Interactive effects of inhibitors of poly(ADP-ribose) polymerase and DNA-dependent protein kinase on cellular responses to DNA damage
Sallyanne Boulton1,
Suzanne Kyle and
Barbara W. Durkacz2
Cancer Research Unit, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
 |
Abstract
|
---|
DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP) are activated by DNA strand breaks and participate in DNA repair. We investigated the interactive effects of inhibitors of these enzymes [wortmannin (WM), which inhibits DNA-PK, and 8-hydroxy-2-methylquinazolin-4-one (NU1025), a PARP inhibitor] on cell survival and DNA double-strand break (DSB) and single-strand break (SSB) rejoining in Chinese hamster ovary-K1 cells following exposure to ionizing radiation (IR) or temozolomide. WM (20 µM) or NU1025 (300 µM) potentiated the cytotoxicity of IR with dose enhancement factors at 10% survival (DEF10) values of 4.5 ± 0.6 and 1.7 ± 0.2, respectively. When used in combination, a DEF10 of 7.8 ± 1.5 was obtained. WM or NU1025 potentiated the cytotoxicity of temozolomide, and an additive effect on the DEF10 value was obtained with the combined inhibitors. Using the same inhibitor concentrations, their single and combined effects on DSB and SSB levels following IR were assessed by neutral and alkaline elution. Cells exposed to IR were post-incubated for 30 min to allow repair to occur. WM or NU1025 increased net DSB levels relative to IR alone (DSB levels of 1.29 ± 0.04 and 1.20 ± 0.05, respectively, compared with 1.01 ± 0.03 for IR alone) and the combination had an additive effect. WM had no effect on SSB levels, either alone or in combination with NU1025. SSB levels were increased to 1.27 ± 0.05 with NU1025 compared with IR alone, 1.02 ± 0.04. The dose-dependent effects of the inhibitors on DSB levels showed that they were near maximal by 20 µM WM and 300 µM NU1025. DSB repair kinetics were studied. Both inhibitors increased net DSB levels over a 3 h time period; when they were combined, net DSB levels at 3 h were identical to DSB levels immediately post-IR. The combined use of DNA repair inhibitors may have therapeutic potential.
Abbreviations: DEF10, dose enhancement factor at 10% survival; DMSO, dimethyl sulfoxide; DNA-PK, DNA-dependent protein kinase; DSB, double-strand break; IR, ionizing radiation; PARP, poly(ADP-ribose) polymerase; RR, relative retention; SSB, single-strand break; WM, wortmannin.
 |
Introduction
|
---|
Ionizing radiation (IR) produces a complex variety of lesions in the DNA which give rise to DNA single-strand breaks (SSBs) and double-strand breaks (DSBs), either by chemical decomposition following free radical attack or as a result of the early steps of DNA repair pathways. Two important enzymes, poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK), bind to, and are activated by, these DNA breaks (for reviews, see refs 13). Mutant cell lines that are defective in either the catalytic subunit (DNA-PKcs) or one of the DNA binding subunits (e.g. Ku80) of DNA-PK are unable to repair IR-induced DNA DSBs, are defective in V(D)J recombination and are highly radiosensitive (e.g. refs 4,5). The fungal metabolite, wortmannin (WM), inhibits DNA-PK and thereby inhibits DSB repair and potentiates IR-induced cytotoxicity (68). Prevention of DSB rejoining by WM has also been demonstrated in cell-free extracts, thus substantiating the direct effect of WM on DSB rejoining (9). Although WM is not a specific inhibitor of DNA-PK, as it also inhibits phosphatidylinositol 3-kinase (PI 3-K) and may potentially inhibit the ataxia telangiectasia gene product (ATM) (both of which share active site homology with DNA-PK) (10,11), its use has identified DNA-PK as a potential target for developing drugs that sensitize cells to IR via inhibition of DNA repair.
Potent PARP inhibitors have already been developed with this aim in mind (1214), and have been shown to potentiate the cytotoxicity of alkylating agents and IR. For example, Boulton et al. (15) demonstrated that the PARP inhibitor 8-hydroxy-2-methylquinazolin-4-one (NU1025) potentiated the cytotoxicity of the monofunctional alkylating agent temozolomide and this correlated with an inhibition of SSB repair.
To date, a large body of evidence has pointed to an involvement of PARP function in the base excision repair pathway, which generates SSBs as repair intermediates. Indeed, the observations of potentiation of the cytotoxicity of DNA damaging agents and inhibition of SSB repair in early studies using inhibitors (e.g. ref. 16) have now been finally confirmed in PARP-deficient cell lines (17). However, the possible function of PARP in DSB repair has been largely neglected, although Benjamin and Gill originally demonstrated in 1980 that PARP was activated by DSBs as well as SSBs (18), and this has more recently been confirmed by Weinfield et al. (19) who showed, using highly purified enzymes, that DSBs activated PARP with almost equal efficiency as SSBs but that DNA-PK could only be activated by DSBs. Moreover, two reports have shown that the rejoining of DSBs induced by the electroporation of restriction enzymes into cells was delayed by the classical PARP inhibitor, 3-aminobenzamide (20,21).
The aim of this study was to investigate the single and combined effects of NU1025 and WM on cytotoxicity and DNA damage repair induced by IR and temozolomide in cell culture. The results provide promising prospects for enhancing the efficacy of radiotherapy and temozolomide via the combined inhibition of mechanistically diverse DNA repair enzymes.
 |
Materials and methods
|
---|
Materials
WM was obtained from Sigma (St Louis, MO). It was dissolved in anhydrous dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM and stored at 20°C. NU1025 was provided by the Newcastle upon Tyne Anticancer Drug Development Initiative (ADDI) (Newcastle upon Tyne, UK) and its synthesis has been described elsewhere (13). Temozolomide was a gift from Professor M.F.G.Stevens (Cancer Research Laboratories, University of Nottingham, UK). NU1025 and temozolomide stock solutions were prepared in DMSO at 100 mM. Solvent concentrations in cell culture experiments were kept constant and at <1% by appropriate additions of DMSO.
Cell culture
CHO-K1 cells were maintained as monolayers in RPMI 1640 medium (supplemented with 10% fetal calf serum, glutamine and antibiotics). HEPES and sodium bicarbonate were added at final concentrations of 18 and 11 mM, respectively. Clonogenic assays were performed as previously described (6). Briefly, cells as monolayers were preincubated ± WM ± NU1025 for 1 h prior to exposure to IR, then post-incubated for 16 h. Cells were then trypsinized and replated for survivors in the absence of drugs. Similarly, following a 1 h incubation with inhibitor(s), cells were treated with temozolomide for 16 h and then trypsinized and replated as above. The data are averaged from at least three independent experiments ± SE. The dose enhancement factors at 10% survival (DEF10) were calculated from the survival curves by taking the ratio of the dose of IR that reduced survival to 10% divided by the dose of IR that reduced survival to 10% in the presence of inhibitor(s).
DNA strand break assays
The filter elution techniques for assaying DSB and SSB levels have been described in detail (22,23), and the radiolabelling, drug treatment, post-incubation conditions and sample preparation used in these experiments were identical to those described by Boulton et al. (6). In all experiments, cells were exposed to either 6 Gy (SSB assay) or 100 Gy (DSB assay). Cell cultures were preincubated ± NU1025 ± WM for 1 h prior to exposure to IR, and the compounds remained in the culture medium during the post-incubation periods. SSB and DSB levels were quantitated as follows. The relative retention (RR) value is the fraction of sample DNA retained on the filter when 50% of the internal standard has eluted. The RR values of DSBs and SSBs in cells treated with inhibitor(s) were expressed relative to the RR values for cells treated with IR alone (Figures 2 and 3
) or to the RR value of unirradiated cells (Figure 4
). In each case, the RR value of the `control' cells was normalized to 1.0, and the sample RR values proportionated accordingly. Thus, a DNA strand break level of 1.0 indicates that there is no difference in DNA strand break levels between the designated `control' cells and sample cells treated with inhibitor(s). Data points represent the mean of at least four independently dosed samples from two or more separate experiments ± SE.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 2. Effects of WM and/or NU1025 on DSB and SSB levels. Cells were exposed to IR (100 Gy for DSB, and 6 Gy for SSB assays), in the presence or absence of WM (20 µM) and NU1025 (300 µM). Cells were post-incubated for 30 min before harvesting for elution assays. (A) DSB assay; (B) SSB assay.
|
|

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 3. Dose-dependent effects of inhibitors on DSB levels. Cells were exposed to 100 Gy IR, and post-incubated for 30 min to allow repair to occur before harvesting cells for neutral elution. (A) Dose-dependent effects of WM; (B) dose-dependent effects of NU1025.
|
|
 |
Results
|
---|
Radiosensitization and chemopotentiation by WM and NU1025
The effects of WM and NU1025 on IR-induced cytotoxicity were investigated. WM (20 µM) or NU1025 (300 µM) alone, neither of which caused loss of clonogenic survival (either per se or in combination), potentiated the cytotoxicity of IR (Figure 1
). When used in combination, at least additive effects on cytotoxicity were observed. The DEF10 values for a range of inhibitor concentrations and combinations are summarized in Table I
. Note the very large DEF10 value (7.8 ± 1.5) obtained for the combination of WM (20 µM) + NU1025 (300 µM). Similar experiments were performed using temozolomide as the cytotoxic agent, and the results are summarized in Table II
. Again, both WM and NU1025 potentiated the cytotoxicity of temozolomide and the combination of inhibitors produced approximately additive effects on the DEF10 values.
View this table:
[in this window]
[in a new window]
|
Table I. Comparison of the DEF10 values derived from IR survival curves using a range of inhibitor concentrations and combinations
|
|
View this table:
[in this window]
[in a new window]
|
Table II. Comparison of the DEF10 values derived from temozolomide survival curves using a range of inhibitor concentrations and combinations
|
|
DNA strand break levels
SSB and DSB levels were assessed in inhibitor-treated cells 30 min post-IR. By this time we have previously established that the majority of DNA strand break rejoining has occurred (6). The results are presented as a histogram in Figure 2
. WM (20 µM) and NU1025 (300 µM) increased relative DSB levels from 1.01 ± 0.03 for IR alone to 1.29 ± 0.04 and 1.20 ± 0.05, respectively (Figure 2A
). When the inhibitors were combined, relative DSB levels increased to 1.61 ± 0.03. In marked contrast, when SSB levels were assessed, WM alone had no effect on SSB levels (0.98 ± 0.04 compared with 1.02 ± 0.04 for IR alone) (Figure 2B
). NU1025 increased SSB levels to 1.27 ± 0.05 and this value was not changed significantly by co-incubation with WM.
Although WM potentiated the cytotoxicity of temozolomide, it was not possible to detect DSBs by neutral elution, even at concentrations of temozolomide as high as 1 mM. We have previously established (15) that NU1025 increases temozolomide-induced SSB levels and therefore no further investigations of the inhibitors on temozolomide-induced DNA strand break production were undertaken here.
A further study comprised a comparison of the dose-dependent effects of the inhibitors on DSB levels 30 min post-IR. The results are shown in Figure 3
. Both WM and NU1025 increased DSB levels in a dose-dependent manner (Figure 3A and B
, respectively), but 300 µM NU1025 was required to achieve an increase comparable to 20 µM WM.
Kinetics of DSB repair
The effects of WM and NU1025 on the kinetics of DSB repair following exposure to IR were compared over a 3 h time period and the results are shown in Figure 4
. In the absence of the inhibitors, DSBs were rejoined rapidly with the majority rejoined by 60 min. Although a small amount of DSB rejoining initially occurred during the first 30 min in the presence of WM (50 µM), DSB levels subsequently increased up to 180 min post-irradiation (1.53 ± 0.04 for IR + WM compared with 1.04 ± 0.01 for IR alone) (Figure 4
). The production of these additional DSBs was not attributable to a direct effect of WM on the integrity of DNA, as we have previously shown that prolonged incubation with WM alone did not cause DSB formation (6). A possible explanation for the formation of additional DSBs observed, in particular since supralethal doses of IR (100 Gy) have to be used to detect DSBs by neutral elution, is the early onset of DNA fragmentation associated with apoptosis. Finally, the inhibitors were combined, in this case with the WM added prior to exposure to IR, and NU1025 added immediately afterwards to preclude possible interactive effects of the drugs on DSB production during IR exposure. (We have found it necessary to add WM prior to exposure to IR to obtain optimum inhibition of DSB repair.) In this case, approximately additive effects on DSB levels were seen throughout the 3 h time period (Figure 4
) such that by 180 min the net level of DSBs was about the same as immediately post-IR, compared with the almost complete rejoining observed in the absence of inhibitors.
 |
Discussion
|
---|
As mentioned in the Introduction, molecular evidence indicates that PARP interacts with DSBs as well as SSBs. PARP has two zinc fingers, both of which are required for SSB binding, but the first alone suffices to bind PARP to a DSB, which also acts as a more potent activator of PARP than a SSB (24). Chung et al. (20) showed that 3-aminobenzamide increased chromosomal aberrations and retarded repair of DNA damage resulting from the electroporation of restriction enzymes into cells. Bryant and Johnston (21) also demonstrated an effect of PARP inhibitors on the repair of restriction enzyme-induced DSBs. Numerous publications have shown that PARP is involved in sister chromatid exchanges and gene amplification. These observations have led to the proposal that PARP may function to prevent spurious recombination events at DSBs in the DNA (2).
The data presented here clearly demonstrate that inhibition of PARP, as well as DNA-PK, retards DSB rejoining. We have considered the possibility that the effect of PARP inhibition in increasing DSB levels could be an artefact of the neutral elution assay allowing the detection of a low level of SSBs, since they are the predominant lesions produced in irradiated DNA. If this were the case, the effect of NU1025, by increasing net SSB levels, would be to apparently increase DSB levels. However, it has been clearly demonstrated that excess SSBs do not interfere with the DSB assay used here (25).
The additional DSBs, as defined by the neutral elution technique, obtained in the presence of NU1025 may arise because of a retardation of DSB rejoining. Aternatively, they may arise from a subset of IR-induced lesions being converted to DSBs. For example, proximal SSBs on complementary strands could be stabilized and repaired by a two-step SSB repair process when PARP is functioning; when PARP is inhibited, these could convert to DSBs. This is a distinct possibility since IR produces localized clusters of multiple damages, which in addition to producing DSBs by direct chemical reaction, will have the potential to convert to DSBs during attempts at repair (26,27).
WM is known to inhibit PI 3-K as well as DNA-PK, and may also inhibit other members of this family of kinases, including the ataxia telangiectasia gene product, ATM, or ATR (for ATM and Rad3-related) (11,28). Mutant ATM cell lines, or ATR cell lines over-expressing kinase-inactive ATR protein, demonstrate similar hypersensitivity to IR as DNA-PK defective cell lines (29,30). Therefore, although WM clearly inhibits DNA-PK and inhibits DNA-PK mediated DSB repair (79), the ability of WM to potentiate IR-induced cytotoxicity could also be mediated, at least in part, via an inhibition of ATM or ATR. The development of specific assays for these enzymes will be required to address these issues. However, we have demonstrated (unpublished data) that potentiation of IR-induced cell killing by WM is largely abolished in the xrs-6 cell line, which is mutated in the Ku80 subunit of DNA-PK (31), supporting the contention that the effects of WM on the cytotoxic response are caused by DNA-PK inhibition.
Although WM potentiated the cytotoxicity of temozolomide, no DSBs were detectable by neutral elution. However, this chemopotentiation is still consistent with an inhibition of DNA-PK, since known DNA-PK defective mutant cell lines have been demonstrated to be hypersensitive to other monofunctional alkylating agents (32). It is probable that inhibition of the repair of very low levels of DSBs, below the relatively insensitive detection limits of the neutral elution assay, would suffice to enhance temozolomide cytotoxicity. As with IR, a useful additive effect on the DEF10 value was obtained when the inhibitors were combined. It should be stressed that although we have shown that NU1025 modulates DSB repair, it also retards SSB repair in IR- and temozolomide-treated cells, and hence it is not possible to ascribe its potentiating effects on cytotoxicity to a single repair pathway.
These data point to cooperation between PARP and DNA-PK at direct DSBs or DSBs that are formed as repair intermediates. As well as regulating DSB and SSB repair in a very similar manner (compare the effects of NU1025 on the kinetics of DSB rejoining presented here with its effects on the kinetics of SSB repair [15]), PARP may also function to promote DNA-PK-mediated non-homologous end-joining by preventing DSB repair by an alternative pathway involving homologous recombination. A role for PARP as an anti-recombinogenic factor has been proposed (2), and this hypothesis is consistent with recent evidence that an additional loss of PARP function in DNA-PK deficient mice can rescue the block in V(D)J recombination that typifies the SCID phenotype (33). An alternative hypothesis is that PARP may function directly to activate DNA-PK, and good evidence for this has recently been published. Ruscetti et al. (34) have shown, using purified enzymes, that the kinase activity of DNA-PK is stimulated by poly(ADP-ribosylation) of its catalytic subunit.
Mice lacking PARP and/or DNA-PK are viable (33,35,36), which is an important consideration in radio- and chemo-therapy, as specific inhibitors of these enzymes should therefore exhibit no systemic toxicity. The most recently developed PARP inhibitors include 2-(4-methoxyphenyl)benzimidazole-4-carboxamide, synthesized as part of the programme of the Newcastle upon Tyne ADDI group. This compound has an IC50 value for inhibition of PARP of 0.06 µM, compared with 0.4 µM for NU1025, and is probably of sufficient potency to be active at physiologically achievable concentrations (14). The evident effectiveness and potency of WM acting as a DNA-PK inhibitor, either alone or in conjunction with NU1025, to potentiate IR- and temozolomide-induced cytotoxicity, indicates that DNA-PK represents another valid repair enzyme target for drug development. The additive effects of the two repair inhibitors on the cytotoxicity of IR and temozolomide may prove powerful tools to enhance their efficacy in cancer therapy.
 |
Acknowledgments
|
---|
This work was supported by funds from the North of England Cancer Research Campaign, and carried out under the auspices of the Anticancer Drug Development Iniative (ADDI), University of Newcastle upon Tyne, Newcastle upon Tyne, UK.
 |
Notes
|
---|
1 Present address: Department of Ophthalmology, Manchester Royal Eye Hospital, Oxford Road, Manchester M13 9WH, UK 
2 To whom correspondence should be addressed Email: b.w.durkacz{at}newcastle.ac.uk 
 |
References
|
---|
-
de Murcia,G. and Ménissier de Murcia,J. (1994) Poly(ADP-ribose) polymerase: a molecular nick-sensor. Trends Biochem. Sci., 19, 172176.[ISI][Medline]
-
Lindahl,T., Satoh,M.S., Poirier,G.G. and Klungland,A. (1995) Post-translational modification of poly(ADP-ribose) polymerase induced by DNA strand breaks. Trends Biochem. Sci., 20, 405411.[ISI][Medline]
-
Jackson,S.P. (1996) DNA damage detection by DNA dependent protein kinase and related enzymes. Cancer Surv., 28, 261279.[ISI][Medline]
-
Jeggo,P.A., Taccioli,G.E. and Jackson,S.P. (1995) Ménage a trois: double strand break repair, V(D)J recombination and DNA-PK. Bioessays, 17, 949957.[ISI][Medline]
-
Roth,D.B., Lindahl,T. and Gellert,M. (1995) How to make ends meet. Current Biol., 5, 496499.[ISI][Medline]
-
Boulton,S., Kyle,S., Yalciintepe,L. and Durkacz,B.W. (1996) Wortmannin is a potent inhibitor of DNA double strand break repair but not single strand break repair in Chinese hamster ovary cells. Carcinogenesis, 17, 22852290.[Abstract]
-
Price,B.D. and Youmell,M. (1996) The PI 3-kinase inhibitor wortmannin sensitizes murine fibroblasts and human tumour cells to radiation and blocks induction of p53 following DNA damage. Cancer Res., 56, 246250.[Abstract]
-
Rosenzweig,K.E., Youmell,M.B., Palayoor,S.T. and Price,B.R. (1997) Radiosensitization of human tumour cells by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 correlates with inhibition of DNA-dependent protein kinase and prolonged G2-M delay. Clin. Cancer Res., 3, 11491156.[Abstract]
-
Gu,X.-Y., Bennet,R.A.O. and Porvik,L.F. (1996) End-joining of free radical-mediated DNA double-strand breaks in vitro is blocked by the kinase inhibitor wortmannin at a step preceding removal of damaged 3' termini. J. Biol. Chem., 271, 1966019663.[Abstract/Free Full Text]
-
Powis,G., Bonjouklian,R., Berggren,M.M. et al. (1994) Wortmannin, a potent and selective inhibitor of phosphatidylinositol 3-kinase. Cancer Res., 54, 24192423.[Abstract]
-
Hartley,K.O., Gell,D., Smith,G.C.M. et al. (1995) DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell, 82, 849856.[ISI][Medline]
-
Suto,M.J., Turner,W.R., Arundel-Suto,C.M., Werbel,L.M. and Sebolt-Leopold,J.S. (1991) Dihydroisoquinolinones: the design and synthesis of a new series of potent inhibitors of poly(ADP-ribose) polymerase. Anticancer Drug Des., 7, 101107.[ISI]
-
Griffin,R.J., Pemberton,L.C., Rhodes,D. et al. (1995) Novel potent inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP). Anticancer Drug Des., 10, 507514.[ISI][Medline]
-
Griffin,R.J., Srinivasan,S., White,A.W., Bowman,K., Calvert,A.H., Curtin,N.J., Newell,D.R. and Golding,B.T. (1996) Novel benzimidazole and quinazolinone inhibitors of the DNA repair enzyme, poly(ADP-ribose) polymerase. Pharmaceut. Sci., 2, 4347.
-
Boulton,S., Pemberton,L.C., Porteous,J.K., Curtin,N.J., Griffin,R.J., Golding,B.T. and Durkacz,B.W. (1995) Potentiation of temozolomide-induced cytotoxicity: a comparative study of the effects of poly(ADP-ribose) polymerase inhibitors. Br. J. Cancer, 72, 849856.[ISI][Medline]
-
Durkacz,B.W., Omidiji,O., Gray,D.A. and Shall,S. (1980) (ADP-ribose)n synthesis participates in DNA repair. Nature, 283, 593596.[ISI][Medline]
-
Trucco,C., Oliver,F.J., de Murcia,G. and Menissier-de Murcia,J. (1998) DNA repair defect in poly(ADP-ribose) polymerase-deficient cell lines. Nucleic Acids Res., 26, 26442649.[Abstract/Free Full Text]
-
Benjamin,R.C. and Gill,D.M. (1980) Poly(ADP-ribose) synthesis in vitro programmed by damaged DNA. A comparison of DNA molecules containing different types of strand breaks. J. Biol. Chem., 255, 1050210508.[Abstract/Free Full Text]
-
Weinfeld,M., Chaudhry,M.A., D'Amours,D., Pelletier,J.D., Poirier,G.G., Povirk,L.F. and Lees-Miller,S.P. (1997) Interaction of DNA-dependent protein kinase and poly(ADP-ribose) polymerase with radiation-induced DNA strand breaks. Radiat. Res., 148, 2228.[ISI][Medline]
-
Chung,H.W., Philips,J.W., Winegar,R.A., Preston,J. and Morgan,W.F. (1991) Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenic and pulsed-field gel analysis. Radiat. Res., 125, 107113.[ISI][Medline]
-
Bryant,P.E. and Johnston,P.J. (1993) Restriction-endonuclease-induced DNA double-strand breaks and chromosomal aberrations in mammalian cells. Mutat. Res., 299, 289296.[ISI][Medline]
-
Kohn,K.W., Ewig,R.A.G. and Zwelling,L.A. (1981) Measurement of strand breaks and crosslinks by alkaline elution. In Friedberg,E.C. and Hanawalt,P.C. (eds) DNA Repair: A Laboratory Manual of Research Procedures, Marcel Dekker, New York and Basel, vol. 2, part B, pp. 379401.Kohn,K.W., Ewig,R.A.G. and Zwelling,L.A. (1981) Measurement of strand breaks and crosslinks by alkaline elution. In Friedberg,E.C. and Hanawalt,P.C. (eds) DNA Repair: A Laboratory Manual of Research Procedures, Marcel Dekker, New York and Basel, vol. 2, part B, pp. 379401.
-
Bradley,M.O. and Kohn,K.W. (1979) X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution. Nucleic Acids Res., 7, 793804.[Abstract]
-
Ikejima,M., Noguchi,S., Yamashita,R., Ogura,T., Sugimura,T., Gill,D.M. and Miwa,M. (1990) The zinc fingers of human poly(ADP-ribose) polymerase are differentially required for the recognition of DNA strand breaks and nicks and consequent enzyme activation. J. Biol. Chem., 255, 2190721913.
-
Johnston,P.J. and Bryant,P.E. (1991) Lack of interference of DNA single-strand breaks with the measurement of double-strand breaks in mammalian cells using the neutral filter elution assay. Nucleic Acids Res., 19, 27352738.[Abstract]
-
Ward,J.F. (1994) The complexity of DNA damage: relevance to biological consequences. Int. J. Radiat., 66, 427432.
-
Holley,W.R. and Chatterjee,A. (1996) Clusters of DNA damage induced by ionising radiation: formation of short DNA fragments. I. Theoretical modelling. Radiat. Res., 145, 188199.[ISI][Medline]
-
Cimprich,K.A., Shin,T.B., Keith,C.T. and Schreiber,S.L. (1996) cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein. Proc. Natl Acad. Sci. USA, 93, 28502855.[Abstract/Free Full Text]
-
Taylor,A.M.R., Harnden,D.G., Arlett,C.F., Harcourt,A.R., Lehmann,A.R., Stevens,S. and Bridges,B.A. (1975) Ataxia telangiectasia, a human mutation with abnormal radiation sensitivity. Nature, 258, 427429.[ISI][Medline]
-
Cliby,W.A., Roberts,C.J., Cimprich,K.A., Stringer,C.M., Lamb,J.R., Schreiber,S.L. and Friend,S.H. (1998) Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints. EMBO J., 17, 159169.[Abstract/Free Full Text]
-
Singleton,B.K., Priestly,A., Steingrimsdottir,H., Gell,D., Blunt,T., Jackson,S.P., Lehmann,A.R. and Jeggo,P.A. (1997) Molecular and biochemical characterisation of xrs mutants defective in Ku80. Mol. Cell. Biol., 17, 12641273.[Abstract]
-
Jeggo,P.A. and Kemp,L.M. (1983) X-ray sensitive mutants of Chinese hamster ovary cell line. Isolation and cross sensitivity to other DNA-damaging agents. Mutat. Res., 112, 313327.[ISI][Medline]
-
Morrison,C., Smith,G.C.M., Sting,L., Jackson,S.P., Wagner,E.F. and Wang,Z.-Q. (1997) Genetic interaction between PARP and DNA-PK in V(D)J recombination and tumorigenesis. Nature Genet., 17, 479482.[ISI][Medline]
-
Ruscetti,T., Lehnert,B.E., Halbrook,J., Trong,H.L., Hoekstras,M.F., Chen,D.J. and Peterson,S.R. (1998) Stimulation of DNA-dependent protein kinase by poly(ADP-ribose) polymerase. J. Biol. Chem., 273, 1446114467.[Abstract/Free Full Text]
-
Wang,Z.-Q., Auer,B., Sting,L., Berghammer,H., Haidacher,D., Schweiger,M. and Wagner,E.F. (1995) Mice lacking ADPRT and poly(ADP-ribosyl)ation develop normally but are susceptible to skin disease. Genes Dev., 9, 509520.[Abstract]
-
Ménissier de Murcia,J., Niedergang,C., Trucco,C. et al. (1997) Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells. Proc. Natl Acad. Sci. USA, 94, 73037307.[Abstract/Free Full Text]
Received July 24, 1998;
revised September 23, 1998;
accepted October 15, 1998.