Application of Biotin, Digoxigenin or Fluorescein Conjugated Deoxynucleotides to Label DNA Strand Breaks for Analysis of Cell Proliferation and Apoptosis Using Flow Cytometry

A flow cytometric method has recently been developed using biotinylated dUTP (b-dUTP) in a reaction catalyzed by terminal deozynucleotidyl transferase (TdT) to identify the endonuclease-induced DNA strand breaks occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the present study, we compared this method with one using digoxigenin-conjugated dUTP (d-dUTP) to label apoptotic cells. The discrimination of apoptotic from nonapoptotic cells was similar when incorporation of d-dUTP was compared with b-dUTP. Both techniques resulted in a 20-30 fold increase in staining of apoptotic over nonapoptotic cells although somewhat less background fluorescence was observed with the d-dUTP. Direct labeling with fluo-resceinated dUTP (f-dUTP) was less sensitive in detecting DNA strand breaks, but had the advantage of simplicity. The principle of labeling DNA strand breaks using TdT was also employed to identify DNA replicating cells. To this end, the cells were incubated in the presence of BrdUrd, then exposed to UV light to selectively photolyse DNA containing the incorporated BrdUrd. DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This approach is an alternative to immunocytochemical detection of BrdUrd incorporation, but unlike the latter does not require prior DNA denaturation, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replication of less than 1% of the cell's genome. The discrimination between apoptotic vs. BrdUrd incorporating-cells is based on different extractability of DNA following cell fixation. This method can be applied to analyze both cell proliferation (DNA replication) and death (by apoptosis) in a single measurement.     

Application of Biotin,Digoxigenin or Fluorescein Conjugated Deoxynucleotides to Label DNA Strand Breaks for Analysis of Cell Proliferation and Apoptosis Using Flow Cytometry

A flow cytometric method has recently been developed using biotinylated dUTP (b-dUTP) in a reaction catalyzed by terminal deozynucleotidyl transferase (TdT) to identify the endonuclease-induced DNA strand breaks occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the present study, we compared this method with one using digoxigenin-conjugated dUTP (d-dUTP) to label apoptotic cells. The discrimination of apoptotic from nonapoptotic cells was similar when incorporation of d-dUTP was compared with b-dUTP. Both techniques resulted in a 20–30 fold increase in staining of apoptotic over nonapoptotic cells although somewhat less background fluorescence was observed with the d-dUTP. Direct labeling with fluo-resceinated dUTP (f-dUTP) was less sensitive in detecting DNA strand breaks, but had the advantage of simplicity. The principle of labeling DNA strand breaks using TdT was also employed to identify DNA replicating cells. To this end, the cells were incubated in the presence of BrdUrd, then exposed to UV light to selectively photolyse DNA containing the incorporated BrdUrd. DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This approach is an alternative to immunocytochemical detection of BrdUrd incorporation, but unlike the latter does not require prior DNA denaturation, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replication of less than 1% of the cell's genome. The discrimination between apoptotic vs. BrdUrd incorporating-cells is based on different extractability of DNA following cell fixation. This method can be applied to analyze both cell proliferation (DNA replication) and death (by apoptosis) in a single measurement.     

Sperm chromatin damage associated with male smoking

Cigarette smoke is a rich source of mutagens and carcinogens; thus, we have investigated the effects of male smoking on the DNA of human sperm. This was performed using the sperm chromatin structure assay (SCSA), which measures the sensitivity of sperm DNA to acid induced denaturation, and the terminal deoxynucleotidyl transferase assay (TdTA), which measures DNA strand breaks by addition of the biotinylated nucleotide dUTP to 3'-OH ends of DNA, sites of DNA breakage, using the enzyme terminal deoxynucleotidyl transferase. Sperm from subjects who smoked were significantly more sensitive to acid induced denaturation than non-smokers (P<0.02) and possessed higher levels of DNA strand breaks (P<0.05). We hypothesise that smoking damages the chromatin structure and produces endogenous DNA strand breaks in human sperm. These changes may result in sperm DNA mutations, that predispose offspring to greater risk of malformations, cancer and genetic diseases.     

Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis

Apoptotic cells in rat thymus were labeled in situ in paraffin-embedded and frozen tissue sections by ligation of double-stranded DNA fragments containing digoxigenin or Texas red. Two forms of double-stranded DNA fragments were prepared using the polymerase chain reaction: one was synthesized using Taq polymerase, which yields products with single- base 3' overhangs, and one using Pfu polymerase, which produces blunt- ended products. Both types of fragment could be ligated to apoptotic nuclei in thymus, indicating the presence in such nuclei of DNA double- strand breaks with single-base 3' overhangs as well as blunt ends. However, in nuclei with DNA damage resulting from a variety of nonapoptotic processes (necrosis, in vitro autolysis, peroxide damage, and heating) single-base 3' overhangs were either nondetectable or present at much lower concentrations than in apoptotic cells. Blunt DNA ends were present in such tissues, but at lower concentrations than in apoptotic cells. In contrast, in all of these forms of DNA damage, nuclei contained abundant 3'-hydroxyls accessible to labeling with terminal deoxynucleotidyl transferase. Thus, although single-base 3' overhangs and blunt ends are present in apoptotic nuclei, the specificity of the in situ ligation of 3'-overhang fragments to apoptotic nuclei indicates that apoptotic cells labeled in this way can readily be distinguished from cells with nonapoptotic DNA damage. These data are consistent with the involvement of an endonuclease similar to DNase I in apoptosis, which is predicted to leave short 3' overhangs as well as blunt ends in digestion of chromatin.     

Comparison of methods based on annexin-V binding, DNA content or TUNEL for evaluating cell death in HL-60 and adherent MCF-7 cells

HL-60 and MCF-7 cells were treated with 0.15 μ M camptothecin (CPT) or with the solvent dimethylsulfoxide (DMSO) for the controls, for 2, 3 and 4 h or for 24, 48 and 72 h, respectively. The apoptotic index (AI) was then evaluated in parallel by the following flow cytometric methods: (1) double staining of unfixed cells with fluoresceinated annexin V and propidium iodide (PI), this after detachment by trypsinization in the case of MCF-7 cultures; (2) prefixation in 70% ethanol, extraction of degraded, low molecular weight DNA with 0.2 M phosphatecitrate buffer and analysis of the DNA content stained with PI; (3) TUNEL, i.e. labelling of DNA strand breaks with biotin-dUTP, followed by staining with streptavidin-fluorescein and counterstaining with PI. In HL-60 cells, the three methods gave similar results for the AI (3–4% in the controls and at 2 h of CPT treatment, and 35–43% at 3 and 4 h after CPT). This indicates that CPT-induced membrane alteration and DNA fragmentation occurred concomitantly in those cells. For MCF-7 cells, CPT-induced apoptosis developed more slowly, the AI, whether based on annexin V or on DNA content, remained unchanged at 24 h, then was increasing to 8% at 48 h and to 25% at 72 h of treatment. In these cells, the TUNEL index did not increase prior to 72 h, and the increase was minor (up to 9% vs. 2–3% in the controls) at 72 h of the treatment. This indicates that in MCF-7 cells DNA strand breaks cannot be effectively labelled, which may be due to inaccessibility of 3-OH ends in the breaks to exogenous terminal deoxynucleotidyl transferase. The mechanism of endonucleolytic DNA fragmentation thus may be different, depending on the cell type.     

Analysis of apoptosis by cytometry using TUNEL assay

Activation of endonucleases that cleave chromosomal DNA preferentially at internucleosomal sections is a hallmark of apoptosis. DNA fragmentation revealed by the presence of a multitude of DNA strand breaks, therefore, is considered to be the gold standard for identification apoptotic cells. Several variants of the methodology that is based on fluorochrome-labeling of 3'-OH termini of DNA strand breaks in situ with the use of exogenous terminal deoxynucleotidyl transferase (TdT), commonly defined as the TUNEL assay, have been developed by us. This Chapter describes the variant based on strand breaks labeling with Br-dUTP that is subsequently detected immunocytochemically with Br-dUAb. Compared with other TUNEL variants the Br-dU-labeling assay offers the greatest sensitivity in detecting DNA breaks. Described also are modifications of the protocol that allow one to use other than Br-dUTP fluorochrome-tagged deoxynucleotides to label DNA breaks. Concurrent staining of DNA with propidium or 4',6-diamidino-2-phenylindole (DAPI) and multiparameter analysis of cells by flow- or laser scanning cytometry enables one to correlate induction of apoptosis with the cell cycle phase.     

Early nuclear events in lymphocyte proliferation : The role of DNA strand break repair and ADP ribosylation

The previously reported extensive DNA strand breakage in resting murine splenic lymphocytes is not an artifact of the extraction or assay procedure. The benzamide inhibitors of poly(ADP ribose) synthetase (pADPRS), such as 5-methoxybenzamide (MBA), had been shown to block the strand break repair occurring within 2 h of activation of splenic lymphocytes by the mitogen concanavalin A (conA); the inhibitors also blocked early events in proliferation, such as blast formation, as well as entry into S phase. Inhibitors of pADPRS blocked lymphocyte proliferation by inhibiting the activity of this enzyme, rather than by non-specific effects. Aphidicolin, an inhibitor of alpha-polymerase, also prevented DNA strand break repair in conA-stimulated cells but, unlike MBA, did not prevent blast formation. DNA strand breaks accumulated in the presence of MBA at the same linear rate (300-400/h) in both resting and conA-treated cells. We and others had hypothesized that this accumulation was due to a continuous production of strand breaks in lymphocytes, leading to their accumulation in presence of repair inhibitors. However, incubation of the cells with aphidicolin at concentrations that inhibited repair did not result in any increase in strand breaks. The hypothesis of continuous cycling of breaks is incorrect; accumulation of breaks was due to some indirect effect of MBA, such as a possible disinhibition of an ADP-ribosylation-sensitive endonuclease described in other cell types. All of the early stages of lymphocyte proliferation, including blast transformation (but not DNA synthesis) require ADP ribosylation. Repair of DNA strand breaks is not a precondition for blast formation, though experiments involving the combined effects of MBA and aphidicolin showed that repair of the breaks is essential in order for the cells to replicate their DNA. Our data are consistent with a model suggesting that DNA strand breaks introduced into differentiated cells act as an additional safety-catch mechanism that restrains them from replicating their genetic material but not from undergoing the early stages of proliferation.     

Degradation of apoptotic cells and fragments in HL-60 suspension cultures after induction of apoptosis by camptothecin and ethanol

Early indicators of apoptosis in mammalian cells are membrane potential breakdown (loss) in mitochondria (MPLM), chromatin condensation, DNA degradation, and phosphatidylserine exposure (PSE) on the outside plasma membrane. One aim of the present study was to determine the kinetics of these characteristics. These changes were measured by flow cytometry using the following methods: membrane potential of mitochondria was analysed using Mito Tracker Green and Red, PSE was analysed using annexin-V-FITC staining simultaneously with propidium iodide (PI) to detect membrane permeability; chromatin condensation was measured using the acid denaturation Acridine Orange (AO) method; DNA degradation was studied by the sub G1 method and the terminal transferase dUTP nick end-labelling (TUNEL) assay (labelling of strand breaks). HL-60 cells were induced to apoptosis by 3% ethanol and 1.5 microM camptothecin (CAM) and the kinetics of the apoptotic cells were measured. The same kinetics were found for chromatin condensation and DNA degradation indicating that these changes appeared at approximately the same time after induction. The MPLM and PSE kinetics showed a considerably later increase indicating that MPLM occurred downstream of DNA degradation and that plasma membrane changes occurred downstream of MPLM. The main aim of the study was to follow the fate of apoptotic cells after the appearance of the initial characteristics. The lifetime of apoptotic cells was studied by chase experiments. The inducing drug was removed after 4 h treatment and the disappearance of apoptoses recorded. An exponential decay was measured with a half life (T(1/2)) of 17.8 h. As a corollary from these experiments, camptothecin was found to induce apoptosis also in G1 and G2 phase cells, however, it took much longer to occur than in S phase cells. Using labelling of the plasma membrane with a fluorescent cell membrane linker, it was possible to show that the majority of apoptotic bodies as well as condensed apoptotic cells contain DNA and membrane. The degradation of these apoptotic bodies follows similar kinetics as those of the condensed apoptotic cells. The membrane remained considerably stable, there was no further loss in the next 7 days, after the first day when the apoptotic characteristics develop. It is concluded that the apoptosis programme is completed within a day and no further steps follow.     

Histochemical Demonstration of DNA Double Strand Breaks by in Situ 3'-tailing Reaction in Apoptotic Endometrium

A new histochemical technique, called in situ 3'-tailing reaction (ISTR), to detect DNA double strand breaks (DSB) was developed and applied to tissue sections of apoptotic endometrium. To demonstrate DSB, biotin-labeled and unlabeled dATPs with terminal deoxynucleotidyl transferase (TdT) were added to the many 3-hydroxyl termini of DNA fragments generated in the apoptotic cells. For an efficient 3'-end labeling, it was necessary to treat the sections with λ-exonuclease (λEx) prior to the TdT reaction to generate 3'-protruding ends. The λEx-TdT reaction specifically labeled nuclear fragments in the apoptotic cells in paraformaldehyde fixed frozen sections. In paraffin sections, pretreatment with proteinase K was effective for 3'-tailing reaction. ISTR should be a useful tool for detecting dying cells in both physiological and pathological states.     

Nonhomologous DNA end rejoining in chromosomal aberration formation

The role of recombination of nonhomologous DNA ends in chromosomal aberration formation was investigated in Chinese hamster ovary cells. Restriction enzymes that produce blunt, 3′ overhanging, or 5′ overhanging DNA double-strand breaks were electroporated into cells in various combinations, and chromosomal aberrations were analyzed at metaphase. For all enzyme combinations tested, there was a significant increase in the frequency of aberrations whose formation requires two breaks in the DNA over the sum obtained when each of the enzymes was tested separately and the aberration frequencies were totaled. No such pattern existed for terminal deletions, which presumably require only one DNA break. The extent of interaction did not depend on the homology in the overhanging sequences or on the combination of ends used, although the largest effect was seen with a combination of two blunt ends. This study shows that nonhomologous DNA double-strand breaks can interact to increase chromosomal aberration formation significantly.     

Characterization of DNA damage in yeast apoptosis induced by hydrogen peroxide, acetic acid, and hyperosmotic shock

Saccharomyces cerevisiae has been reported to die, under certain conditions, from programmed cell death with apoptotic markers. One of the most important markers is chromosomal DNA fragmentation as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. We found TUNEL staining in S. cerevisiae to be a consequence of both single- and double-strand DNA breaks, whereas in situ ligation specifically stained double-strand DNA breaks. Cells treated with hydrogen peroxide or acetic acid staining positively for TUNEL assay stained negatively for in situ ligation, indicating that DNA damage in both cases mainly consists of single-strand DNA breaks. Pulsed field gel electrophoresis of chromosomal DNA from cells dying from hydrogen peroxide, acetic acid, or hyperosmotic shock revealed DNA breakdown into fragments of several hundred kilobases, consistent with the higher order chromatin degradation preceding DNA laddering in apoptotic mammalian cells. DNA fragmentation was associated with death by treatment with 10 mM hydrogen peroxide but not 150 mM and was absent if cells were fixed with formaldehyde to eliminate enzyme activity before hydrogen peroxide treatment. These observations are consistent with a process that, like mammalian apoptosis, is enzyme dependent, degrades chromosomal DNA, and is activated only at low intensity of death stimuli.     

DNA strand breakage during physiological apoptosis of the embryonic chick lens: Free 3' OH end single strand breaks do not accumulate even in the presence of a cation-independent deoxyribonuclease

Epithelial cells from the lens equator differentiate into elongated fiber cells. In the final steps of differentiation, the chromatin appears quite condensed and chromatin breakdown into nucleosmes occurs. DNA breaks due to an endodeoxyribonuclease activity corresponding to at least two polypeptides of 30 and 40 kDa have been identified. To identify the nature and the developmental appearance of initial breaks, nick translation reaction was followed both biochemically and in situ in fiber and epithelial cells from chick embryonic lenses. There is no accumulation of single-strand breaks (SSB) with 3'OH ends in lens fiber cells during embryonic development. Such damage can be increased in these cells by treatment with DNAase I indicating the absence of an inhibitor of the nick translation reaction in fiber cells. However, there are indications of the presence of DNA breaks with blocked termini when the phosphatase activity of nuclease P1 is used. The presence of breaks is also indicated by the large amounts of (ADP-ribose)_n found in lens fibers particularly at 11 days of embryonic development (E11) as ADP-ribosyl transferase binds to and is activated by DNA strand breaks. Incubation of lens cells in vitro, which causes nucleosomal fragmentation only in fiber cells, produces SSB with 3'OH ends in both epithelia and fibers. Incubation for short periods, observed in experiments in situ, induces SSB first in the central fiber nuclei, which are late in differentiation. This may indicate that these SSB play a physiological role. Long incubations produce larger numbers of SSB in epithelia than fibers. The SSB in the fibers may have been converted into double-strand breaks (D SB), seen as nucleosomal fragments, and therefore no longer act as substrates for nick translation. The nuclease activity responsible for SSB production is independent of divalent cations and could be implicated in lens terminal differentiation. © 1994 Wiley-Liss, Inc.     

Mechanism of protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide- and menadione-induced DNA single strand breaks in Caco-2 cells

Protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide (tert-BOOH)- and menadione-induced DNA single strand breaks was investigated in Caco-2 cells. Both tert-BOOH and menadione induced DNA single strand breaks in a concentration-dependent manner. Pre-incubation of Caco-2 cells with either quercetin or rutin for 24 h significantly decreased the formation of DNA single strand breaks evoked by tert-BOOH (P <.05). Iron chelators, 1,10-phenanthroline (o-Phen) and deferoxamine mesylate (DFO), also protected against tert-BOOH-induced DNA damage, whereas butylated hydroxytoluene (BHT) had no effect. Quercetin, and not rutin, decreased the extent of menadione-induced DNA single strand breaks. DFO and BHT, and not o-Phen, protected against menadione-induced DNA strand break formation (P <.05). From the results of this study, iron ions were involved in tert-BOOH-induced DNA single strand break formation in Caco-2 cells, whereas DNA damage evoked by menadione was far more complex. We demonstrated that the flavonoids, quercetin and rutin, protected against tert-BOOH-induced DNA strand breaks by way of their metal ion chelating mechanism. However, quercetin, and not rutin, protected against menadione-induced DNA single strand breaks by acting as both a metal chelator and radical scavenger.     

Laser scanning cytometer (LSC) analysis of fraction of labelled mitoses (FLM)

Abstract. In this report we describe the successful application of a novel microscope-based multiparameter laser scanning cytometer (LSC) to measure duration of different phases of cell cycle in HL-60 human leukaemic cell lines by the fraction of labelled mitoses (FLM) method. Exponentially growing cells were harvested after various time intervals following pulse-labelling with 5'-bromo-2'-deoxyuridine (BrdUrd), cytocentrifuged, fixed in ethanol, and then exposed to UV light to induce DNA strand breaks at the sites of incorporated BrdUrd. The 3'OH termini of the photolytically generated DNA strand breaks were labelled with BrdUTP in the reaction catalysed by exogenous terminal deoxynucleotidyl transferase (TdT), followed by FITC-labelled BrdUrd antibodies. DNA was counterstained with propidium iodide (PI). Due to differences in chromatin structure between the interphase and mitotic cells, the LSC identified the latter by virtue of their higher red (PI) fluorescence intensity values among all pixels over the measured cell. To confirm that the cells selected were indeed cells in mitosis, predominantly in metaphase, the recorded X-Y coordinates of selected cells were used to re-position the cell for their visual examination. From the time lapse analysis of percentage BrdUrd-labelled cells progressing through mitosis it was possible to calculate the duration of individual phases of the cell cycle. The duration of S (T_s) and G₂+ M (T_G2+M) was 8 and 3 h, respectively, and the minimal duration of G₂ (T_G2) was 2 h. The cell cycle time (T_c) estimated for the cohort of the most rapidly progressing cells was 13 h. The ability to automatically and rapidly discriminate mitotic cells combined with the possibility of their subsequent identification by image analysis makes LSC the instrument of choice for the FLM analysis.     

Synergistic decrease of DNA single-strand break repair rates in mouse neural cells lacking both Tdp1 and aprataxin

Ataxia oculomotor apraxia-1 (AOA1) is an autosomal recessive neurodegenerative disease that results from mutations of aprataxin (APTX). APTX associates with the DNA single- and double-strand break repair machinery and is able to remove AMP from 5′-termini at DNA strand breaks in vitro. However, attempts to establish a DNA strand break repair defect in APTX-defective cells have proved conflicting and unclear. We reasoned that this may reflect that DNA strand breaks with 5′-AMP represent only a minor subset of breaks induced in cells, and/or the availability of alternative mechanisms for removing AMP from 5′-termini. Here, we have attempted to increase the dependency of chromosomal single- and double-strand break repair on aprataxin activity by slowing the rate of repair of 3′-termini in aprataxin-defective neural cells, thereby increasing the likelihood that the 5′-termini at such breaks become adenylated and/or block alternative repair mechanisms. To do this, we generated a mouse model in which APTX is deleted together with tyrosyl DNA phosphodiesterase (TDP1), an enzyme that repairs 3′-termini at a subset of single-strand breaks (SSBs), including those with 3′-topoisomerase-1 (Top1) peptide. Notably, the global rate of repair of oxidative and alkylation-induced SSBs was significantly slower in Tdp1^?/?/Aptx^?/? double knockout quiescent mouse astrocytes compared with Tdp1^?/? or Aptx^?/? single knockouts. In contrast, camptothecin-induced Top1-SSBs accumulated to similar levels in Tdp1^?/? and Tdp1^?/?/Aptx^?/? double knockout astrocytes. Finally, we failed to identify a measurable defect in double-strand break repair in Tdp1^?/?, Aptx^?/? or Tdp1^?/?/Aptx^?/? astrocytes. These data provide direct evidence for a requirement for aprataxin during chromosomal single-strand break repair in primary neural cells lacking Tdp1.     

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.     

Defective repair of DNA single- and double-strand breaks in the bleomycin- and X-ray-sensitive Chinese hamster ovary cell mutant, BLM-2

Using filter elution techniques, we have measured the level of induced single- and double-strand DNA breaks and the rate of strand break rejoining following exposure of two Chinese hamster ovary (CHO) cell mutants to bleomycin or neocarzinostatin. These mutants, designated BLM-1 and BLM-2, were isolated on the basis of hypersensitivity to bleomycin and are cross-sensitive to a range of other free radical-generating agents, but exhibit enhanced resistance to neocarzinostatin. A 1-h exposure to equimolar doses of bleomycin induces a similar level of DNA strand breaks in parental CHO-K1 and mutant BLM-1 cells, but a consistently higher level is accumulated by BLM-2 cells. The rate of rejoining of bleomycin-induced single- and double-strand DNA breaks is slower in BLM-2 cells than in CHO-K1 cells. BLM-1 cells show normal strand break repair kinetics. The level of single- and double-strand breaks induced by neocarzinostatin is lower in both BLM-1 and BLM-2 cells than in CHO-K1 cells. The rate of repair of neocarzinostatin-induced strand breaks is normal in BLM-1 cells but retarded somewhat in BLM-2 cells. Thus, there is a correlation between the level of drug-induced DNA damage in BLM-2 cells and the bleomycin-sensitive, neocarzinostatin resistant phenotype of this mutant. Strand breaks induced by both of these agents are also repaired with reduced efficiency by BLM-2 cells. The neocarzinostatin resistance of BLM-1 cells appears to be a consequence of a reduced accumulation of DNA damage. However, the bleomycin-sensitive phenotype of BLM-1 cells does not apparently correlate with any alteration in DNA strand break induction or repair, as analysed by filter elution techniques, suggesting an alternative mechanism of cell killing.     

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.     

The actions of Neurospora endo-exonuclease on double strand DNAs

Neurospora crassa endo-exonuclease, an enzyme implicated in recombinational DNA repair, was found previously to have a distributive endonuclease activity with a high specificity for single strand DNA and a highly processive exonuclease activity. The activities of endo-exonuclease on double strand DNA substrates have been further explored. Endo-exonuclease was shown to have a low bona fide endonuclease activity with completely relaxed covalently closed circular DNA and made site-specific breaks in linear double strand DNA at a low frequency while simultaneously generating a relatively high level of single strand breaks (nicks) in the DNA. Sequencing at nicks induced by endo-exonuclease in pBR322 restriction fragments showed that the highest frequency of nicking occurred at the mid-points of two sites with the common sequence, p-AGCACT-OH. In addition, sequencing revealed less frequent nicking at identical or homologous hexanucleotide sequences in all other 54 cases examined where these sequences either straddled the break site itself or were within a few nucleotides on either side of the break site. The exonucleolytic action of endo-exonuclease on linear DNA showed about 100-fold preference for acting in the 5' to 3' direction. Removal of the 5'-terminal phosphates substantially reduced this activity, internal nicking, and the ability of endo-exonuclease to generate site-specific double strand breaks. On the other hand, nicking of the dephosphorylated double strand DNA with pancreatic DNase I stimulated the exonuclease activity by almost 5-fold, but no stimulation was observed when the DNA was nicked by Micrococcal nuclease. Thus, 5'-p termini either at double strand ends or at nicks in double strand DNA are entry points to the duplex from which endo-exonuclease diffuses linearly or "tracks" in the 5' to 3' direction to initiate its major endo- and exonucleolytic actions. The results are interpreted to show how it is possible for endo-exonuclease to generate single strand DNA for switching into a homologous duplex either at a nick or while remaining bound at a double strand break in the DNA. Such mechanisms are consistent with current models for recombinational double strand break repair in eukaryotes.     

Structure of bleomycin-induced DNA double-strand breaks: predominance of blunt ends and single-base 5' extensions

In order to examine the structure of bleomycin-induced DNA double-strand breaks, defined-sequence DNA was labeled in each strand at a single restriction site and treated with bleomycin. Various double-stranded fragments resulting from bleomycin-induced double-strand breaks were isolated, denatured, and run on sequencing gels to determine the sites of cleavage in each strand. For virtually every double-strand break, the cleavage site in one strand was a pyrimidine in a G-Py sequence, reflecting a specificity similar to that of bleomycin-induced single-strand cleavage. However, the cleavage site in the complementary strand was seldom a G-Py sequence, and was usually a site where single-strand cleavage was infrequent. When the sequence at the double-strand break was G-Py-Py', the break at Py was usually accompanied by a break at the base directly opposite Py, resulting in blunt ends. When the sequence was G-Py-Pu, the break at Py was usually accompanied by a break at the base opposite Pu, resulting in single-base 5' extensions. Double-strand breaks with 3' extensions, such as would result from cleavage of two C residues in a self-complementary G-C sequence, were conspicuously absent. These data provide further evidence that bleomycin-induced double-strand breaks do not result from coincidence of independent site-specific single-strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)