Inhibition of DNA excision repair and the repair of X-ray-induced DNA damage by cytosine arabinoside and hydroxyurea

Pharmac. Ther. Vol. 31, pp. 165-176, 1985 Printed in Great Britain. All rights reserved

0163-7258/85 $0.00 +0.50 Copyright © 1987 Pergamon Journals Ltd

Specialist Subject Editor: L. E. ERICKSON

INHIBITION OF DNA EXCISION THE REPAIR OF X-RAY-INDUCED BY CYTOSINE ARABINOSIDE AND

REPAIR AND DNA DAMAGE HYDROXYUREA

R. J. FRAM* and D. W. KUVEt *Departments of Medicine and Pharmacology, University of Massachusetts Medical School, and University of Massachusetts Medical Center, Worcester, MA 01605, U.S.A. tLaboratory of Clinical Pharmacology, Dana Father Cancer Institute and Harvard Medical School, Boston, MA 02115, U.S.A.

1. INTRODUCTION Insights into the mechanisms underlying DNA repair have been gained by the study of the effects of inhibitors of replicative DNA synthesis. The purpose of this review is to contrast the effects of cytosine arabinoside (ara-C), aphidicolin (Apc) and hydroxyurea (HU) on replicative DNA synthesis with the effects of these inhibitors on the repair of u.v.and X-ray-induced DNA damage. The interaction of inhibitors of DNA synthesis with these processes is discussed after briefly reviewing the mechanisms by which these agents inhibit replicative DNA synthesis and the repair of DNA damage by u.v. and X-ray.

2. MECHANISMS UNDERLYING THE INHIBITION OF REPLICATIVE DNA SYNTHESIS The mechanisms underlying the inhibition of DNA synthesis by ara-C, aphidicolin (Apc) and hydroxyurea (HU) are distinct. HU, in contrast to both ara-C and Apc, does not interact with DNA polymerase ct and exerts its effects on DNA synthesis by lowering intracellular pools of deoxyribonucleotides through reversible inhibition of ribonucleotide reductase (Thelander and Reichard, 1979). Apc solely inhibits DNA polymerase 0t and does not incorporate into DNA (Pedrale-Noy and Spadari, 1979; Huberman, 1981; Ikegami et al., 1978). The inhibition of polymerase • by Apc is competitive with respect to dCTP and not other deoxyribonucleotides in cell free extracts (Ikegami et al., 1978). However, in isolated nuclei, perhaps owing to the interaction of DNA polymerase ~twith other enzymes to form a replication complex, the inhibition of polymerase ~ by Apc is noncompetitive with regard to all four deoxyribonucleoside triphosphates (Oguro et al., 1980). The mechanisms underlying the inhibition of DNA synthesis by ara-C are more complex as a result of its effects on DNA polymerase ~ and its incorporation into DNA. After transport across the cell membrane, ara-C is phosphorylated to the 5'-monophosphate by deoxycytidine kinase (Plagemann et al., 1978). Ara-CMP is then sequentially phosphorylated to ara-CTP by pyrimidine nucleoside monophosphate kinase and nucleoside diphosphokinase. While prior studies emphasized the competitive inhibition of polymerase by ara-CTP as a means of inhibiting replicative DNA synthesis (Momparler et al., 1968; Furth and Cohen, 1968; Graham and Whitmore, 1970a,b), kinetic studies have demonstrated that ara-CTP is a weak competitive inhibitor of this enzyme and that this competition does not completely explain the effect of this agent on DNA synthesis (Momparler, 1982). Other reports have demonstrated that ara-C is incorporated in internucleotide linkage (Momparler, 1969) as well as at the chain terminus (Graham and Whitmore, 1970a). These studies, however, did not correlate the extent of incorporation with either inhibition of DNA synthesis or cytotoxic effects. J.P.r 31/3-^

165

166

R . J . FRAM and D. W. KUFE INCORPORATION OF [=HI A R A - C

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FIG. 1. Incorporation of ara-C into HL-60 DNA. HL-60 cells in logarithmic growth at 5 x 105 cells per ml were incubated with [3H]ara-C (13 pCi/ml) and [32P]H2PO4 (5/~Ci/ml) for 3(A), 12(B) and 24(C) hr. The total cellular nucleic acids were purified and analyzed by cesium sulfate density centrifugation. The [3H]ara-C and 32p counts banding in the RNA region (between 1.62 and 1.68 g/ml) and DNA region (between 1.42 and 1.48 g/ml) of the gradients were determined and used as a measure of the incorporation of ara-C into DNA and of the relative synthetic rates of both RNA and DNA. (From Major et al., 1981.)

Recent studies have shown that ara-C is exclusively incorporated into DNA rather than RNA (Fig. 1) and that this incorporation is concentration and time dependent (Kufe et al., 1980; Major et al., 1981; Major et al., 1982; Kufe et al., 1984a). Further, the degree of inhibition of DNA synthesis is correlated with the extent of incorporation of ara-C into DNA (Fig. 2). The mechanisms by which the incorporation of ara-C into DNA results in inhibition of DNA synthesis are unclear. That ara-C behaves as a poor primer for chain elongation, however, is suggested by experiments in which exposure of L1210 cells to increasing concentrations of ara-C is associated with an increasing proportion of ara-CMP residues at the chain terminus (Table 1). Also consistent with this view is the irreversible nature

EFFECT OF ARA-C INCORPORATION ON INHIBITION OF DNA SYNTHESIS 100 A. 3Hrs. 1000 B. 6Hrs.

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lO-r 10-6 10-5 10-4 10-3 10-7 10-6 lO-S 10-4 10-~ [A~-C] M FIG. 2. Effect of ara-C incorporation on inhibition of DNA synthesis. LI210 cells in logarithmic growth phase at a concentration of 1 x 106 cells/ml were incubated with [3H]ara-C (10-7-10 -3 M) for 3 and 6 hr. The total cellular nucleic acids were purified and analyzed by cesium sulfate density gradient centrifugation. The [3H]ara-C counts banding in the DNA region of the gradients were determined and used as a measure of the incorporation of ara-C in DNA (O). Similar experiments were performed by exposing LI210 cells to ara-C (10-7-10 -3 M) for 3 and 6 hr with the addition of 2 juCi/ml [3H]dThd during last 30 min of each incubation. The amount of [3H]dThd incorporated in DNA (©) was then determined as a measure of DNA synthesis. (From Major et al., 1982.)

Cytosine arabinoside and hydroxyurea

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TABLE1. Positioning of [3H]ara-C and [3H]dThd residues at different concentrations of ara-C* Ara-C dThd Ara-C concn (r,I) Internucleotide Chainterminus Internucleotide Chainterminus Control 97.0 + 1.7 3.0 +__1.7 10 -8 100.0 + 0.0 0.0 + 0.0 96.8 + 1.3 3.2 _+1.7 10 -7 92.0+2.3 8.0+2.3 97.0-1- 1.1 3.0+ 1.1 10-6 87.5 + 3.2 12.5 + 3.2 98.1 + 0.5 1.9 + 0.5 10-5 71.5 -t- 1.9 28.5 _ 1.9 99.4 + 0.5 0.6 _ 0.5 10-4 70.5 +__2.2 29.5 + 2.2 99.8 _ 0.2 0.2 + 0.2 *Values are expressed as mean% +__SD. (From Major et al., 1982.)

of the effects of ara-C on D N A synthesis at high concentrations. L1210 murine leukemia cells exposed to 10-SM ara-C for 3 hr, washed, and then incubated in 10-SM dCyd demonstrate less than 10% recovery of control D N A synthesis after 12 hr (Kufe et al., 1984b). This effect is not explainable by persistence of intracellular ara-CTP pools (Kufe et al., 1984b) and is probably due to the more absolute chain terminator effect of ara-C following exposure to higher concentrations of drug. Lastly, the introduction of ara-C at 3' termini of D N A oligomers with defined sequence abrogates the addition of succeeding nucleotides by HeLa cell Pol 2 holoenzyme (Beardsley and Mikita, 1986). 3. B I O L O G I C A L A N D B I O C H E M I C A L E F F E C T S OF I N C O R P O R A T I O N OF ARA-C Several biological and biochemical events occur as a consequence of the incorporation of ara-C into DNA. Incorporation of ara-C into D N A is closely correlated with a reduction in clonogenic survival in both L1210 murine leukemia and HL-60 human promyeloblast cell lines as well as in blasts obtained from patients with acute myelogenous leukemia (Kufe et al., 1980; Major et al., 1981). While such a correlation between cytotoxicity and incorporation of ara-C in D N A most probably results from the inhibition of D N A synthesis and the irreversibility of this inhibition at high concentrations of ara-C, other effects including D N A fragmentation and terminal differentiation (Fram and Kufe, 1982; Griffin et al., 1982; Luisi-Deluca et al., 1984) may also contribute to cytotoxicity. 4. R E P A I R OF U.V. A N D X-RAY I N D U C E D D N A D A M A G E U.V. irradiation-induced D N A damage is the most completely studied form of D N A damage that undergoes excision repair. Since this subject is extensively reviewed elsewhere, only those features of the repair of u.v. and X-ray-induced D N A damage are mentioned that are relevant to the subsequent discussion (Friedberg, 1984; Hall and Mount, 1981; Hanawalt et al., 1979). U.V. irradiation induces a variety of photoproducts in DNA. Among these photoproducts are cyclobutane-type dipyridmidine dimers (Kittler and L6ber, 1977; Smith and Hanawalt, 1969), pyrimidine adducts (Kittler and LSber, 1977), pyrimidinepyrimidine(6-4) lesions (Lipke et al., 1981), and monomeric pyrimidine base damage (Yamane et al., 1967; Demple and Linn, 1982). While the principal cytotoxic lesion is the pyrimidine dimer, the primary lesion with respect to mutagenic events is the pyrimidine-pyrimidine(6-4) lesion (Brash and Haseltine, 1982; Wood et al., 1984). U.V. damage, specifically pyrimidine dimers, are repaired via two basic biochemical processes: (1) monomerization in the presence of D N A photolyase and near u.v. light (Cook, 1970; Rupert and Harm, 1966; Rupert, 1975; Sutherland, 1977, 1978; Werben, 1977); and (2) dark repair reactions. Among the latter, at least three mechanisms may contribute. First, damage-specific incising endonuclease makes a nick 5' to the dimer. Excision then occurs via a 5'-3' exonuclease followed by D N A repair synthesis and

168

R.J. FRAMand D. W. KUFE

ligation. This set of events defines the classical model for the repair of pyrimidine dimers in both prokaryotes and eukaryotes (Hanawalt et al., 1979; Friedberg, 1984). An alternative repair pathway is suggested by data from E. coli (Rupp et al., 1982; Sancar and Rupp, 1983). In this scheme, nicks are made 5' and 3' to the dimer and about 12 nucleotides are removed with the dimer. Repair synthesis and ligation then follow. A third pathway for repair of DNA dimers involves pyrimidine dimer DNA glycosylase which specifically cleaves the phosphodiester bond 5' to dimers; this pathway, however, appears limited as yet to M . luteus and bacteriophage T4 (Friedberg, 1984). X-ray damage is principally limited to base damage (Ward, 1975; Hems, 1960). X-ray causes single strand DNA breaks via cleavage of phosphodiester linkages and of deoxyribose rings (Ward, 1975). These effects appear to a great extent mediated by the direct effects of ionizing radiation as well as by free radicals (Ward, 1975). While single strand DNA breaks are poorly correlated with cytotoxicity by X-ray, these lesions likely mediate formation of double strand DNA breaks; the latter are more formidable lesions to repair and are correlated with cytotoxicity (Ritter et al., 1977; Weibezahn et al., 1980). The type of DNA lesion caused by ionizing radiation appears important in determining the repair mechanism(s) that is initiated. When a synthetic DNA oligonucleotide is subjected to gamma irradiation both 3' phosphoryl and glycolate moieties are identified (Henner et al., 1983). While the former is readily repaired by polynucleotide kinase, the latter must be repaired by another mechanism (Henner et al., 1983). This suspicion is confirmed by several observations. First, the kinetics of repair of DNA double strand breaks in both eukaryotes and prokaryotes is biphasic, consisting of an early, rapid as well as a slower second phase, thus suggesting two processes (Weibezahn and Coquerelle, 1981). Secondly, E. coli which are temperature sensitive for DNA ligase lack the initial rapid repair phase after induction of double strand DNA breaks by gamma irradiation, while recA E. coli are defective in the slower, second phase. The latter are fourfold more sensitive to X-ray suggesting that repair of these lesions is of biological significance (Weibezahn and Coquerelle, 1981). X-ray in contrast to u.v. irradiation is associated with the induction of shorter repair patches, on the order of 3-4 bases compared to 30-100 bases (Regan and Setlow, 1974; Frances et al., 1981). DNA repair synthesis, however, does occur in eukaryotic cells exposed to X-ray when doses in excess of 10 Gy are used (Painter and Young, 1971). Such a finding may occur because of the induction of larger numbers of repair patches at higher doses of X-ray.

5. EFFECTS OF DNA EXCISION REPAIR BY INHIBITORS OF REPLICATIVE DNA SYNTHESIS Ara-C inhibits excision repair of DNA damage induced by u.v., alkylators, and to a lesser extent, X-rays (Collins, 1977; Hiss and Preston, 1977; Dunn and Regan, 1979; Erixon and Ahnstrom, 1979). The inhibition by ara-C of DNA repair following u.v. damage occurs at the resynthesis step and does not appear to affect incision over a short period, since the breaks introduced enzymatically in the damaged DNA accumulate in the presence of ara-C. This accumulation does not occur in incicion-deficient fibroblasts from patients with xeroderma pigmentosum (Erixon and Aknstrom, 1979; Snyder et al., 1981; Squires et al., 1982). The kinetics of break accumulation, however, are saturated during more prolonged incubations of human fibroblasts with ara-C/HU following u.v. irradiation (Snyder et al., 1981). Kinetics of break accumulation are also affected by u.v. dose and lower u.v. doses are associated with a slower rate of accumulation (Snyder et al., 1981). These findings are consistent with the hypothesis that limited numbers of DNA polymerase molecules are sequestered at repair sites. Sequestration of DNA polymerase could result from stuttering by polymerase after encountering ara-C moieties incorporated into the DNA repair patch; the latter causing helical distortion through interference with hydrogen bonding. Thus, a

Cytosine arabinoside and hydroxyurea

169

maximal number of repair sites would remain open at a given time and larger numbers of repair sites would be available at higher u.v. doses, resulting in a more rapid initial accumulation of strand breaks. Studies assessing the kinetics of pyrimidine dimer excision from u.v. irradiated human fibroblasts reveal a block in dimer excision at 6-48 hr in cells incubated with ara-C/HU (Snyder et al., 1981). Thus, excision as well as incision may be affected during more prolonged incubations. The association of endonuclease, polymerase and possibly other repair enzymes in a repair complex would explain deficiencies in these functions following sequestration of polymerase. Intracellular levels of deoxyribonucleotides, particularly dCTP, influence the extent of inhibition of DNA repair by ara-C. This is evident by reversal with dCyd of DNA single strand breaks that accumulate after u.v. irradiation in the presence of ara-C (Hiss and Preston, 1977). Further, HU, by inhibiting ribonucleotide reductase, depletes dCTP pools and enhances the formation of single strand breaks by ara-C after u.v. irradiation (Dunn and Regan, 1979; Snyder et al., 1984). HU (10 -2 M) also inhibits the repair of u.v.-induced DNA damage in HeLa cells and human skin fibroblasts (Collins, 1977; Frances et al., 1979; Ben-Hur and Ben-Ishi, 1971). At least one study did not demonstrate strand break accumulation following u.v. irradiation of bovine cells, although this may have occurred partly because lower doses of hydroxyurea were employed (Cleaver et al., 1972). The effects of hydroxyurea appear mediated by depletion of substrates for DNA repair synthesis since both changes in chromosomal morphology and accumulation of single strand breaks are reversed by deoxyribonucleosides (Ben-Hur and Ben-Ishi, 1971; Collins 1977; Collins and Johnson, 1979). This conclusion, however, remains controversial since hydroxyurea either increased or did not affect DNA repair synthesis in Chinese hamster cells, human fibroblasts, SV-40 transformed human fibroblasts, and HeLa cells (Cleaver, 1969a,b; Smith and Hanawalt, 1976; Lampidis and Little, 1977; Clarkson, 1978). Technical considerations may prove relevant since both nucleotide pool size and concentration of hydroxyurea appear important. Thus, the inhibitory effect of this agent on DNA repair synthesis in HeLa cells was found to depend on the concentration of thymidine in the medium (Collins, 1977). We recently confirmed that ara-C is incorporated into DNA undergoing repair of u.v. damage (Kufe et al., 1984c). Since monitoring of ara-C incorporation into DNA undergoing repair requires the use of cells that undergo density-dependent growth arrest to avoid significant incorporation of ara-C in DNA engaged in semi-conservative synthesis, these studies were performed using confluent human foreskin diploid fibroblasts (AG1522) subjected to density-dependent growth arrest. The extent of [3H]ara-C incorporation into AG1522 DNA was determined by CsC1 gradient centrifugation, which separates DNA undergoing repair and replicative synthesis. Figure 3 illustrates the incorporation of [3H]ara-C in AG1522 DNA during u.v. excision repair. There was little detectable incorporation of ara-C in the growth-arrested AG1522 cells not treated with u.v. irradiation. In contrast, significant amounts of ara-C were incorporated into DNA following treatment of growth-arrested ceils with 5 or 20 J/m 2 u.v. light. The pattern of incorporation (Fig. 3) indicated that over 90% of the detectable radioactivity is present in DNA undergoing repair. Furthermore, the extent of ara-C incorporation during DNA repair was dependent upon drug concentration (Fig. 4) and time of exposure. Lastly, labeled DNA digested to nucleotides and analyzed by high pressure liquid chromatography demonstrated that tritium radioactivity represented [3H]ara-C (Kufe et aI., 1984c). The relationship of ara-C incorporation of AG1522 DNA undergoing DNA repair to drug-induced cytotoxicity was studied by comparing the amount of ara-C incorporated with loss of clonogenic survival. The effect of ara-C on clonogenic survival of untreated and u.v.-treated AG1522 cells was determined by exposure to concentrations of 10 -7 M to 10 -4 M for 3, 6, 12 and 24 hr. The results are illustrated in Fig. 5. While there was little difference between untreated and u.v.-treated cells following exposure to ara-C for 3 hr (Fig. 5), a progressive enhancement of cell lethality occurred with increasing time of incubation. The loss of clonogenic survival for both untreated and u.v.-treated cells was

170

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FIG. 3. Incorporation of [3H]ara-C into DNA following u.v. treatment. Growth-arrested Ag1522 ceils were treated with 10-si BrdUrd and 1 0 - r i FdUrd for 2hr and then irradiated with 0, 5 or 20 J/m 2 u.v. The ceils were then incubated with 2 x 10-3M hydroxyurea; 10-rM FdUrd, 10-SM BrdUrd and 10-7 M[3H]ara-C for 24 hr. The DNA was then purified and analyzed by CsC1gradient centrifugation (20). Cells treated with 20 J/m 2 u.v. and ara-C (O), 5 J/m 2 u.v. and ara-C (A), or ara-C alone (0). Under these conditions, the DNA undergoing repair bands at density of 1.700 g/ml, and the DNA undergoing semiconservativesynthesis bands at 1.751 g/ml. (From Kufe et al., 1984c.)

also d e p e n d e n t u p o n drug c o n c e n t r a t i o n . These results are in a g r e e m e n t with prior studies that d e m o n s t r a t e synergistic e n h a n c e m e n t by a r a - C o f u . v . - i n d u c e d cytotoxicity in e u k a r y o t i c cells (Collins a n d J o h n s o n , 1979). The r e l a t i o n s h i p between i n c o r p o r a t i o n o f a r a - C into D N A u n d e r g o i n g u.v. repair synthesis a n d loss o f c l o n o g e n i c survival was d e t e r m i n e d by m e a s u r i n g the a m o u n t o f [3H]ara-C i n c o r p o r a t e d into D N A u n d e r the same c o n d i t i o n s used for studies m o n i t o r i n g c l o n o g e n i c survival. I n c o r p o r a t i o n studies were p e r f o r m e d at [3H]ara-C c o n c e n t r a t i o n s

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A,a-C [M] FIG. 5. Clonogenic survival of AG 1522 cells exposed to u.v. irradiation and varying concentrations of ara-C. Growth-arrested Ag1522 ceils were treated with 5 J/m 2u.v. irradiation and then incubated with varying concentrations (10-7-10 -4) of ara-C for 3, 6, 12 or 24 hr. After drug exposure, the cells were washed, trypsinized and resuspended in drug-free medium. Clonogenic survival was determined after 7-14 days by scoring colonies containing more than 20 cells. O, Cells treated with u.v. and ara-C; O, cells treated with ara-C alone. (From Kufe et al., 1984c.)

ranging from 10 -7 to 10-4M during incubation periods of 3, 6, 12 and 24 hr. Clonogenic survival at each ara-C concentration was obtained from the cloning data shown in Fig. 6. The extent of ara-C incorporation into DNA undergoing repair synthesis correlated significantly, by probit analysis (Kufe et al., 1984c), with loss of clonogenic survival. The probit analysis of this relationship as determined by a computer-assisted program is illustrated in Fig. 6. Inhibition of the resynthesis step could occur as a result of ara-C incorporation into the repair patch. Thus, an ara-C residue incorporated into the repair patch could act as in the case of replication of DNA synthesis, as a relative chain terminator and force the polymerase to stutter at this site. Furthermore, the inhibition of the resynthesis step byara-C coul d sequester the polymerase at a repair site and make it or repair enzymes associated With it unavailable to perform excision functions at other damaged DNA sites. In addition to incorporation of ara-C into DNA undergoing repair synthesis, competitive inhibition of DNA polymerases undergoing repair also may contribute to the inhibition of DNA repair synthesis. Ara-C is a competitive inhibitor of DNA polymerase ct and to a lesser extent polymerase fl (Yoshida et al., 1977). And while DNA polymerase

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172

R.J. FRAMand D. W. KUFE

fl is often implicated in DNA repair synthesis, DNA polymerase ~ may also be involved since Apc, an agent that specifically inhibits polymerase ~, also inhibits DNA repair synthesis after u.v. irradiation and alkylator induced DNA damage (Berger et al., 1979; Ciarrocchi et al., 1979; Natarajan et al., 1982; Snyder and Regan, 1981; Miller and Chinault, 1982; Hanaoka et al., 1979). While Apc is not invariably found to inhibit the repair of u.v.-induced DNA damage, such disparate observations may be related to differences in cell type and in experimental approach (Giulotto and Mondello, 1981). Further, depending on the type of DNA damage, polymerase 0~and fl appear to influence DNA repair synthesis to varying degrees. Thus, polymerase ct appears more involved in the repair of DNA damage induced by M N N G (N-methyl-N-nitro-N-nitrosoguanidine) or M N U (N-methyl-N-nitrosourea) and is subject to greater inhibition by ara-CTP and Apc. In contrast, DNA damage induced by bleomycin and neocarzinostatin is more sensitive to inhibitors of polymerase fl (Miller and Chinault, 1982). While no clear basis for the varying involvement of polymerase ct is yet apparent, this enzyme, in contrast to polymerase fl, would have greater affinity for relatively large template regions made available by excision, and differences in patch size are known to result from exposure of DNA to different agents (Regan and Setlow, 1974; Frances et al., 1981). The precise interaction of ara-C residues incorporated into DNA undergoing repair with DNA excision repair processes remains unclear and raises fundamental questions as to our current concepts of excision repair in eukaryotes. While in some cell systems ara-C and aphidicolin clearly inhibit DNA repair synthesis, these findings are not observed in all systems (Snyder et aL, 1984). Little or no inhibition of DNA repair synthesis is observed in rapidly proliferating cells probably owing to high levels of dCTP and polymerase 0t. In confluent fibroblasts 10 -4 M ara-C inhibits DNA repair synthesis by 75% following an 8 hr incubation with drug after irradiation with 50Jm -2 u.v. (Smith, 1984). Because no alteration in repair patch size was evident, it was concluded that repair patches were reduced in number rather than size in the presence of ara-C (Smith, 1984). Such findings are consistent with: (1) Inhibition of exonuclease by incorporated ara-C residues; exonuclease is required to excise bases 5' to the pyrimidine dimer following incision by endonuclease in the classical model of excision repair (see Section 4), permitting DNA repair synthesis; and/or (2) sequestration of repair enzymes associated with polymerase 0t, resulting in the initiation of only a limited number of repair patches. As a primary event in inhibition excision repair, interference of incorporated ara-C residues with repair ligase, although a possibility, seems less likely. The [3H]ara-C residues incorporated into DNA undergoing repair persist indefinitely, while single strand breaks arising after u.v. irradiation become readily ligated upon removal of inhibitor (Smith, 1984; Dunn and Regan, 1979). Further, at least some ligation occurs in the presence of either ara-C or aphidicolin following u.v. irradiation as assessed by the sensitivity of labeled thymidine incorporated into DNA undergoing repair synthesis to Alteromonas nuclease, an enzyme that digests single-stranded DNA (Smith, 1984). Under these circumstances polymerase fl, which is relatively insensitive to these inhibitors may complete a small portion of DNA repair synthesis which is then followed by a ligation step. Initial studies suggested that nearly all labeled thymidine incorporated during repair synthesis after u.v. irradiation of fibroblasts and in the presence of ara-C was restricted to the 3' terminus or was single-stranded in nature as assessed by digestion with S1 nuclease and exonuclease III (Cleaver, 1981). Subsequent studies, on the other hand, employing more appropriate levels of S1 nuclease demonstrated that less than 50% of repair label was associated with single stranded DNA or near the 3' terminus (Bodell et aL, 1982; Cleaver, 1984). The latter findings are more consistent with a model of excision repair similar to that of E. coli in which incisions are made 5' and 3' to the pyrimidine dimer followed by spontaneous excision, DNA synthesis and ligation. The issue remains unresolved since in one other system employing thymidine incorporated into DNA during DNA repair synthesis after u.v. irradiation, 63-75% of labeled thymidine is released following exposure to Altermonas nuclease, a nuclease which exhibits endonuclease activity against single-stranded DNA (Smith, 1984).

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6. EFFECTS ON THE REPAIR OF X-RAY INDUCED DNA LESIONS BY ARA-C, HU, AND APHIDICOLIN In contrast to the inhibition of repair of u.v.-induced DNA damage by ara-C, the repair of X-ray-induced single strand DNA breaks is only partially inhibited by ara-C and hydroxyurea in human leukemic blasts and HeLa cells (Fram and Kufe, 1985; Ward et al., 1984; Hiss and Preston, 1977). Further, unlike repair after u.v. irradiation in the presence of labeled [3H]ara-C, DNA repair after X-irradiation is not accompanied by detectable incorporation of [3H]ara-C in the repair patch (Fram and Kufe, 1985). These findings may be related to the small repair patch associated with X-ray induced damage and thus lower the probability of ara-C misincorporation. Furthermore, the difference in incorporation of ara-C into DNA undergoing repair synthesis of u.v. induced damage as compared to X-ray-induced damage could be related to the polymerase involved in these repair processes. Semiconservative synthesis and long patch repair utilize polymerase ~t, while short patch repair synthesis probably involves DNA polymerase ft. In contrast to DNA polymerase ~, DNA polymerase fl is inhibited only by high concentrations of ara-CTP. Data is limited on the biological significance of inhibiting X-ray repair with ara-C. In HeLa ceils, this agent significantly enhances radiosensitivity (Ward et al., 1984). Although the basis for this finding is unclear, the slowing of repair of X-ray induced single strand breaks may enhance the yield of double strand breaks, a lesion that is more likely irreversible and cytotoxic. The accumulation of chromosomal aberrations by DNA synthesis inhibitors is a finding of potential biological relevance and one that as with effects on X-ray and u.v. repair appears related to the slowing of DNA repair kinetics. Ara-C and Apc both accumulate DNA strand breaks after DNA damage and enhance the induction of chromosomal gaps and deletions after u.v. irradiation, X-ray and alkylating agent induced DNA damage (Natarajan et al., 1982; Preston, 1980; Preston, 1982a,b). Thus, the inhibition of repair may potentially contribute to increased numbers of DNA lesions that can interact to form chromosomal aberrations. A finding consistent with this hypothesis is that agents, such as alkylators and X-rays, which differ substantially in the rates of repair of their respective DNA lesions, synergistically induce chromosomal aberrations in the presence of ara-C, while their effect is only additive in the absence of ara-C (Preston, 1982a,b). Ara-C incorporated into DNA undergoing long patch repair may also contribute to mutagenesis independently of its effects on repair kinetics since ara-C as well as other nucleoside analogs incorporated into DNA are mutagenic (Bubley et al., 1985). 7. CONCLUSIONS Inhibitors of replicative DNA synthesis inhibit repair of DNA damage induced by u.v. irradiation and X-ray. The inhibition of repair by these agents has provided a means for studying repair kinetics after u.v. irradiation. Differences in the effects of these agents also has provided insights into mechanisms underlying the repair of these types of DNA damage. The effects of DNA synthesis inhibitors on DNA repair raise several fundamental questions. Perhaps most important is the need to identify clearly the critical similarities and differences that underlie replicative and repair DNA synthesis in mammalian cells. For example, an issue currently under active investigation includes the extent and mechanisms by which the structure and size of a DNA lesion contribute to determining the kind of polymerase which participates in its repair. Although ara-C is incorporated in DNA repair segments, whether the effects of the incorporated residue on DNA repair synthesis are entirely analogous to replicative synthesis also requires clarification. The basis underlying an inhibitory effect by ara-C and other DNA synthesis inhibitors on the repair of X-ray-induced DNA damage and whether these effects occur as a result of inhibition of apurinic endonuclease, repair ligase, effects on polymerases involved in repair synthesis also requires further study. Lastly, although effects on DNA repair synthesis are implicated

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as t h e b a s i s f o r t h e i n h i b i t i o n b y a r a - C o f r e p a i r a f t e r u . v . - i n d u c e d D N A d a m a g e , t h e i s s u e is n o t c o m p l e t e l y r e s o l v e d a n d o t h e r effects, s u c h as i n h i b i t i o n o f e x o n u c l e a s e a c t i v i t y , s e q u e s t r a t i o n o f r e p a i r e n z y m e s a s s o c i a t e d w i t h p o l y m e r a s e ~ b y a r a - C i n c o r p o r a t e d in DNA are also potentially important.

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