 01 [02 ] 0 4 2 0 0 2[0 4] 0 8
d a m a g e sites f r o m c o n t i n u i n g d u r i n g a br ief
pos t -u l t r av io l e t i n c u b a t i o n (as is d e m o n s t r a t e d by
the a c c u m u l a t e d breaks) , it can have an effect over
a p e r i o d of several hours ; S n y d e r and R e g a n [10]
f o u n d that m the p r e sence of aph ld l co l i n (4 ~ g / m l )
fewer d imer s were r e m o v e d ove r a 12 o r 24 h
p e r i o d af ter u l t rav io le t i r r ad i a t i on o f n o r m a l hu-
m a n f ibroblas ts . It is l ikely that the a c c u m u l a t i o n
o f i n c o m p l e t e repa i r sites exer ts a f eedback inhib i -
tory ef fec t on the ear l ie r s teps in the repa i r pa th -
way. G w e n m T a b l e I a re the resul ts of an exper t -
m e n t wi th H e L a cells, g iven low doses of ul t ra-
v io le t and i n c u b a t e d for 26 h w~th or w i thou t
a p h l d l c o h n . In the con t ro l cells, abou t 80% of
d i m e r s are r e m o v e d in this t ime, whereas m the
p r e s e n c e of aph ldxcohn abou t ha l f the d lmer s re-
ma in . Thus , the e f f ecuvenes s of aph ld l co l i n at
m h i b i t i n g repa i r m H e L a cells is c o n f i r m e d .
D i s c u s s i o n
The difference between HeLa and normal human cells
T h e fact that a tugher c o n c e n t r a t i o n of apht-
d i co l in is requxred to d e m o n s t r a t e i nh ib i t i on of D N A repaxr (m the D N A b reak a c c u m u l a t i o n
assay) in H e L a cells c o m p a r e d w~th n o r m a l h u m a n
345
fibroblasts suggests that HeLa cells may contain a higher concentration of dCTP to compete with aphidlcolin for the binding site on DNA poly- merase a. Alternative explanations, such as a dif- ference between HeLa and normal cells in the efficiency of transport of aphidicolin to the nucleus, or a difference in polymerase a susceptibility, are hard to reconcile with the apparently normal in- hibition of replicative DNA synthesis by aphi- dicolin in HeLa cells. If there is indeed a pool difference between these cell types, it may at first sight seem odd that this is not revealed in a differential effect of aphidicolin on replicative synthesis, too. But the replicative DNA poly- merase a is part of an enzyme complex [22] and relatively isolated from the free deoxyribonucleo- side tnphosphate pool. The potentiation of the inhibition of repair in HeLa cells by hydroxyurea, which blocks ribonucleotide reductase [23], may result from depletion of a limiting triphosphate m the DNA precursor pool.
Comparative data on deoxyrlbonucleos~de tn- phosphate concentrations in HeLa cells and in normal human fibroblasts are not avadable. But similar arguments invoking differences in dCTP pool size have been used to explain different re- sponses of DNA repair to aphidicolin m compari- sons of proliferating and confluent human fibrob- lasts [15,24].
In one respect, the results of Snyder and Regan [15] are at variance with those reported here. These authors found proliferating human cells to be very refractory to inhibition of DNA repair by apha- dlcolin (assayed by break accumulation or dimer removal) compared with confluent cells, so that with aphidicolin at 10 ~ g / m l hardly any effect was seen. Although a comparison with confluent cells has not been done in the present work, aphi- dicolin appears to elicit a maximal (and substan- tial) response in proliferating cells at only 2 #g /ml .
Repatr DNA synthests continues in the presence of aphldwohn
The data reported here clearly show that DNA repair m HeLa cells is severely disrupted by apha- dicolin, but it is equally clear that measurement of repair synthesis does not reflect this disruption; the incorporation of [3H]dThd into repaired DNA in HeLa cells is unaffected or even stimulated by aphidicolin [16,24]. In this latter respect, HeLa cells resemble proliferating (but not quiescent or
G 0) normal human fibroblasts [24]. The paradox of an inhibition of DNA repair
which is not revealed as a reduced level of repair synthesis is not unique to aphidicolin. Hydroxy- urea, too, causes repair-related breaks to accu- mulate in HeLa cells, and yet it stimulates the incorporation of [3H]dThd due to repair [24,25]. And the combination of hydroxyurea and 1-fl-D- arabinofuranosylcytosine (ara C), very potent at causing breaks to accumulate after ultraviolet irradiation, inhibits repair incorporation in con- fluent, non-dividing human cells, but stimulates incorporation in proliferating cells, whether HeLa, or normal diploid human cells [26].
Incorporation of [3H]dThd into DNA is likely to be an unreliable index of DNA synthesis, since the amount of 3H incorporated is a function of the specific activity of [3H]dThd in the cellular pool and will therefore be affected by fluctuations in the pool likely to result from the presence of inhibitors and from ultraviolet-induced changes in cell metabolism. We have shown the importance of this effect [27]. But even if estimates of repair synthesis are not entirely trustworthy, it cannot be denied that some repair synthesis does occur in the presence of inhibitors.
In the following model, which is summarized in Fig. 3, I attempt to reconcile these uncomfortable facts about inhibited repair, in a way which is consistent with certain other features;
Fig 3. Excision repair; a model to explain continuing repair synthesis at 'blocked' excision sites. ~; D N A with les~on endo = lesion-specific endonuclease, exo = exonuclease: pol = polymerase, hg = ligase The tluck hne denotes the repaar patch m the DNA. The normal pathway is shown along the top of the diagram. The lower section (' plus inhibitor') shows the poly- merase workang over a prolonged period but at a reduced rate through lack of precursors or direct mlubmon
346
(1) the involvement of polymerase a in repair of ultraviolet-reduced damage:
(2) the length of repair patch syntheslsed, esti- mated in the range 30-100 nucleotides [28,29]:
(3) the requirement of DNA polymerase c~ for a stretch of single-stranded template DNA longer than about 25 nucleotides for it to attach and begin processive polymerization [30]'
(4) the estimated 3-10 rain required for com- plete repair, i.e., from incision to hgation, at a single ultraviolet damage site [31] For the number of nucleotides inserted, this is a very long time, since in replicative synthesis in the same time interval several thousand nucleotides would be polymensed.
I propose that under normal conditions (without lnhibltors present), once incision has occurred at a damage site, the rate-limiting step in the comple- tion of repair is excision. Exonuclease action even- tually uncovers a region of single-stranded D N A long enough for DNA polymerase a to bind and start synthesis. Polymerization will occur at a rate exceeding the rate at which exonuclease removes nucleotides, so that the repair patch will become contiguous with the end of the DNA strand left by the exonuclease; a substrate will thus be provided for hgation, and the patch will be fully integrated in the DNA. In this scheme, DNA polymerase a might operate at a rate approaching the rate of rephcattve synthesis, but for a very short time; the long period for which a repair site remains open would be accounted for by the process of excision. The relatively rapid 'zipping-up' of the patch by polymerase a would imply a close correspondence between the size of template required by poly- merase a and the final patch size, since little further excision would occur once the polymerase had bound. Patch size and template size are clearly similar, but the figures are not precise enough to deduce the true relationship.
In an in vitro system, D N A polymerase a was found to operate at large single-strand gaps in DNA, but the gaps were not completely filled unless polymerase /3 were also present [32]. Whether this division of function applies to exci- sion repair in vivo is not known, but sequential involvement of both species of enzyme could read- dy be accommodated by the model.
Turning to the performance of repair under inhibitory conditions, we can suppose that aphl- dlcolin inhibits DNA polymerase a to an extent which would virtually abolish replicatlve DNA synthesis, but in repair, the reduced rate might still be sufficient for the polymerase slowly to synthe- stse a repair patch: instead of zipping up, the polymerase would stagger along. When repair synthesis is measured, it is normally done over a period much longer than the 10 min which is the highest estimate of the time required for repair at one site, so repair synthesis occurring in the pres- ence of lnhibltors at a reduced rate but over a longer period might give no indication of inhibi- tion in terms of nucleotldes incorporated. Further- more, if the reduced rate of polymensation is now less than the rate of excision, the gap and the patch will continue to grow, since hgatlon is im- possible. There is some evidence of enlarged repair patches in the presence of lnhibltors [33,34]. In time, repair sites are completed even in the pres- ence of lnhibltors [13,35], perhaps because the exonuclease, if acting processively, eventually dis- sociates from DNA, or because the reduced rate of polymerlsatlon is not significantly less than the rate of excision, or because the other species of polymerase takes over from the inhabited one
Acknowledgements
This work was supported by the Cancer Re- search Campaign. I thank Dr R.T. Johnson for reading the manuscript, Mrs. J. Whybrow and Mrs. P. Pawley for technical assistance, and ICI for the gift of aphidicohn.
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