DNA Rejoining Research Articles

Pretreatment with heat does not affect double-strand breaks DNA rejoining in Chlamydomonas reinhardtii

We aimed to clarify if heat pretreatment could protect Chlamydomonas reinhardtii cells from gamma rays DNA damaging action. It was studied whether: (1) heat pretreatment could accelerate DNA DSB rejoining; (2) chloroplast chaperones (HSP70B, HSP90C) could be involved in protection from radiation-induced DNA DSB. It was obtained that heat pretreatment (37–42 °C) induced minor DNA DSB levels which might be insufficient as signals for DNA DSB repair induction. No correlation between chaperones overproduction and DNA DSB rejoining was shown. These are probably the first data that HSP70B and HSP90C do not protect DNA against radiation-induced damage in a plant model system.

Topoisomerase II: a fitted mechanism for the chromatin landscape

The mechanism by which type-2A topoisomerases transport one DNA duplex through a transient double-strand break produced in another exhibits fascinating traits. One of them is the fine coupling between inter-domainal movements and ATP usage; another is their preference to transport DNA in particular directions. These capabilities have been inferred from in vitro studies but we ignore their significance inside the cell, where DNA configurations markedly differ from those of DNA in free solution. The eukaryotic type-2A enzyme, topoisomerase II, is the second most abundant chromatin protein after histones and its biological roles include the decatenation of newly replicated DNA and the relaxation of polymerase-driven supercoils. Yet, topoisomerase II is also implicated in other cellular processes such as chromatin folding and gene expression, in which the topological transformations catalysed by the enzyme are uncertain. Here, some capabilities of topoisomerase II that might be relevant to infer the enzyme performance in the context of chromatin architecture are discussed. Some aspects addressed are the importance of the DNA rejoining step to ensure genome stability, the regulation of the enzyme activity and of its putative structural role, and the selectively of DNA transport in the chromatin milieu.

Absence ofKu70Gene Obliterates X-Ray-InducedlacZMutagenesis of Small Deletions in Mouse Tissues

With the goal of understanding the role of non-homologous end-joining repair in the maintenance of genetic information at the tissue level, we studied mutations induced by radiation and subsequent repair of DNA double-strand breaks in Ku70 gene-deficient lacZ transgenic mice. The local mutation frequencies and types of mutations were analyzed on a lacZ gene that had been chromosomally integrated, which allowed us to monitor DNA sequence alterations within this 3.1-kbp region. The mutagenic process leading to the development of the most frequently observed small deletions in wild-type mice after exposure to 20 Gy of X rays was suppressed in Ku70(-/-) mice in the three tissues examined: spleen, liver and brain. Examination of DNA break rejoining and the phosphorylation of histone H2AX in Ku70-deficient and -proficient mice revealed that Ku70 deficiency decreased the frequency of DNA rejoining, suggesting that DNA rejoining is one of the causes of radiation-induced deletion mutations. Limited but statistically significant DNA rejoining was found in the liver and brain of Ku70-deficient mice 3.5 days after irradiation, showing the presence of a DNA double-strand break repair system other than non-homologous end joining. These data indicate a predominant role of non-homologous end joining in the production of radiation-induced mutations in vivo.

SU-GG-T-408: Computational Analysis of the Dose and Dose-Rate Dependence of DNA Double-Strand Break Repairs

Purpose: Computational quantification of the dose and dose‐rate dependence of radiation‐induced DNA double‐strand break (DSB) repair, and the significance of high dose and high dose‐rate limits pertinent to stereotactic body radiation therapy(SBRT) fractionation regimens. Method and Materials: The mass‐action biochemical kinetics approach was used to describe the binding of repair enzymes to DSBs through the formation of intermediate repair complexes which lead to DNA rejoining. To quantify the formation of intermediate repair complexes, coupled non‐linear ordinary differential equations (ODEs) which govern the dynamics of DNADSB non‐homologous end‐joining (NHEJ) repair were used. Biochemical kinetics parameters for human fibroblast cells were used and the ODEs were numerically solved to predict the number of DSB complexes for doses in the range of 2–30 Gy and dose‐rates in the range of 1–100 Gy/h. Results: Our computational analysis predicts that the number of DSB repair complexes formed in the high dose and high dose‐rate limit can be several orders of magnitude higher than those in the low dose and low dose‐rate limit. Conclusion: This work indicates that SBRT can lead to significantly higher therapeutic ratios in radiation therapy, if normal tissue toxicity can be maintained at levels comparable to those of conventional fractionation regimens. Since our computational analysis directly incorporates the quantification of cellular biochemical processes, its predictions are considerably more tractable in the high dose and high dose‐rate regime than models such as the linear‐quadratic model. Contributors: Ramin M. Abolfath, Ph.D., Robert D. Timmerman, M.D., Timothy D. Solberg, Ph.D., Debabrata Saha, Ph.D., Michael Story, Ph.D., Paul Derose, M.D., Homayoun Hamidian, Ph.D.

Insertions in xanthene dehydrogenase genes suppress cell death due to stabilization of topoisomerase IA covalent complex

Bacterial type IA or IIA topoisomerases modify DNA topology by transiently cleaving and rejoining either one or two strands of DNA respectively. When topoisomerases cleave the DNA, a covalent complex between the cleaved DNA and the enzyme is formed. Stabilization of the type IIA topoisomerase complex by antibacterial drugs such as fluoroquinolones leads to decrease in DNA rejoining, accumulation of DNA strand breaks which in turn leads to cell death. There is an urgent need for new antibiotics due to the emerging drug resistance and topoisomerase IA is a promising target. It has been shown that a recombinant type IA Yersinia pestis topoisomerase with a conserved glycine residue in the TOPRIM motif mutated to serine accumulates the covalent complex and causes rapid bacterial cell killing. The main goal of this work is to study the mechanism of cell killing resistance for the stabilized type IA topoisomerase covalent complex. Insertional mutagenesis was used to identify and characterize the genes in Escherichia coli that influence the cell killing by the mutant Y. pestis topoisomerase I. We observed that insertions in the xanthene dehydrogenase operon (xdhABC) and homolog xdhD resulted in resistance to topoisomerase IA covalent complex mediated cell killing by ~4 log10 fold. Currently, studies are being conducted to elucidate the effect of these insertions on SOS response and other DNA damaging agents.

Local changes of higher-order chromatin structure during DSB-repair

We show that double-strand breaks (DSBs) induced in DNA of human cells by γ-radiation arise mainly in active, gene-rich, decondensed chromatin. We demonstrate that DSBs show limited movement in living cells, occasionally resulting in their permanent clustering, which poses a risk of incorrect DNA rejoining. In addition, some DSBs remain unrepaired for several days after irradiation, forming lesions repairable only with difficulty which are hazardous for genome stability. These ‘late’ DSBs colocalize with heterochromatin markers (dimethylated histone H3 at lysine 9, HP1 and CENP-A proteins), despite the low density of the surrounding chromatin. This indicates that there is epigenetic silencing of loci close to unrepaired DSBs and/or stabilization of damaged decondensed chromatin loops during repair and post-repair reconstitution of chromatin structure.

Mutation adjacent to the active site tyrosine can enhance DNA cleavage and cell killing by the TOPRIM Gly to Ser mutant of bacterial topoisomerase I

The TOPRIM DXDXXG residues of type IA and II topoisomerases are involved in Mg(II) binding and the cleavage-rejoining of DNA. Mutation of the strictly conserved glycine to serine in Yersinia pestis and Escherichia coli topoisomerase I results in bacterial cell killing due to inhibition of DNA religation after DNA cleavage. In this study, all other substitutions at the TOPRIM glycine of Y. pestis topoisomerase I were examined. While the Gly to Ala substitution allowed both DNA cleavage and religation, other mutations abolished DNA cleavage. DNA cleavage activity retained by the Gly to Ser mutant could be significantly enhanced by a second mutation of the methionine residue adjacent to the active site tyrosine. Induction of mutant topoisomerase with both the TOPRIM glycine and active site region methionine mutations resulted in up to 40-fold higher cell killing rate when compared with the single TOPRIM Gly to Ser mutant. Bacterial type IA topoisomerases are potential targets for discovery of novel antibiotics. These results suggest that compounds that interact simultaneously with the TOPRIM motif and the molecular surface around the active site tyrosine could be highly efficient topoisomerase poisons through both enhancement of DNA cleavage and inhibition of DNA rejoining.

Impact of the comet assay in radiobiology

Until the development of single cell gel electrophoresis methods in the 1980s, measurement of radiation-induced DNA strand breaks in individual cells was limited to detection of micronuclei or chromosome breaks that measured the combined effects of exposure and repair. Development of methods to measure the extent of migration of DNA from single cells permitted detection of initial radiation-induced DNA breaks present in each cell. As cells need not be radiolabeled, there were new opportunities for analysis of radiation effects on cells from virtually any tissue, provided a single cell suspension could be prepared. The comet assay (as this method was subsequently named) was able to measure, for the first time, the fraction of radiobiologically hypoxic cells in mouse and human tumors. It was used to determine that the rate of rejoining of DNA breaks was relatively homogenous within an irradiated population of cells. Because individual cells were analyzed, heavily damaged or apoptotic cells could be identified and eliminated from analysis to determine "true" DNA strand break rejoining rates. Other examples of applications of the comet assay in radiobiology research include analysis of the inter-individual differences in response to radiation, effect of hypoxia modifying agents on tumor hypoxic fraction, the role of cell cycle position during DNA break induction and rejoining, non-targeted effects on bystander cells, and effects of charged particles on DNA fragmentation patterns.

Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli

Modulation of bacterial chromosomal supercoiling is a function of DNA gyrase-catalyzed strand breakage and rejoining. This reaction is exploited by both antibiotic and proteic gyrase inhibitors, which trap the gyrase molecule at the DNA cleavage stage. Owing to this interaction, double-stranded DNA breaks are introduced and replication machinery is arrested at blocked replication forks. This immediately results in bacteriostasis and ultimately induces cell death. Here we demonstrate, through a series of phenotypic and gene expression analyses, that superoxide and hydroxyl radical oxidative species are generated following gyrase poisoning and play an important role in cell killing by gyrase inhibitors. We show that superoxide-mediated oxidation of iron–sulfur clusters promotes a breakdown of iron regulatory dynamics; in turn, iron misregulation drives the generation of highly destructive hydroxyl radicals via the Fenton reaction. Importantly, our data reveal that blockage of hydroxyl radical formation increases the survival of gyrase-poisoned cells. Together, this series of biochemical reactions appears to compose a maladaptive response, that serves to amplify the primary effect of gyrase inhibition by oxidatively damaging DNA, proteins and lipids.

Topo IIβ Gets to Work

DNA topoisomerases regulate conformational changes in DNA topology by catalyzing DNA strand breakage and rejoining. Topoisomerase IIβ (Topo IIβ) alters DNA conformation near gene promoters and associates with sequence-specific transcription factors and chromatin modifying and remodeling factors. Ju et al. now show that DNA Topo IIβ generates double-strand breaks at transcriptional promoters when these genes are activated in a signal-dependent manner. Subsequently, DNA repair enzymes are activated and there is a resultant exchange in histone composition and local chromatin structure. B.-G. Ju, V. V. Lunyak, V. Perissi, I. Garcia-Bassets, D. W. Rose, C. K. Glass, M. G. Rosenfeld, A topoisomerase IIβ-mediated dsDNA break required for regulated transcription. Science 312 , 1798-1802 (2006). [Abstract] [Full Text] J.-F. Haince, M. Rouleau, G. G. Poirier, Gene expression needs a break to unwind before carrying on. Science 312 , 1752-1753 (2006). [Summary] [Full Text]

An Inhibitor of DNA Recombination Blocks Memory Consolidation, But Not Reconsolidation, in Context Fear Conditioning

Genomic recombination requires cutting, processing, and rejoining of DNA by endonucleases, polymerases, and ligases, among other factors. We have proposed that DNA recombination mechanisms may contribute to long-term memory (LTM) formation in the brain. Our previous studies with the nucleoside analog 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP), a known inhibitor of DNA ligases and polymerases, showed that this agent blocked consolidation of conditioned taste aversion without interfering with short-term memory (STM). However, because polymerases and ligases are also essential for DNA replication, it remained unclear whether the effects of this drug on consolidation were attributable to interference with DNA recombination or neurogenesis. Here we show, using C57BL/6 mice, that ara-CTP specifically blocks consolidation but not STM of context fear conditioning, a task previously shown not to require neurogenesis. The effects of a single systemic dose of cytosine arabinoside (ara-C) on LTM were evident as early as 6 h after training. In addition, although ara-C impaired LTM, it did not impair general locomotor activity nor induce brain neurotoxicity. Importantly, hippocampal, but not insular cortex, infusions of ara-C also blocked consolidation of context fear conditioning. Separate studies revealed that context fear conditioning training significantly induced nonhomologous DNA end joining activity indicative of DNA ligase-dependent recombination in hippocampal, but not cortex, protein extracts. Finally, unlike inhibition of protein synthesis, systemic ara-C did not block reconsolidation of context fear conditioning. Our results support the idea that DNA recombination is a process specific to consolidation that is not involved in the postreactivation editing of memories.

Phosphorylated Histone H2AX Foci Persist on Rejoined Mitotic Chromosomes in Normal Human Diploid Cells Exposed to Ionizing Radiation

Histone H2AX is phosphorylated and forms foci in response to exposure to ionizing radiation. It has been thought that phosphorylated histone H2AX foci reflect unrepaired DNA double-strand breaks; however, we report here the localization of phosphorylated histone H2AX foci at the site of rejoined DNA double-strand breaks. We observed that phosphorylated histone H2AX foci remained even 96 h after exposure to X rays in interphase cells. To clarify the localization of residual phosphorylated histone H2AX foci, we examined localization of focus formation on mitotic chromosomes irradiated with X rays. We found that phosphorylated histone H2AX foci were located not only on chromosomal fragments but also on intact metaphase chromosomes without fragments. In anaphase cells, chromosomal bridges, which resulted from illegitimate rejoining of DNA broken ends, had phosphorylated histone H2AX foci. These foci were detected as individual small spots 30 min after X irradiation, but foci detected 20 or 96 h after X irradiation were clustered along the chromosomal bridges. These results indicate that phosphorylated histone H2AX foci persist if DNA breaks are rejoined. It is suggested that "residual" foci indicate an aberrant chromatin structure by illegitimate rejoining but not a DNA double-strand break itself.

新しい現象「遅発性分裂遅延」： アルファ線およびX線照射後生き残ったHeLa細胞の無限増殖能を保持した子孫におけるDNA二重鎖切断と再結合を示唆する証拠

新しい現象「遅発性分裂遅延」： アルファ線およびX線照射後生き残ったHeLa細胞の無限増殖能を保持した子孫におけるDNA二重鎖切断と再結合を示唆する証拠

Sex, Not Genotype, Determines Recombination Levels in Mice

Recombination, the precise physical breakage and rejoining of DNA between homologous chromosomes, plays a central role in mediating the orderly segregation of meiotic chromosomes in most eukaryotes. Despite its importance, the factors that control the number and placement of recombination events within a cell remain poorly defined. The rate of recombination exhibits remarkable species specificity, and, within a species, recombination is affected by the physical size of the chromosome, chromosomal location, proximity to other recombination events (i.e., chiasma interference), and, intriguingly, the sex of the transmitting parent. To distinguish between simple genetic and nongenetic explanations of sex-specific recombination differences in mammals, we compared recombination in meiocytes from XY sex-reversed and XO females with that in meiocytes from XX female and XY male mice. The rate and pattern of recombination in XY and XO oocytes were virtually identical to those in normal XX females, indicating that sex, not genotype, is the primary determinant of meiotic recombination patterns in mammals.

Nature of DNA lesions induced in human hepatoma cells, human colonic cells and human embryonic lung fibroblasts by the antiretroviral drug 3′-azido-3′-deoxythymidine

This study tried to clarify the question if nuclear genotoxicity played a role in 3′-azido-3′-deoxythymidine (AZT) toxicity. We investigated cytotoxic and DNA-damaging effects of AZT on human hepatoma HepG2 and human colonic CaCo-2 cells as well as on human diploid lung fibroblasts HEL. The amount of induced DNA damage was measured by standard alkaline single cell gel electrophoresis (SCGE). The nature of induced DNA lesions was evaluated (1) by modified SCGE, which includes treatment of lysed cells with DNA repair enzymes Endo III and Fpg and enables to recognize oxidized bases of DNA, and (2) by SCGE processed in parallel at pH 13.0 (standard technique) and pH 12.1, which enables to recognize alkali labile DNA lesions and direct DNA strand breaks. Cytotoxicity of AZT was evaluated by the trypan blue exclusion technique. Our findings showed that 3-h treatment of cells with AZT decreased the viability of all cell lines studied. SCGE performed in the presence of DNA repair enzymes proved that AZT induced oxidative lesions to DNA in all cell types. In hepatoma HepG2 cells and embryonic lung fibroblasts HEL the majority of AZT-induced DNA strand breaks were pH-independent, i.e. they were identified at both pH values (12.1 and 13.0). These DNA lesions represented direct DNA breaks. In colonic Caco-2 cells DNA lesions were converted to DNA strand breaks particularly under strong alkaline conditions (pH > 13.0), which is characteristic for alkali-labile sites of DNA. DNA strand break rejoining was investigated by the standard comet assay technique during 48 h of post-AZT-treatment in HepG2 and Caco-2 cells. The kinetics of DNA rejoining, considered an indicator of DNA repair, revealed that AZT-induced DNA breaks were repaired in both cell types slowly, though HepG2 cells seemed to be more repair proficient with respect to AZT-induced DNA lesions.

Stability of the Topoisomerase II Closed Clamp Conformation May Influence DNA-stimulated ATP Hydrolysis

Type II DNA topoisomerases catalyze changes in DNA topology and use nucleotide binding and hydrolysis to control conformational changes required for the enzyme reaction. We examined the ATP hydrolysis activity of a bisdioxopiperazine-resistant mutant of human topoisomerase II alpha with phenylalanine substituted for tyrosine at residue 50 in the ATP hydrolysis domain of the enzyme. This substitution reduced the DNA-dependent ATP hydrolysis activity of the mutant protein without affecting the relaxation activity of the enzyme. A similar but stronger effect was seen when the homologous mutation (Tyr28 --> Phe) was introduced in yeast Top2. The ATPase activities of human TOP2alpha(Tyr50 --> Phe) and yeast Top2(Tyr28 --> Phe) were resistant to both bisdioxopiperazines and the ATPase inhibitor sodium orthovanadate. Like bisdioxopiperazines, vanadate traps the enzyme in a salt-stable closed conformation termed the closed clamp, which can be detected in the presence of circular DNA substrates. Consistent with the vanadate-resistant ATPase activity, salt-stable closed clamps were not detected in reactions containing the yeast or human mutant protein, vanadate, and ATP. Similarly, ADP trapped wild-type topoisomerase II as a closed clamp, but could not trap either the human or yeast mutant enzymes. Our results demonstrate that bisdioxopiperazine-resistant mutants exhibit a difference in the stability of the closed clamp formed by the enzyme and that this difference in stability may lead to a loss of DNA-stimulated ATPase. We suggest that the DNA-stimulated ATPase of topoisomerase II is intimately connected with steps that occur while the N-terminal domain of the enzyme is dimerized.

Involvement of Poly(ADP-ribose) Polymerase-1 and XRCC1/DNA Ligase III in an Alternative Route for DNA Double-strand Breaks Rejoining

The efficient repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In mammalian cells, the nonhomologous end-joining process that represents the predominant repair pathway relies on the DNA-dependent protein kinase (DNA-PK) and the XRCC4-DNA ligase IV complex. Nonetheless, several in vitro and in vivo results indicate that mammalian cells use more than a single end-joining mechanism. While searching for a DNA-PK-independent end-joining activity, we found that the pretreatment of DNA-PK-proficient and -deficient rodent cells with an inhibitor of the poly(ADP-ribose) polymerase-1 enzyme (PARP-1) led to increased cytotoxicity of the highly efficient DNA double-strand breaking compound calicheamicin gamma1. In addition, the repair kinetics of the DSBs induced by calicheamicin gamma1 was delayed both in PARP-1-proficient cells pretreated with the PARP-1 inhibitor and in PARP-1-deficient cells. In order to get new insights into the mechanism of an alternative route for DSBs repair, we have established a new synapsis and end-joining two-step assay in vitro, operating on DSBs with either nuclear protein extracts or recombinant proteins. We found an end-joining activity independent of the DNA-PK/XRCC4-ligase IV complex but that actually required a novel synapsis activity of PARP-1 and the ligation activity of the XRCC1-DNA ligase III complex, proteins otherwise involved in the base excision repair pathway. Taken together, these results strongly suggest that a PARP-1-dependent DSBs end-joining activity may exist in mammalian cells. We propose that this mechanism could act as an alternative route of DSBs repair that complements the DNA-PK/XRCC4/ligase IV-dependent nonhomologous end-joining.

Radiation Response in Vitro of Fibroblasts from a Fanconi Anemia Patient with Marked Clinical Radiosensitivity

. Fanconi anemia (FA) is an autosomal recessive chromosome instability disorder characterized by progressive pancytopenia and cancer susceptibility. The risks of radiation therapy in FA patients who have cancer remain to be investigated. Recently, Marcou et al. (2001) reported a case of severe clinical radiosensitivity in a female FA patient with a tonsillar squamous cell carcinoma treated by radiotherapy. By contrast, her in vitro irradiated skin fibroblasts revealed nearly normal radiosensitivity as determined by the colony survival assay. . In view of this discrepancy, the radiation response of this particular FA fibroblast strain (designated 425BR) was further analyzed in the present study by means of the alkaline single-cell gel electrophoresis (Comet) assay, and also by the cytochalasin-blocked micronuclei (MN) test. In addition, the expression levels of DNA repair proteins, hMre11, Rad50, and Rad51, were investigated using Western blot and foci immunofluorescence staining. . The Comet assay revealed that the initial DNA fragmentation in irradiated FA cells was two times higher and the DNA rejoining process was three times slower than that in control (1BR3) fibroblasts. Moreover, although the baseline level of MNs was lower in FA cells than in controls, the FA fibroblasts were more prone (about two times) to MN production than control cells when irradiated with 2-4 Gy. Western blot analysis of the DNA repair proteins (hMre11, Rad50, and Rad51) did not reveal any abnormalities in protein expression levels or their migration patterns in the fibroblasts derived from an FA patient either before or after irradiation. At the same time, in vitro irradiated cells from the FA patient exhibited a significantly reduced number of nuclei with focally concentrated DNA repair Rad51 protein than in control cells. . The increased DNA damage and MN induction in irradiated FA fibroblasts, and the reduction of the formation of DNA repair foci containing Rad51 suggest a possible link to the profound clinical radiosensitivity reported earlier for this FA patient. The findings on this particular FA cell strain presented in the study point toward the difficulties involved in the prediction of the radiation response of cell lines and tumors based solely on the colony survival test.

Additive interaction of gefitinib (‘Iressa’, ZD1839) and ionising radiation in human tumour cells in vitro

Cultures of human carcinoma A-431, A-549 and HeLa cells were challenged with γ-rays without or with concomitant exposure to gefitinib, a potent inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR). The outcome of treatment was determined from cell and colony count, cell cycle progression and DNA double-strand break formation and rejoining. Apoptosis was measured in parallel from hypodiploid DNA and using an annexin V assay. Gefitinib developed a cytostatic effect in all cell lines, with drug sensitivity correlating the level of EGFR expression. A weak cytotoxicity of gefitinib was observed in HeLa cells only, although the drug was unable to induce significant cell cycle redistribution in this cell line. In contrast, substantial G1 block and S-phase depletion was observed in A-431 and A-549 cells exposed to gefitinib. The drug brought about additive to subadditive interaction with radiation with regard to growth inhibition, clonogenic death and induction of apoptosis. Consistently, gefitinib did not hinder the rejoining of radiation-induced DNA double-strand breaks in any cell line. The results demonstrate that gefitinib may elicit cytotoxicity at high concentration, but does not act as a radiosensitiser in vitro in concomitant association with radiation.

Inhibition of repair of carboplatin-induced DNA damage by 9-β- d-arabinofuranosyl-2-fluoroadenine in quiescent human lymphocytes

Previous studies including ours have demonstrated that DNA repair is one of the important targets of fludarabine. The aim of this study is to clarify a mechanistic interaction of carboplatin and F-ara-A, from the perspective of F-ara-A-mediated inhibition of DNA repair initiated by carboplatin. Using human quiescent lymphocytes, we focused on DNA repair, since these cells provide a model of dormant cells. To evaluate the carboplatin-induced DNA incision and its repair, we used the alkaline comet assay. When lymphocytes were incubated with carboplatin, a dose-dependent increase in the tail-moment was observed. Then, tail-moment decreased in proportion to the incubation period in fresh media and recovered to the control level at 4 h. DNA rejoining was completely inhibited by F-ara-A at 10 μM through 0 to 6 h after washing out of these drugs and this F-ara-A-induced inhibition was concentration-dependent. Cellular damage after drug exposure was evaluated with the induction of apoptosis as well as cytotoxic effect. Exposure to carboplatin alone did not induce any apparent cellular damage in quiescent lymphocytes. In contrast, a more than additive induction of apoptosis as well as an enhancement of cytotoxic action was observed in cells treated with a combination of carboplatin and F-ara-A. In the CEM cell line, there was no enhancement of the cytotoxic action of these drugs, despite the clear demonstration of an inhibitory effect on DNA repair. These results indicate that chemotherapy with carboplatin opened a new target for F-ara-A by initiating DNA repair in quiescent cells.