Alkylation base damage is converted into repairable double-strand breaks and complex intermediates in G2 cells lacking AP endonuclease

DNA double-strand breaks (DSBs) are potent sources of genome instability. While there is considerable genetic and molecular information about the disposition of direct DSBs and breaks that arise during replication, relatively little is known about DSBs derived during processing of single-strand lesions, especially for the case of single-strand breaks (SSBs) with 3'-blocked termini generated in vivo. Using our recently developed assay for detecting end-processing at random DSBs in budding yeast, we show that single-strand lesions produced by the alkylating agent methyl methanesulfonate (MMS) can generate DSBs in G2-arrested cells, i.e., S-phase independent. These derived DSBs were observed in apn1/2 endonuclease mutants and resulted from aborted base excision repair leading to 3' blocked single-strand breaks following the creation of abasic (AP) sites. DSB formation was reduced by additional mutations that affect processing of AP sites including ntg1, ntg2, and, unexpectedly, ogg1, or by a lack of AP sites due to deletion of the MAG1 glycosylase gene. Similar to direct DSBs, the derived DSBs were subject to MRX (Mre11, Rad50, Xrs2)-determined resection and relied upon the recombinational repair genes RAD51, RAD52, as well as on the MCD1 cohesin gene, for repair. In addition, we identified a novel DNA intermediate, detected as slow-moving chromosomal DNA (SMD) in pulsed field electrophoresis gels shortly after MMS exposure in apn1/2 cells. The SMD requires nicked AP sites, but is independent of resection/recombination processes, suggesting that it is a novel structure generated during processing of 3'-blocked SSBs. Collectively, this study provides new insights into the potential consequences of alkylation base damage in vivo, including creation of novel structures as well as generation and repair of DSBs in nonreplicating cells.     

Endogenous DNA abasic sites cause cell death in the absence of Apn1, Apn2 and Rad1/Rad10 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, mutations in APN1, APN2 and either RAD1 or RAD10 genes are synthetic lethal. In fact, apn1 apn2 rad1 triple mutants can form microcolonies of approximately 300 cells. Expression of Nfo, the bacterial homologue of Apn1, suppresses the lethality. Turning off the expression of Nfo induces G(2)/M cell cycle arrest in an apn1 apn2 rad1 triple mutant. The activation of this checkpoint is RAD9 dependent and allows residual DNA repair. The Mus81/Mms4 complex was identified as one of these back-up repair activities. Furthermore, inactivation of Ntg1, Ntg2 and Ogg1 DNA N-glycosylase/AP lyases in the apn1 apn2 rad1 background delayed lethality, allowing the formation of minicolonies of approximately 10(5) cells. These results demonstrate that, under physiological conditions, endogenous DNA damage causes death in cells deficient in Apn1, Apn2 and Rad1/Rad10 proteins. We propose a model in which endogenous DNA abasic sites are converted into 3'-blocked single-strand breaks (SSBs) by DNA N-glycosylases/AP lyases. Therefore, we suggest that the essential and overlapping function of Apn1, Apn2, Rad1/Rad10 and Mus81/Mms4 is to repair 3'-blocked SSBs using their 3'-phosphodiesterase activity or their 3'-flap endonuclease activity, respectively.     

Apurinic and apyrimidinic DNA endonuclease of extremely thermophilic Thermothrix thiopara.

An endonuclease specific for apurinic, apyrimidinic (AP) sites in DNA was purified nearly to homogeneity from the extremely thermophilic bacterium Thermothrix thiopara. The enzyme has a molecular weight of approximately 26,000. It cleaves neither native nor UV- or gamma-irradiated DNAs and has no contaminating exonuclease or uracil-DNA glycosylase activities. The enzyme has no cofactor requirement and is not inhibited by EDTA or N'-ethylmaleimide. It shows maximal activity at 70 degrees C and a pH between 7.5 and 9.0. The Arrhenius activation energy of the reaction is 17 kJ/mol, and the apparent Km for AP sites is 38 nM. The rate of heat inactivation of the enzyme followed first-order kinetics, with a half-life of 10 min at 70 degrees C but about 150 min in the presence of 0.5 M ammonium sulfate or 0.5 mg of bovine serum albumin per ml at the same temperature. One cell of T. thiopara contains sufficient AP endonuclease activity for hydrolysis of about 10(6) phosphodiester bonds per h at 70 degrees C. An extract of these bacteria does not contain detectable Mg-dependent AP endonuclease activity, and the above-mentioned enzyme appears to be the main AP endonuclease of T. thiopara.     

Elaboration of cellular DNA breaks by hydroperoxides.

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.     

Induced thermotolerance to apoptosis in a human T lymphocyte cell line.

A brief exposure to elevated temperatures elicits, in all organisms, a transient state of increased heat resistance known as thermotolerance. The mechanism for this thermotolerant state is unknown primarily because it is not clear how mild hyperthermia leads to cell death. The realization that cell death can occur through an active process of self destruction, known as apoptosis, led us to consider whether thermotolerance provides protection against this mode of cell death. Apoptosis is a common and essential form of cell death that occurs under both physiological and pathological conditions. This mode of cell death requires the active participation of the dying cell and in this way differs mechanistically from the alternative mode of cell death, necrosis. Here we show that mild hyperthermia induces apoptosis in a human leukemic T cell line. This is evidenced by chromatin condensation, nuclear fragmentation and the cleavage of DNA into oligonucleosome size units. DNA fragmentation is a biochemical hallmark of apoptosis and requires the activation of an endogenous endonuclease. The extent of DNA fragmentation was proportional to the severity of heat stress for cells heated at 43 degrees C from 30 to 90 minutes. A brief conditioning heat treatment induced a resistance to apoptosis. This was evident as a resistance to DNA fragmentation and a reduction in the number of apoptotic cells after a heat challenge. Resistance to DNA fragmentation developed during a recovery period at 37 degrees C and was correlated with enhanced heat shock protein (hsp) synthesis. This heat-induced resistance to apoptosis suggests that thermotolerant cells have gained the capacity to prevent the onset of this pathway of self-destruction. An examination of this process in heated cells should provide new insights into the molecular basis of cellular thermotolerance.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. V. The correlation of DNA strand breaks and base damage to chromosomal aberrations and sister-chromatid exchanges induced by X-irradiation

The X-ray-sensitive Chinese hamster ovary (CHO) mutant cell lines xrs 5 and xrs 6 were used to study the relation between X-ray-induced DNA lesions and biological effects. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE) were determined in wild-type CHO-K1 as well as mutants xrs 5 and xrs 6 cells following X-irradiation under aerobic and anaerobic conditions. Furthermore, we used a newly developed immunochemical method (based on the binding of a monoclonal antibody to single-stranded DNA) to assay DNA single-strand breaks (SSBs) induced by gamma-rays in these CHO cells, after a repair time of up to 4 h. For all cell lines tested the frequency of X-ray-induced chromosomal aberrations was strongly increased after irradiation in air compared with hypoxic conditions. When compared to the wild-type line, the xrs mutants known to have a defect in repair of DNA double-strand breaks (DSBs) exhibited a markedly enhanced sensitivity to aerobic irradiation, and a high OER (oxygen enhancement ratio) of 2.8-3.5, compared with 1.8-2 in CHO-K1 cells. The induction of SCE by X-rays was relatively little affected in CHO-K1 irradiated in air compared with hypoxic conditions (OER = 0.8), and in xrs 5 (OER = 0.7). A dose-dependent increase in the frequency of SCEs was obtained in xrs 6 cells treated with X-rays in air, and a further increase by a factor of 2 was evident under hypoxic conditions (OER = 0.4). With the immunochemical assay of SSB following gamma-irradiation, no difference was found between wild-type and mutant strains in the number of SSBs induced. The observed rate of rejoining of SSBs was also the same for all cell lines studied.     

Effect of the DNA replication velocity on the response of Salmonella typhimurium to mild heating

Changes in the DNA replication velocity of Salmonella typhimurium following mild heat stress (52 degrees C) were studied independently of the major physiological parameter of growth rate, using thymine-requiring mutant strains derived from Salm. typhimurium LT2. The isolated mutant strains BM1 or BM2, grown either as batch or chemostat cultures, showed a greater sensitivity to 52 degrees C heat stress when grown on a minimal medium containing near-limiting concentrations of thymine, compared with growth in the presence of excess thymine. Radiolabelling experiments provided evidence for alterations in the velocity of DNA replication upon growth on different thymine concentrations, independent of the growth rate. Thus, replicating DNA was implicated as the major site of injury after mild moist heat stress.     

Purification and properties of 2-aminoadipate: 2-oxoglutarate aminotransferase from bovine kidney.

Escherichia coli endonuclease IV hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free deoxyribose. It also hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free 2',3'-unsaturated sugar produced by nicking 3' to an AP (apurinic or apyrimidinic) site by beta-elimination; this explains why the unproductive end produced by beta-elimination is converted by the enzyme into a 3'-OH end able to prime DNA synthesis. The action of E. coli endonuclease IV on an internal AP site is more complex: in a first step the C(3')-O-P bond 5' to the AP site is hydrolysed, but in a second step the 5'-terminal base-free deoxyribose 5'-phosphate is lost. This loss is due to a spontaneous beta-elimination reaction in which the enzyme plays no role. The extreme lability of the C(3')-O-P bond 3' to a 5'-terminal AP site contrasts with the relative stability of the same bond 3' to an internal AP site; in the absence of beta-elimination catalysts, at 37 degrees C the half-life of the former is about 2 h and that of the latter 200 h. The extreme lability of a 5'-terminal AP site means that, after nicking 5' to an AP site with an AP endonuclease, in principle no 5'----3' exonuclease is needed to excise the AP site: it falls off spontaneously. We have repaired DNA containing AP sites with an AP endonuclease (E. coli endonuclease IV or the chromatin AP endonuclease from rat liver), a DNA polymerase devoid of 5'----3' exonuclease activity (Klenow polymerase or rat liver DNA polymerase beta) and a DNA ligase. Catalysts of beta-elimination, such as spermine, can drastically shorten the already brief half-life of a 5'-terminal AP site; it is what very probably happens in the chromatin of eukaryotic cells. E. coli endonuclease IV also probably participates in the repair of strand breaks produced by ionizing radiations: as E. coli endonuclease VI/exonuclease III, it is a 3'-phosphoglycollatase and also a 3'-phosphatase. The 3'-phosphatase activity of E. coli endonuclease VI/exonuclease III and E. coli endonuclease IV can also be useful when the AP site has been excised by a beta delta-elimination reaction.     

Incomplete rejoining of DNA double-strand breaks in unstimulated normal human lymphocytes

We investigated the repair kinetics of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in unstimulated normal human peripheral blood lymphocytes (HPBL). SSBs and DSBs induced by gamma-irradiation (at 0 degree C) were assayed without radiolabel by alkaline and neutral filter elution, respectively. Incubation of irradiated cells at 37 degrees C for various lengths of time demonstrated that the percent DNA rejoined increased until it reached a plateau at approximately 60 min; this repair plateau underwent no substantial change when incubation continued for 20-24 h. The level of the plateau indicated how closely the elution profile of DNA from cells irradiated and incubated (experimental) resembled the elution profile of DNA from unirradiated cells (control). After 6 Gy and 60 min incubation, the alkaline elution profile of DNA from experimental cells from 5 donors was indistinguishable from that seen in DNA from control cells, suggesting that rejoining of SSBs was complete. In contrast after 100 Gy and 60 min incubation the neutral elution profile of DNA from cells from the same donors demonstrated that, compared to DNA from control cells, rejoining of DSBs was approximately two-thirds complete. In the range of 2-8 Gy, 85-104% of SSBs were rejoined after 60 min incubation; in the range of 30-120 Gy, 46-80% of DSBs were rejoined after 60 min incubation. These unexpected results stand in contrast to our previous studies with confluent normal human diploid fibroblasts (HDF), in which rejoining of both SSBs and DSBs was greater than 90% complete by 60 min repair incubation and 100% complete after 18-24 h.     

Microinjection of T4 endonuclease V produced by a synthetic denV gene stimulates unscheduled DNA synthesis in both xeroderma pigmentosum and normal cells

A structural gene for T4 endonuclease V was constructed by ligating synthetic oligonucleotides. The endonuclease V was overproduced in E. coli under control of the E. coli tryptophan promoter and purified to apparent homogeneity. The product had comparable DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease activities to the natural enzyme in vitro. When this endonuclease V was microinjected into the cytoplasm of xeroderma pigmentosum (XP) cells of complementation group A, B, C, D, F, G or H, unscheduled DNA synthesis (UDS) above the residual level was detected in all the cells at a dose of about 10(3) molecules following UV irradiation. The gain numbers of UDS in these XP cells increased with increase in the dose of enzyme and reached a plateau at the normal cell level on introduction of about 10(4) molecules. Introduction of more enzyme into either XP cells or normal human cells did not increase the grain number under regular labelling conditions (2.5 h, 37 degrees C). In normal mouse cells, introduction of the enzyme increased the grain number more than 4-fold under the same conditions during at least 8.5 h following UV irradiation. Furthermore, with a labelling time of 30 min, the enzyme more than doubled the grain number even in normal human cells.     

No change in DNA damage or repair of single- and double-strand breaks as human diploid fibroblasts age in vitro

Using the in vitro human diploid fibroblast model, we tested theories of aging which hypothesize that either accumulation of DNA damage or decreased DNA repair capacity is causally related to cellular senescence. Between population doubling level (PDL) 32 and 71, fetal lung-derived normal diploid human fibroblasts (IMR 90) were assayed for both DNA single-strand breaks (SSBs, spontaneous and induced by 6 Gy) and DNA double-strand breaks (DSBs, spontaneous and induced by 100 Gy). After gamma-irradiation cells were kept on ice unless undergoing repair incubation at 37 degrees C for 7.5-120 min or 18-24 h. To assay DNA strand breaks we used the filter elution technique in conjunction with a fluorometric determination of DNA which is not biased in favor of proliferating aging cells as are radioactive labelling methods. We found no change with in vitro age in the accumulation of spontaneous SSBs or DSBs, nor in the kinetics or completeness of DNA strand rejoining after gamma-irradiation. Cells at varying PDLs rejoined approx. 90% of SSBs and DSBs after 60 min repair incubation and 100% after 18-24 h repair incubation. We conclude that aging and senescence as measured by proliferative lifespan in IMR 90 cells are neither accompanied nor caused by accumulation of DNA strand breaks or by diminished capacity to rejoin gamma-radiation-induced SSBs or DSBs in DNA.     

Excision of 8-oxoguanine within clustered damage by the yeast OGG1 protein

Clustered damages are formed in DNA by ionising radiation and radiomimetic anticancer agents and are thought to be biologically severe. 7,8-dihydro-8-oxoguanine (8-oxoG), a major DNA damage resulting from oxidative attack, is highly mutagenic leading to a high level of G·C→T·A transversions if not previously excised by OGG1 DNA glycosylase/AP lyase proteins in eukaryotes. However, 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell. The ability of yeast OGG1 to excise 8-oxoG was determined when another type of damage [dihydrothymine, uracil, 8-oxoG, abasic (AP) site or various types of single-strand breaks (SSBs)] is present on the complementary strand 1, 3 or 5 bases 5′ or 3′ opposite to 8-oxoG. Base damages have little or no influence on the excision of 8-oxoG by yeast OGG1 (yOGG1) whereas an AP site has a strong inhibitory effect. Various types of SSBs, obtained using either oligonucleotides with 3′- and 5′-phosphate termini around a gap or through conversion of an AP site with either endonuclease III or human AP endonuclease 1, strongly inhibit excision of 8-oxoG by yOGG1. Therefore, this large inhibitory effect of an AP site or a SSB may minimise the probability of formation of a double-strand break in the processing of 8-oxoG within clustered damages.     

Excision repair of pyrimidine dimers from simian virus 40 minichromosomes in vitro

The ability of DNA repair enzymes to carry out excision repair of pyrimidine dimers in SV40 minichromosomes irradiated with 16 to 64 J/m2 of UV light was examined. Half of the dimers were substrate for the DNA glycosylase activity of phage T4 UV endonuclease immediately after irradiation, but this limit decreased to 27% after 2 h at 0 degrees C. Moreover, the apyrimidinic (AP) endonuclease activity of the enzyme did not incise all of the AP sites created by glycosylase activity, although all AP sites were substrate for HeLa AP endonuclease II. The initial rate of the glycosylase was 40% that upon DNA. After incision by the T4 enzyme, excision was mediated by HeLa DNase V (acting with an exonuclease present in the chromatin preparation). Under physiological salt conditions, excision did not proceed appreciably beyond the damaged nucleotides in DNA or chromatin. With chromatin, about 70% of the accessible dimers were removed, but at a rate slower than for DNA. Finally, HeLa DNA polymerase beta was able to fill the short gaps created after dimer excision, and these patches were sealed by T4 DNA ligase. Overall, roughly 30% of the sites incised by the endonuclease were ultimately sealed by the ligase. The resistance of some sites was due to interference with the ligase by the chromatin structure, as only 30-40% of the nicks created in chromatin by pancreatic DNase could be sealed by T4 or HeLa DNA ligases. The overall excision repair process did not detectably disrupt the chromatin structure, since the repair label was recovered in Form I DNA present in 75 S condensed minichromosomes. Although other factors might stimulate the rate of this repair process, it appears that the enzymes utilized could carry out excision repair of chromatin to a limit near that observed at the initial rate in mammalian cells in vivo.     

Inhibitory Effects of Trientine, a Copper-Chelating Agent, on Induction of DNA Strand Breaks in Kidney Cells of Long-Evans Cinnamon (LEC) Rats.

The effects of treatment with trientine, a specific copper-chelating agent, on the accumulation of copper and induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. Copper accumulated in the kidneys of LEC rats in an age-dependent manner from 12 to 18 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, renal copper contents did not increase and were maintained at the same levels as those in 4-week-old LEC rats. Estimation of the amounts of DNA single-strand breaks (SSBs) by comet assay showed that SSBs of DNA were induced in a substantial population of LEC rat renal cortex cells around 12 weeks of age and that the amounts of SSBs increased in an age-dependent manner from 12 to 18 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, the observed number of cells with DNA damage decreased, suggesting that induction of SSBs of DNA was inhibited and/or SSBs were repaired during the period of treatment with trientine. The results show that SSBs of DNA in LEC rat kidney cells are induced prior to occurrence of clinical signs of hepatic injury and that treatment of LEC rats with trientine decreases the number of DNA strand breaks.     

T cell receptor-mediated DNA fragmentation and cell death in T cell hybridomas

Activation of Ag-specific T cell hybridomas with a high density of immobilized anti-CD3 antibody resulted in not only secretion of IL-2 but also cell death of up to 60 to 80% in selected hybridomas after 14 h. Similar results were obtained with V beta 8+ T cell hybridomas stimulated with cross-linked F23.1 antibody. In these activated hybridomas, we found that DNA was fragmented into 180- to 200-bp multiples. DNA fragmentation was not observed when T cells were maintained after killing with anti-Thy-1 plus C or with heat treatment at 45 degrees C, nor when T cells were incubated with fixed anti-CD4 antibody. Furthermore, fragmentation was detectable at 6 h after incubation when almost all of the cells were still viable as evaluated by trypan blue dye exclusion test. Cell death was prevented by addition of EGTA, cycloheximide, actinomycin D, and zinc, suggesting that the induction of cell death requires Ca2+ influx, newly synthesized protein(s), and involvement of endonuclease.     

Drosophila apurinic/apyrimidinic DNA endonucleases. Characterization of mechanism of action and demonstration of a novel type of enzyme activity

Two species of apurinic/apyrimidinic (AP) endonuclease have been purified approximately 400-fold from extracts of Drosophila embryos. AP endonuclease I, which flows through phosphocellulose columns, has an apparent subunit molecular weight of 66,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas AP endonuclease II, which is retained by phosphocellulose, has a subunit molecular weight of 63,000. The molecular weight determinations were made possible in part by the finding that both Drosophila enzymes, along with Escherichia coli endonuclease IV, cross-react with an antibody prepared toward a human AP endonuclease (Kane, C. M., and Linn, S. (1981) J. Biol. Chem. 256, 3405-3414). The nature of phosphodiester bond breaks produced by the two partially purified AP endonucleases from Drosophila have been investigated. Nicks introduced into partially depurinated PM2 DNA by Drosophila AP endonuclease I did not support DNA synthesis by E. coli DNA polymerase I, whereas nicks created by AP endonuclease II were able to support DNA synthesis, but at a rate far less than that observed for nicks introduced by E. coli endonuclease IV. The priming activity of DNA incised by either of the Drosophila enzymes can be enhanced, however, by an additional incubation with E. coli endonuclease IV, which is known to cleave depurinated DNA on the 5'-side of an apurinic site. These results suggest that the Drosophila enzymes cleave depurinated DNA on the 3'-side of the apurinic site. This suggestion was strengthened by the observation that the combined action of AP endonuclease II and E. coli endonuclease IV resulted in the removal of [32P]dAMP from partially depyrimidinated [dAMP-5'-32P,uracil-3H]poly(dA-dT). Taken together, these results propose that Drosophila AP endonuclease II produces 3'-deoxyribose and 5'-phosphomonoester nucleotide termini. Conversely, the absolute inability to detect priming activity for DNA cleaved by AP endonuclease I alone suggested a different mechanism, possibly the formation of a deoxyribose-3'-phosphate terminus. When apurinic DNA cleaved by AP endonuclease I was subsequently treated with bacterial alkaline phosphatase, DNA synthesis was now detected at levels similar to that observed for AP endonuclease II alone. Additionally, DNA nicked by AP endonuclease I was susceptible to 5'-end labeling by polynucleotide T4 kinase without prior phosphomonoesterase treatment. These results suggest that AP endonuclease I forms deoxyribose 3'-phosphate and 5'-OH termini upon cleaving depurinated DNA.     

Early Detection of DNA Strand Breaks in the Brain After Transient Focal Ischemia: Implications for the Role of DNA Damage in Apoptosis and Neuronal Cell Death

Abstract: Using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single-strand breaks (SSBs) detected by PANT were present in neurons after as little as 1 min of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double-strand breaks (DSBs) detected by TUNEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB-containing cells was greater than that of DSB-containing cells at all time points tested. SSB-containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16–72 h of reperfusion, many SSB- and DSB-containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis.     

In situ detection of neuronal DNA strand breaks using the Klenow fragment of DNA polymerase I reveals different mechanisms of neuron death after global cerebral ischemia

Ischemic cell injury in the brain may involve a cascade of programmed cell death. DNA damage may be either a catalyst or a consequence of this cascade. Therefore, the induction of DNA strand breaks in the rat brain following transient global ischemia was examined using (a) the Klenow labeling assay, identifying DNA single-strand breaks (SSBs) or double-strand breaks (DSBs) with protruding 5' termini, and (b) terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), detecting DNA DSBs with protruding 3' termini or blunt ends. Klenow-positive staining occurred within 2 h of reperfusion and increased with increasing durations of reperfusion. DNA damage detected with the Klenow labeling assay preceded that of TUNEL expression in the caudate putamen, reticular thalamus, thalamus, and cortex. However, in CA1, DNA SSBs were not detected until 72 h of reperfusion and occurred simultaneously with DSBs. Thus, the time course and fragmentation characteristics of DNA damage differ between the hippocampal CA1 and other selectively vulnerable brain regions. This distinct pattern suggests that the delayed neuronal death in CA1 following transient global ischemia may occur via an apoptotic mechanism different from that of other brain regions.     

Effect of apurinic/apyrimidinic endonucleases and polyamines on DNA treated with bleomycin and neocarzinostatin: specific formation and cleavage of closely opposed lesions in complementary strands

Bleomycin and neocarzinostatin induce modified apurinic/apyrimidinic (AP) sites by oxidation of the sugar moiety in DNA. In order to quantitatively assess the susceptibility of these lesions to repair endonucleases, drug-treated 3H-labeled colE1 DNA was mixed with 14C-labeled heat-depurinated DNA, and endonuclease-susceptible sites in the mixture were titrated with various AP endonucleases or with polyamines. Single- and double-strand breaks were quantitated by determining the fractions of supercoiled, nicked circular, and linear molecules. Exonuclease III and endonucleases III and IV of Escherichia coli, as well as putrescine, produced a nearly 2-fold increase in single-strand breaks in bleomycin-treated DNA, indicating cleavage of drug-induced AP sites. The bleomycin-induced AP sites were comparable to heat-induced sites in their sensitivity to E. coli endonucleases III and IV but were cleaved by exonuclease III only at high concentrations. Bleomycin-induced AP sites were much more sensitive to cleavage by putrescine than heat-induced sites. Treatment with putrescine or very high concentrations of endonuclease III also increased the number of double-strand breaks in bleomycin-treated DNA, suggesting a minor class of lesion consisting of an AP site accompanied by a closely opposed break in the complementary strand. These complex lesions were resistant to cleavage by endonuclease IV. However, when colE1 DNA was treated with neocarzinostatin, subsequent treatment with putrescine, endonuclease IV, or very high concentrations of endonuclease III produced a dramatic increase in double-strand breaks but no detectable increase in single-strand breaks. These results suggest that virtually all neocarzinostatin-induced AP sites are accompanied by a closely opposed strand break.(ABSTRACT TRUNCATED AT 250 WORDS)