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International workshop on inhibition of DNA repair
75
Mutation Research, 112 (1983) 75-83 DNA Repair Reports
Elsevier BiomedicalPress
International Workshop on Inhibition of D N A Repair K i n g ' s College, C a m b r i d g e , U . K . , 29th J u n e - 2 n d J u l y 1982 C. S t e p h e n D o w n e s , A n d r e w R.S. Collins a n d R o b e r t T. J o h n s o n Cancer Research Campaign Mammalian Cell DNA Repair Group, Department of Zoology, Universityof Cambridge.
(Received 17 August 1982) (Accepted21 October 1982)
Mammalian DNA repair systems have been subjected to quite detailed genetic analysis over the past decade. Complementation groups have been established for repair-deficient syndromes, and more recently cell lines hyper- or hypo-sensitive to DNA damaging agents have been created; but unlike the bacterial analogues, the plethora of genetically defective cell cultures has not yet given much insight into the undoubtedly complex nature of the response to DNA damage. The complementary biochemical approach of using selective inhibitors to dissect the different stages of the repair pathway has proved increasingly rewarding; this meeting was concerned with the modes of action and possible uses of a variety of inhibitors. [A book dealing with the topics discussed is being prepared (edited by A.R.S. Collins, C.S. Downes and R.T. Johnson) and will be published by Marcel Dekker.] Much of the workshop was concerned with the action of inhibitors of DNA synthesis. As pointed out by R.T. Johnson (C.R.C. Group, Cambridge) in his introductory talk, until very recently it was almost universally held that inhibitors of DNA replication are without effect on D N A repair; only a small number of cytogeneticists thought otherwise. This view has now been tacitly abandoned, and the consensus is now that hydroxyurea, cytosine (or adenine) arabinoside, aphidicolin and dideoxythymidine can all inhibit replication and repair alike. At the end of the meeting tables were compiled to summarise the observed effects of the more widely used inhibitors, and these are presented with this report, together with a diagram illustrating the known sites of inhibitor action (Fig. 1). Hydroxyurea (HU), an inhibitor of ribonucleotide reductase and so indirectly of DNA polymerisation, was relatively neglected. C.M. Pearson (I.C.I. Laboratories, Macclesfield) described parallel effects of HU, in UV-irradiated HeLa cells held in a 'stationary' state, in inhibiting DNA repair synthesis and in causing the accumulation of D N A strand breaks at sites where enzymic incision is not followed by repair synthesis and ligation. R.S. Day (N.I.H., Bethesda) reported that HU potentiates the killing of MNNG-treated adenovirus in mer- cells that repair MNNG damage only 0167-8817/83/0000-0000/$03.00 © 1983 ElsevierSciencePublishers
76 J~i i r i i
J
Chemicot alteration (e.g. base damage, dimerization, cross-tinking)
Strand breakaq~ (X rays, etc.)
ribonucteotide reductase Jb tacked
: ;=11 T 1 i
J [iJ
NAD
~" ~ ~i~;A~)B~( ~ - ~
~.btocked by novobiocin, Incision = "na[idixic acid
._L ~;[i' Excision ~~
Limited
by HU DNA /nrecursor suRpJY
~
polyme, r-
---
=
' I I I
AI~
/ ~
ization blocked aro A
poty (AOP-ribose)
= by
I II
;merase~ ~ pal / ¢¢ po~ymerose
I I
i / i li / ~e [ P_.9.JvmerizotionI /"" z ,'g, ==~=bradded by | btocked , ~ ara A, ara C,
=7=bY ddT
,:~'~
."
~gation
[igas
J ] i E[ ] J F i g . 1. S i m p l i f i e d d i a g r a m o f repair p r o c e s s e s , s h o w i n g sites o f i n h i b i t i o n . B r o k e n lines i n d i c a t e i n t e r a c t i o n s s u c h as a c t i v a t i o n or c o m p e t i t i o n .
by a nucleotide excision process. And the spectrum of reported effects (Table 1) leaves no doubt that repair synthesis, while less sensitive than replicative synthesis to the depletion of the deoxyribonucleotide pool that HU causes, is frequently affected. The popular technique of measuring repair synthesis through incorporation of labelled thymidine in the presence of H U (to suppress replication) may therefore be misleading; but it is quick, cheap and simple and will doubtless remain widely used. The arabinosides have similar but more marked effects, potentiated by HU. D.W. Kufe (Sidney Farber Cancer Institute, Boston) showed how cell killing and inhibition of D N A synthesis by cytosine arabinoside (ara C) correlate with incorporation of ara C into cell DNA; this incorporation occurs at chain termini and at internal sites. The internal arabinose groups are alkali-labile, which may possibly account for some of the 'strand breaks' detected in alkaline-lysed D N A from cells given ara C during repair. The kinetics of accumulation of such breaks in human diploid cells were described by R.D. Snyder (Johns Hopkins University, Baltimore), who showed that
77 TABLE 1
:
:
:
S U M M A R Y O F R E P O R T E D EFFECTS OF H Y D R O X Y U R E A ON D N A R E P A I R Observations contributed by V. Bohr, A.R.S. Collins, C.S. Downes, F. Hanaoka, B.A. Kihlman, C.M. Pearson, C.A. Smith, S. Squires, R. Waters and A.A. van Zeeland. In most cases the damaging agent used was UV light. Cell type
Repair assay Repair replication: BUdR incorporation, CsC1 sedimentation
Transformed (human, hamster)
Unscheduled D N A synthesis: [ 3H]thymidine incorporation
No effect, or increased
Normal proliferating (human,
In vitro repair: [ 3 H]dTTP incorporation
Accumulation of repair-related D N A breaks
Chromosome damage
Slightly reduced
+
+
+ (human)
+
Variable
+
Vicia faba ) Normal quiescent (human)
No effect
Variable
continuous incubation with H U and ara C after UV damage produces UV dose-dependent, stable, long-lived D N A strand breaks, the formation of which saturates after 2-5 h as if the excision-repair enzymes were immobilised at the break sites. Post-irradiation pulses o f . H U and ara C produced levels of breaks which rapidly decreased over a few hours. However, pulses of inhibitors after treatment with EMS or MMS showed a more complex pattern of break formation, with an initial decline followed by a second peak 20-40 h after treatment indicating the presence of a slowly repaired minor lesion. The kinetics of break accumulation in mouse cells undergoing UV repair, as described by G.C. Elliott (C.R.C. Group, Cambridge) are significantly different from human kinetics; breaks accumulate over a much longer period but at a much lower rate. Ara C was also shown, by A. Fornace (N.I.H., Bethesda) to accumulate strand breaks in cells repairing D N A cross-links induced by chromate, 'trans-platinum or formaldehyde; since these breaks occur to a lesser extent in xeroderma pigmentosum cells (in the case of damage by trans-platinum or formaldehyde), an excision process related to that which removes UV damage appears to be involved in repair of crosslinks in DNA. R. Waters (University College, Swansea) has developed the concept of ara C-sensitive repair into a screening assay to differentiate between the kinds of D N A damage inflicted by closely related chemical agents of the nitroquinoline oxide series. The question as to how far such break accumulation can be used as a quantitative
78
measurement of repair remained undecided. C.A. Smith (Stanford University) presented evidence, based on the sensitivity to nuclease digestion of labelled repair patches formed in the presence of ara C, that the majority of UV repair events occur at sites where the repair patch size is normal but HU and ara C together prevent ligation; with a minor ara C-insensitive component of repair that saturates at low UV doses, possibly due to polymerase fl acting at some repair sites. J.E. Cleaver (University of California, San Francisco) argued, from similar experiments, that no more than 50% of UV repair events give rise to ara C strand breaks. Previous publications on this subject may have been too optimistic about nuclease specificity. A different system described by C.S. Downes (C.R.C. Group, Cambridge), using chromatographic separation of repair-labelled DNA, also indicates that ara C-induced breaks may represent no more than 50% of UV repair, with greater ara C insensitivity at low UV doses. A comparison by Snyder of break formation and removal of labelled methyl groups showed that only a minor component of the repair of alkylated DNA is ara C-sensitive. Cleaver also described the effects of ara C on chromatin structure at the repairing site, as detected by nuclease digestion of repair-labelled regions in permeabilised
TABLE 2 SUMMARY OF REPORTED EFFECTS OF CYTOSINE ARABINOSIDE ON DNA REPAIR Observations contributed by A.R.S. Collins, C.S. Downes, G.C. Elliott, F. Hanaoka, A.M. Mullinger, A.T. Natarajan, G.P. van der Schans, C.A. Smith, R.D. Snyder and S. Squires. In most cases the damaging agent used was UV light. Square brackets indicate the presence of HU with ara C. Cell type
Transformed (human, hamster,
Repair assay Repair replication: BUdR incorporation, CsC1 sedimentation
Unscheduled DNA synthesis: [ 3H]thymidine incorporation
In vitro repair: [3H]dTTP incorporation
Accumulation of repair-related DNA breaks
[Slightly increased]
No effect
Slightly reduced [Further reduced]
+
[No effect]
i/louse) Normal proliferating (human, hamster, mouse)
No effect
Normal quiescent (human, hamster, mouse)
[+ + ]
[Reduced]
[Reduced in mouse, no effect in others]
+ [+ + ]
Reduced
Reduced
+
[Reduced]
[+ ]
Chromosome damage
79 UV-irradiated cells. The well-established decrease i n sensitivity of repair label to staphylococcal nuclease as cells are incubated after pulse-labelling, which may be due to nucleosomal rearrangement at the repair site, is partially blocked by ara C; and at the ara C-blocked sites, about half the DNA of repair patches is protected by protein while the rest is accessible to 3'-5' exonuclease. Chromatin changes at such inhibited sites can have long-range effects on nuclear structure, as was clearly seen in the micrographs of prematurely condensed chromosomes shown by W.N. Hittelman (Texas Medical Center, Houston); when repair of nitrogen mustard damage is blocked in G j phase cells by HU and ara C, the G j PCC become progressively more elongated, diffuse and fragmented. Table 2 shows the summary of reported ara C effects. Although, as Snyder reported, adenine arabinoside (ara A) can inhibit UV repair as effectively as ara C (though at a higher concentration), ara A has been used mainly in studies of X-ray repair. P.E. Bryant (G.S.F., Frankfurt/Main) reported that ara A inhibits essentially all of the slow component of X-ray-induced DNA strand break repair, which involves repair of double-strand breaks, and 70% of the fast repair of single-strand breaks. (Possibly the ara A-insensitive repair of strand breaks involves only the ligation of breaks between deoxyribose and phosphate without insertion of nucleotides by polymerase.) This degree of inhibition removes the shoulder from the X-ray survival curve. Aphidicolin, which specifically inhibits DNA polymerase a, has marked effects on DNA repair. G. Pedrali-Noy (University of Pavia) reiterated that in some circumstances UV-induced repair synthesis may appear resistant to aphidicolin; but in many cases repair is undoubtedly inhibited (see Table 3). M.R. Mattern (N.I.H., Bethesda) described the block to repair of UV- or MNNG-induced lesions, as measured by the sensitive assay of nucleoid sedimentation rates; Day showed how it, like other polymerase inhibitors, reduces host cell reactivation of UV-irradiated adenovirus; C.A. Smith and Cleaver described aphidicolin-sensitive and insensitive UV repair sites, the latter at least a respectable minority; and F. Hanaoka (University of Tokyo) showed how a HeLa homogenate system could be used to discriminate between the different types of DNA polymerase a. It appears that the aphidicolin-sensitive UV repair in this system depends on the PII polymerase selectively extractable in 0.2 M KC1, which is present throughout the cell cycle, and not on the S-phase-associated PI polymerase. But aphidicolin, though a repair inhibitor, actually increases the survival of cells X-irradiated in S phase. G. Iliakis (G.S.F., Frankfurt/Main) argued that since this effect is not seen in plateau-phase cultures, aphidicolin must in growing cultures inhibit S phase progression far more thari it inhibits repair, and so permit recovery from potentially lethal damage. However, P.J. Smith (M.R.C. Centre, Cambridge) found that inhibiting S phase with aphidicolin did not improve the survival of y-irradiated ataxia telangiectasia cells to the level seen in normal cells, at ,/-ray doses where the radiation damage strongly inhibits replication in normal but not ataxia cells. Dideoxythyrnidine (ddT), the specific inhibitor of DNA polymerase fl, might be expected to have a complementary effect to aphidicolin if both ct and fl polymerases
Inhibited
Normal quiescent (human) Reduced
Reduced [Reduced]
No effect
Unscheduled D N A synthesis: [3H]thymidine incorporation
a Higher concentration of aphidicolin needed in proliferating compared with quiescent cells.
Reduced [Further reduced]
N o effect or slightly increased [Reduced[
Inhibited
Normal proliferating (human)
Repair replication: BUdR incorporation, CsCI sedimentation
Increased [Reduced]
Removal of lesion
Repair assay
Transformed (human, hamster)
Cell type
Reduced [Greatly reduced]
In vitro repair: [3H]dTTP incorporation
+ + [+ + ]
+ [+ + ] a
+ [+ + ]
Accumulation of repair-related D N A breaks
+
Chromosome damage
Observations contributed by V. Bohr, P.K. Botcherby, A.R.S. Collins, F. Hanaoka, C.A. Smith, P.J. Smith, R.D. Snyder, R. Waters and A.A. van Zeeland. In most cases the damaging agent used was UV light. Square brackets indicate the presence of HU with aphidicolin.
S U M M A R Y OF R E P O R T E D EFFECTS OF A P H I D I C O L I N ON D N A R E P A I R
TABLE 3
81 are involved in repair. Mattern indeed reported t h a t though ara C, aphidicolin or ddT alone had no effect on or even enhanced repair of potentially lethal UV or M N N G damage, and provided a transient accumulation of inhibited repair sites, ddT in combination with either ara C or aphidicolin increased killing and caused lasting inhibition: as if either polymerase can substitute for the other in the repair of DNA lesions. But Cleaver's studies of nuclease sensitivity of repair label indicate that inhibition of synthesis and accumulation of inhibited sites are never complete, even with ara C and ddT together. One point clearly emerging from the meeting was that the effect of all these polymerase inhibitors on DNA repair is critically dependent on the growth state of the cells studied. The effects of aphidicolin in particular are dramatically less in exponentially growing than in quiescent cells, as was clearly shown by results presented by A.R.S. Collins (C.R.C. Group, Cambridge), C.A. Smith and Snyder. A 200-fold difference in the aphidicolin concentration necessary to achieve the same level of break accumulation in UV-irradiated quiescent and growing human fibroblasts was reported by Snyder. Ara C-induced breaks also tend to be fewer in growing than in quiescent cells, and Snyder reported that HU elevates the break frequency in growing but not in quiescent cells. Collins found that HU strikingly potentiates aphidicolin in causing break accumulation in UV-irradiated proliferating CHO or HeLa cells, which are otherwise rather insensitive to aphidicolin. The general ability of HU to enhance the activity of other inhibitors, probably by reducing intracellular levels of competing deoxyribonucleotides, suggests that variations in the response to inhibitors between different cell types and culture conditions are most likely due to differences in the size of deoxyribonucleotide pools. M. Meuth (Institut des Recherches Cliniques, Montreal) reviewed pool effects and showed how the pool sizes could be incidentally altered by added deoxyribonucleosides or their analogues, and described CHO thymidine auxotrophs whose deoxyribonucleotide pool imbalance leads to a high rate of spontaneous mutation; the implication being that some cellular effects of DNA polymerase inhibitors may be very indirect. A separate class of inhibitors, exemplified by 3-aminobenzamide (3AB), act on DNA repair via an inhibition of poly(ADP-ribose) synthesis. S. Shall (University of Sussex, Brighton) described the evidence for an involvement of this process in repair. A decrease in cellular levels of NAD, the precursor of ADP-ribose, is a general response to DNA damage, and is inhibited by 3AB. 3AB blocks the activation, by ADP-ribosylation, of DNA ligase II. The effect of 3AB on repair is seen as an inhibition of DNA-strand break rejoining, but only after certain kinds of DNAdamaging treatment. A.R. Lehmann (M.R.C. Cell Mutation Unit, Brighton) showed that 3AB only affects DNA repair in situations where DNA ligase activity, rather than some earlier step, is the rate-limiting factor. In fibroblasts this is the case for repair of alkylation lesions, but not for UV or X-ray repair. 3AB in alkylated cells causes DNA-strand breaks to persist at unligated repair sites, with a concurrent stimulation of DNA-repair synthesis (due to the unligated repair patches being longer), inhibition of recovery of replicative DNA synthesis and increased cytotoxicity. A.T. Natarajan (University of Leiden) also showed that these strand breaks, like strand breaks induced by repair polymerase inhibitors, have effects at the chro-
82 mosome level; chiefly in formation of sister chromatid exchanges. But the effects of 3AB vary between cell types. Cleaver warned that some of its effects may occur via the 1-carbon pool; in HeLa cells, though not in CHO, labelling of this pool is greatly decreased and the alkylating effect of MMS increased by 3AB; though indeed this may only reflect the cytotoxicity of MMS plus 3AB. And lymphocytes differ from fibroblasts; V. Bohr (University of Copenhagen) found that in lectin-stimulated human lymphocytes 3AB increases both UV-induced DNA repair synthesis (measured in the presence of HU) and the ligation of MNNG-induced strand breaks, the opposite effects being seen in L1210 cells. A.A. van Zeeland (University of Leiden) could find no effect of 3AB on UV-repair replication measured in the absence of HU, in either fibroblasts or lymphocytes; Cleaver confirmed that its apparent effects on UV repair replication are indeed HU-dependent. This seems odd, for HU as an indirect polymerisation inhibitor would not be expected to make tigation the rate-limiting step. W.A. Cramp (M.R.C. Cyclotron Unit, Hammersmith Hospital, London) reported the appearance of single-stranded fragments of DNA when cells replicate DNA after ionising irradiation, representing a delayed ligation of nascent DNA into the growing chain. Acetylated 3AB potentiates the effect of radiosensitisers in increasing the extent of single-stranded DNA. Two reports raised the interesting prospect that several clinical syndromes may be connected with spontaneous inhibition, or deficiencies, of repair ligase. A survey conducted by P.D. Lipetz (Ohio State University, Columbus) suggests that about 5% of the population may have lymphocytes that are seriously deficient in the ability to repair X-ray-induced strand breaks; and the defect in cells from 46BR, a unique clinical case which involves immunodeficiency and photosensitivity with no defect in UV-induced repair synthesis or post-replication recovery, was shown by S. Squires (C.R.C. Group, Cambridge) to involve spontaneous DNA-strand break accumulation in UV-irradiated cells, similar to that induced by ara C in normal cells. Topoisomerase inhibitors may well affect a pre-incision step; but much of the work has been concerned with novobiocin around which there remains a penumbra of doubt. This drug has rather widespread activity as an ATPase inhibitor; and Snyder reported that it causes a 5-fold increase in DNA-protein crosslinking, in the absence of DNA damage. This seriously complicates experiments measuring DNA repair through changes in DNA sedimentation; it is not clear how much of the reported decrease by novobiocin in DNA-strand breaks after UV plus ara C is due to crosslinking. But Snyder also reported a novobiocin inhibition of UV-induced repair synthesis and dimer removal that cannot be a crosslinking artefact; and Mattern showed that nalidixic acid, a more specific topoisomerase inhibitor than novobiocin, has similar effects in inhibiting repair of UV and M N N G damage. Lipetz also described a n original system using Paramecium cultures in which novobiocin increases the mutagenicity of photo-reactivable UV damage. Lastly, caffeine, which has been known for decades to be a repair inhibitor of some sort, remains obscure in its actions. B.A. Kihlman (Uppsala University) surveyed its diverse effects in producing chromosome aberrations in plant and mammalian cells emphasising, however, that caffeine may exert its damaging effect
83 in part by eliminating cell cycle lag after DNA:darnage. J.J. Roberts (Chester Beatty Research Institute, Chalfont St. Giles) described its reversal of the overall inhibition of DNA replication by DNA damage, which is combined with a production of local blocks at damage sites in replicating regions. A further complication in caffeine's effects on the replication of damaged DNA was introduced by G. Ahnstrt~m (Stockholm University), who found that pre-exposure of human fibroblasts to a small dose of UV will accelerate the recovery of replicon completion from the inhibitory effects of a second UV dose, and found also that caffeine removes the effect of pre-exposure. C.A. Waldren (Eleanor Roosevelt Institute, Denver, Colorado) discussed caffeine effects on purine and pyrimidine metabolism, where it blocks both de novo and salvage pathways. The actual fate of caffeine inside cells was disputed. Perhaps the general message from all parts of the workshop was that anyone studying DNA repair should also have an HPLC system for measuring intracellular pools of small molecules which we neglect at our peril. We are grateful to the Cancer Research Campaign for providing substantial support, and also to B.D.H. Chemicals Ltd., Glaxo (1972) Charity Trust, Imperial Chemical Industries Ltd., Shell Research Ltd., Sigma London, Unilever Research and the Wellcome Trust for generous financial help for this Workshop.
Mutation Research, 112 (1983) 75-83 DNA Repair Reports
Elsevier BiomedicalPress
International Workshop on Inhibition of D N A Repair K i n g ' s College, C a m b r i d g e , U . K . , 29th J u n e - 2 n d J u l y 1982 C. S t e p h e n D o w n e s , A n d r e w R.S. Collins a n d R o b e r t T. J o h n s o n Cancer Research Campaign Mammalian Cell DNA Repair Group, Department of Zoology, Universityof Cambridge.
(Received 17 August 1982) (Accepted21 October 1982)
Mammalian DNA repair systems have been subjected to quite detailed genetic analysis over the past decade. Complementation groups have been established for repair-deficient syndromes, and more recently cell lines hyper- or hypo-sensitive to DNA damaging agents have been created; but unlike the bacterial analogues, the plethora of genetically defective cell cultures has not yet given much insight into the undoubtedly complex nature of the response to DNA damage. The complementary biochemical approach of using selective inhibitors to dissect the different stages of the repair pathway has proved increasingly rewarding; this meeting was concerned with the modes of action and possible uses of a variety of inhibitors. [A book dealing with the topics discussed is being prepared (edited by A.R.S. Collins, C.S. Downes and R.T. Johnson) and will be published by Marcel Dekker.] Much of the workshop was concerned with the action of inhibitors of DNA synthesis. As pointed out by R.T. Johnson (C.R.C. Group, Cambridge) in his introductory talk, until very recently it was almost universally held that inhibitors of DNA replication are without effect on D N A repair; only a small number of cytogeneticists thought otherwise. This view has now been tacitly abandoned, and the consensus is now that hydroxyurea, cytosine (or adenine) arabinoside, aphidicolin and dideoxythymidine can all inhibit replication and repair alike. At the end of the meeting tables were compiled to summarise the observed effects of the more widely used inhibitors, and these are presented with this report, together with a diagram illustrating the known sites of inhibitor action (Fig. 1). Hydroxyurea (HU), an inhibitor of ribonucleotide reductase and so indirectly of DNA polymerisation, was relatively neglected. C.M. Pearson (I.C.I. Laboratories, Macclesfield) described parallel effects of HU, in UV-irradiated HeLa cells held in a 'stationary' state, in inhibiting DNA repair synthesis and in causing the accumulation of D N A strand breaks at sites where enzymic incision is not followed by repair synthesis and ligation. R.S. Day (N.I.H., Bethesda) reported that HU potentiates the killing of MNNG-treated adenovirus in mer- cells that repair MNNG damage only 0167-8817/83/0000-0000/$03.00 © 1983 ElsevierSciencePublishers
76 J~i i r i i
J
Chemicot alteration (e.g. base damage, dimerization, cross-tinking)
Strand breakaq~ (X rays, etc.)
ribonucteotide reductase Jb tacked
: ;=11 T 1 i
J [iJ
NAD
~" ~ ~i~;A~)B~( ~ - ~
~.btocked by novobiocin, Incision = "na[idixic acid
._L ~;[i' Excision ~~
Limited
by HU DNA /nrecursor suRpJY
~
polyme, r-
---
=
' I I I
AI~
/ ~
ization blocked aro A
poty (AOP-ribose)
= by
I II
;merase~ ~ pal / ¢¢ po~ymerose
I I
i / i li / ~e [ P_.9.JvmerizotionI /"" z ,'g, ==~=bradded by | btocked , ~ ara A, ara C,
=7=bY ddT
,:~'~
."
~gation
[igas
J ] i E[ ] J F i g . 1. S i m p l i f i e d d i a g r a m o f repair p r o c e s s e s , s h o w i n g sites o f i n h i b i t i o n . B r o k e n lines i n d i c a t e i n t e r a c t i o n s s u c h as a c t i v a t i o n or c o m p e t i t i o n .
by a nucleotide excision process. And the spectrum of reported effects (Table 1) leaves no doubt that repair synthesis, while less sensitive than replicative synthesis to the depletion of the deoxyribonucleotide pool that HU causes, is frequently affected. The popular technique of measuring repair synthesis through incorporation of labelled thymidine in the presence of H U (to suppress replication) may therefore be misleading; but it is quick, cheap and simple and will doubtless remain widely used. The arabinosides have similar but more marked effects, potentiated by HU. D.W. Kufe (Sidney Farber Cancer Institute, Boston) showed how cell killing and inhibition of D N A synthesis by cytosine arabinoside (ara C) correlate with incorporation of ara C into cell DNA; this incorporation occurs at chain termini and at internal sites. The internal arabinose groups are alkali-labile, which may possibly account for some of the 'strand breaks' detected in alkaline-lysed D N A from cells given ara C during repair. The kinetics of accumulation of such breaks in human diploid cells were described by R.D. Snyder (Johns Hopkins University, Baltimore), who showed that
77 TABLE 1
:
:
:
S U M M A R Y O F R E P O R T E D EFFECTS OF H Y D R O X Y U R E A ON D N A R E P A I R Observations contributed by V. Bohr, A.R.S. Collins, C.S. Downes, F. Hanaoka, B.A. Kihlman, C.M. Pearson, C.A. Smith, S. Squires, R. Waters and A.A. van Zeeland. In most cases the damaging agent used was UV light. Cell type
Repair assay Repair replication: BUdR incorporation, CsC1 sedimentation
Transformed (human, hamster)
Unscheduled D N A synthesis: [ 3H]thymidine incorporation
No effect, or increased
Normal proliferating (human,
In vitro repair: [ 3 H]dTTP incorporation
Accumulation of repair-related D N A breaks
Chromosome damage
Slightly reduced
+
+
+ (human)
+
Variable
+
Vicia faba ) Normal quiescent (human)
No effect
Variable
continuous incubation with H U and ara C after UV damage produces UV dose-dependent, stable, long-lived D N A strand breaks, the formation of which saturates after 2-5 h as if the excision-repair enzymes were immobilised at the break sites. Post-irradiation pulses o f . H U and ara C produced levels of breaks which rapidly decreased over a few hours. However, pulses of inhibitors after treatment with EMS or MMS showed a more complex pattern of break formation, with an initial decline followed by a second peak 20-40 h after treatment indicating the presence of a slowly repaired minor lesion. The kinetics of break accumulation in mouse cells undergoing UV repair, as described by G.C. Elliott (C.R.C. Group, Cambridge) are significantly different from human kinetics; breaks accumulate over a much longer period but at a much lower rate. Ara C was also shown, by A. Fornace (N.I.H., Bethesda) to accumulate strand breaks in cells repairing D N A cross-links induced by chromate, 'trans-platinum or formaldehyde; since these breaks occur to a lesser extent in xeroderma pigmentosum cells (in the case of damage by trans-platinum or formaldehyde), an excision process related to that which removes UV damage appears to be involved in repair of crosslinks in DNA. R. Waters (University College, Swansea) has developed the concept of ara C-sensitive repair into a screening assay to differentiate between the kinds of D N A damage inflicted by closely related chemical agents of the nitroquinoline oxide series. The question as to how far such break accumulation can be used as a quantitative
78
measurement of repair remained undecided. C.A. Smith (Stanford University) presented evidence, based on the sensitivity to nuclease digestion of labelled repair patches formed in the presence of ara C, that the majority of UV repair events occur at sites where the repair patch size is normal but HU and ara C together prevent ligation; with a minor ara C-insensitive component of repair that saturates at low UV doses, possibly due to polymerase fl acting at some repair sites. J.E. Cleaver (University of California, San Francisco) argued, from similar experiments, that no more than 50% of UV repair events give rise to ara C strand breaks. Previous publications on this subject may have been too optimistic about nuclease specificity. A different system described by C.S. Downes (C.R.C. Group, Cambridge), using chromatographic separation of repair-labelled DNA, also indicates that ara C-induced breaks may represent no more than 50% of UV repair, with greater ara C insensitivity at low UV doses. A comparison by Snyder of break formation and removal of labelled methyl groups showed that only a minor component of the repair of alkylated DNA is ara C-sensitive. Cleaver also described the effects of ara C on chromatin structure at the repairing site, as detected by nuclease digestion of repair-labelled regions in permeabilised
TABLE 2 SUMMARY OF REPORTED EFFECTS OF CYTOSINE ARABINOSIDE ON DNA REPAIR Observations contributed by A.R.S. Collins, C.S. Downes, G.C. Elliott, F. Hanaoka, A.M. Mullinger, A.T. Natarajan, G.P. van der Schans, C.A. Smith, R.D. Snyder and S. Squires. In most cases the damaging agent used was UV light. Square brackets indicate the presence of HU with ara C. Cell type
Transformed (human, hamster,
Repair assay Repair replication: BUdR incorporation, CsC1 sedimentation
Unscheduled DNA synthesis: [ 3H]thymidine incorporation
In vitro repair: [3H]dTTP incorporation
Accumulation of repair-related DNA breaks
[Slightly increased]
No effect
Slightly reduced [Further reduced]
+
[No effect]
i/louse) Normal proliferating (human, hamster, mouse)
No effect
Normal quiescent (human, hamster, mouse)
[+ + ]
[Reduced]
[Reduced in mouse, no effect in others]
+ [+ + ]
Reduced
Reduced
+
[Reduced]
[+ ]
Chromosome damage
79 UV-irradiated cells. The well-established decrease i n sensitivity of repair label to staphylococcal nuclease as cells are incubated after pulse-labelling, which may be due to nucleosomal rearrangement at the repair site, is partially blocked by ara C; and at the ara C-blocked sites, about half the DNA of repair patches is protected by protein while the rest is accessible to 3'-5' exonuclease. Chromatin changes at such inhibited sites can have long-range effects on nuclear structure, as was clearly seen in the micrographs of prematurely condensed chromosomes shown by W.N. Hittelman (Texas Medical Center, Houston); when repair of nitrogen mustard damage is blocked in G j phase cells by HU and ara C, the G j PCC become progressively more elongated, diffuse and fragmented. Table 2 shows the summary of reported ara C effects. Although, as Snyder reported, adenine arabinoside (ara A) can inhibit UV repair as effectively as ara C (though at a higher concentration), ara A has been used mainly in studies of X-ray repair. P.E. Bryant (G.S.F., Frankfurt/Main) reported that ara A inhibits essentially all of the slow component of X-ray-induced DNA strand break repair, which involves repair of double-strand breaks, and 70% of the fast repair of single-strand breaks. (Possibly the ara A-insensitive repair of strand breaks involves only the ligation of breaks between deoxyribose and phosphate without insertion of nucleotides by polymerase.) This degree of inhibition removes the shoulder from the X-ray survival curve. Aphidicolin, which specifically inhibits DNA polymerase a, has marked effects on DNA repair. G. Pedrali-Noy (University of Pavia) reiterated that in some circumstances UV-induced repair synthesis may appear resistant to aphidicolin; but in many cases repair is undoubtedly inhibited (see Table 3). M.R. Mattern (N.I.H., Bethesda) described the block to repair of UV- or MNNG-induced lesions, as measured by the sensitive assay of nucleoid sedimentation rates; Day showed how it, like other polymerase inhibitors, reduces host cell reactivation of UV-irradiated adenovirus; C.A. Smith and Cleaver described aphidicolin-sensitive and insensitive UV repair sites, the latter at least a respectable minority; and F. Hanaoka (University of Tokyo) showed how a HeLa homogenate system could be used to discriminate between the different types of DNA polymerase a. It appears that the aphidicolin-sensitive UV repair in this system depends on the PII polymerase selectively extractable in 0.2 M KC1, which is present throughout the cell cycle, and not on the S-phase-associated PI polymerase. But aphidicolin, though a repair inhibitor, actually increases the survival of cells X-irradiated in S phase. G. Iliakis (G.S.F., Frankfurt/Main) argued that since this effect is not seen in plateau-phase cultures, aphidicolin must in growing cultures inhibit S phase progression far more thari it inhibits repair, and so permit recovery from potentially lethal damage. However, P.J. Smith (M.R.C. Centre, Cambridge) found that inhibiting S phase with aphidicolin did not improve the survival of y-irradiated ataxia telangiectasia cells to the level seen in normal cells, at ,/-ray doses where the radiation damage strongly inhibits replication in normal but not ataxia cells. Dideoxythyrnidine (ddT), the specific inhibitor of DNA polymerase fl, might be expected to have a complementary effect to aphidicolin if both ct and fl polymerases
Inhibited
Normal quiescent (human) Reduced
Reduced [Reduced]
No effect
Unscheduled D N A synthesis: [3H]thymidine incorporation
a Higher concentration of aphidicolin needed in proliferating compared with quiescent cells.
Reduced [Further reduced]
N o effect or slightly increased [Reduced[
Inhibited
Normal proliferating (human)
Repair replication: BUdR incorporation, CsCI sedimentation
Increased [Reduced]
Removal of lesion
Repair assay
Transformed (human, hamster)
Cell type
Reduced [Greatly reduced]
In vitro repair: [3H]dTTP incorporation
+ + [+ + ]
+ [+ + ] a
+ [+ + ]
Accumulation of repair-related D N A breaks
+
Chromosome damage
Observations contributed by V. Bohr, P.K. Botcherby, A.R.S. Collins, F. Hanaoka, C.A. Smith, P.J. Smith, R.D. Snyder, R. Waters and A.A. van Zeeland. In most cases the damaging agent used was UV light. Square brackets indicate the presence of HU with aphidicolin.
S U M M A R Y OF R E P O R T E D EFFECTS OF A P H I D I C O L I N ON D N A R E P A I R
TABLE 3
81 are involved in repair. Mattern indeed reported t h a t though ara C, aphidicolin or ddT alone had no effect on or even enhanced repair of potentially lethal UV or M N N G damage, and provided a transient accumulation of inhibited repair sites, ddT in combination with either ara C or aphidicolin increased killing and caused lasting inhibition: as if either polymerase can substitute for the other in the repair of DNA lesions. But Cleaver's studies of nuclease sensitivity of repair label indicate that inhibition of synthesis and accumulation of inhibited sites are never complete, even with ara C and ddT together. One point clearly emerging from the meeting was that the effect of all these polymerase inhibitors on DNA repair is critically dependent on the growth state of the cells studied. The effects of aphidicolin in particular are dramatically less in exponentially growing than in quiescent cells, as was clearly shown by results presented by A.R.S. Collins (C.R.C. Group, Cambridge), C.A. Smith and Snyder. A 200-fold difference in the aphidicolin concentration necessary to achieve the same level of break accumulation in UV-irradiated quiescent and growing human fibroblasts was reported by Snyder. Ara C-induced breaks also tend to be fewer in growing than in quiescent cells, and Snyder reported that HU elevates the break frequency in growing but not in quiescent cells. Collins found that HU strikingly potentiates aphidicolin in causing break accumulation in UV-irradiated proliferating CHO or HeLa cells, which are otherwise rather insensitive to aphidicolin. The general ability of HU to enhance the activity of other inhibitors, probably by reducing intracellular levels of competing deoxyribonucleotides, suggests that variations in the response to inhibitors between different cell types and culture conditions are most likely due to differences in the size of deoxyribonucleotide pools. M. Meuth (Institut des Recherches Cliniques, Montreal) reviewed pool effects and showed how the pool sizes could be incidentally altered by added deoxyribonucleosides or their analogues, and described CHO thymidine auxotrophs whose deoxyribonucleotide pool imbalance leads to a high rate of spontaneous mutation; the implication being that some cellular effects of DNA polymerase inhibitors may be very indirect. A separate class of inhibitors, exemplified by 3-aminobenzamide (3AB), act on DNA repair via an inhibition of poly(ADP-ribose) synthesis. S. Shall (University of Sussex, Brighton) described the evidence for an involvement of this process in repair. A decrease in cellular levels of NAD, the precursor of ADP-ribose, is a general response to DNA damage, and is inhibited by 3AB. 3AB blocks the activation, by ADP-ribosylation, of DNA ligase II. The effect of 3AB on repair is seen as an inhibition of DNA-strand break rejoining, but only after certain kinds of DNAdamaging treatment. A.R. Lehmann (M.R.C. Cell Mutation Unit, Brighton) showed that 3AB only affects DNA repair in situations where DNA ligase activity, rather than some earlier step, is the rate-limiting factor. In fibroblasts this is the case for repair of alkylation lesions, but not for UV or X-ray repair. 3AB in alkylated cells causes DNA-strand breaks to persist at unligated repair sites, with a concurrent stimulation of DNA-repair synthesis (due to the unligated repair patches being longer), inhibition of recovery of replicative DNA synthesis and increased cytotoxicity. A.T. Natarajan (University of Leiden) also showed that these strand breaks, like strand breaks induced by repair polymerase inhibitors, have effects at the chro-
82 mosome level; chiefly in formation of sister chromatid exchanges. But the effects of 3AB vary between cell types. Cleaver warned that some of its effects may occur via the 1-carbon pool; in HeLa cells, though not in CHO, labelling of this pool is greatly decreased and the alkylating effect of MMS increased by 3AB; though indeed this may only reflect the cytotoxicity of MMS plus 3AB. And lymphocytes differ from fibroblasts; V. Bohr (University of Copenhagen) found that in lectin-stimulated human lymphocytes 3AB increases both UV-induced DNA repair synthesis (measured in the presence of HU) and the ligation of MNNG-induced strand breaks, the opposite effects being seen in L1210 cells. A.A. van Zeeland (University of Leiden) could find no effect of 3AB on UV-repair replication measured in the absence of HU, in either fibroblasts or lymphocytes; Cleaver confirmed that its apparent effects on UV repair replication are indeed HU-dependent. This seems odd, for HU as an indirect polymerisation inhibitor would not be expected to make tigation the rate-limiting step. W.A. Cramp (M.R.C. Cyclotron Unit, Hammersmith Hospital, London) reported the appearance of single-stranded fragments of DNA when cells replicate DNA after ionising irradiation, representing a delayed ligation of nascent DNA into the growing chain. Acetylated 3AB potentiates the effect of radiosensitisers in increasing the extent of single-stranded DNA. Two reports raised the interesting prospect that several clinical syndromes may be connected with spontaneous inhibition, or deficiencies, of repair ligase. A survey conducted by P.D. Lipetz (Ohio State University, Columbus) suggests that about 5% of the population may have lymphocytes that are seriously deficient in the ability to repair X-ray-induced strand breaks; and the defect in cells from 46BR, a unique clinical case which involves immunodeficiency and photosensitivity with no defect in UV-induced repair synthesis or post-replication recovery, was shown by S. Squires (C.R.C. Group, Cambridge) to involve spontaneous DNA-strand break accumulation in UV-irradiated cells, similar to that induced by ara C in normal cells. Topoisomerase inhibitors may well affect a pre-incision step; but much of the work has been concerned with novobiocin around which there remains a penumbra of doubt. This drug has rather widespread activity as an ATPase inhibitor; and Snyder reported that it causes a 5-fold increase in DNA-protein crosslinking, in the absence of DNA damage. This seriously complicates experiments measuring DNA repair through changes in DNA sedimentation; it is not clear how much of the reported decrease by novobiocin in DNA-strand breaks after UV plus ara C is due to crosslinking. But Snyder also reported a novobiocin inhibition of UV-induced repair synthesis and dimer removal that cannot be a crosslinking artefact; and Mattern showed that nalidixic acid, a more specific topoisomerase inhibitor than novobiocin, has similar effects in inhibiting repair of UV and M N N G damage. Lipetz also described a n original system using Paramecium cultures in which novobiocin increases the mutagenicity of photo-reactivable UV damage. Lastly, caffeine, which has been known for decades to be a repair inhibitor of some sort, remains obscure in its actions. B.A. Kihlman (Uppsala University) surveyed its diverse effects in producing chromosome aberrations in plant and mammalian cells emphasising, however, that caffeine may exert its damaging effect
83 in part by eliminating cell cycle lag after DNA:darnage. J.J. Roberts (Chester Beatty Research Institute, Chalfont St. Giles) described its reversal of the overall inhibition of DNA replication by DNA damage, which is combined with a production of local blocks at damage sites in replicating regions. A further complication in caffeine's effects on the replication of damaged DNA was introduced by G. Ahnstrt~m (Stockholm University), who found that pre-exposure of human fibroblasts to a small dose of UV will accelerate the recovery of replicon completion from the inhibitory effects of a second UV dose, and found also that caffeine removes the effect of pre-exposure. C.A. Waldren (Eleanor Roosevelt Institute, Denver, Colorado) discussed caffeine effects on purine and pyrimidine metabolism, where it blocks both de novo and salvage pathways. The actual fate of caffeine inside cells was disputed. Perhaps the general message from all parts of the workshop was that anyone studying DNA repair should also have an HPLC system for measuring intracellular pools of small molecules which we neglect at our peril. We are grateful to the Cancer Research Campaign for providing substantial support, and also to B.D.H. Chemicals Ltd., Glaxo (1972) Charity Trust, Imperial Chemical Industries Ltd., Shell Research Ltd., Sigma London, Unilever Research and the Wellcome Trust for generous financial help for this Workshop.