Journal of Experimental Botany, Vol. 53, No. 378, pp. 2151-2158,
November 1, 2002
© 2002 Oxford University Press
Recovery of tobacco cells from cadmium stress is accompanied by DNA repair and increased telomerase activity
Received 14 December 2001; Accepted 3 July 2002
ková2
í Fajkus1,2
Kovaík3,1
1 Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, Brno, CZ-612 65, Czech Republic
2 Department of Functional Genomics and Proteomics, Masaryk University Brno, CZ-612 65, Czech Republic
Abbreviations: dNTP, deoxynucleotide triphosphate; DTT, 1,4,-dithio-DL-threitol; FDA, fluorescein diacetate; Mb, megabase; MES, 2[N-morpholino]ethanesulphonic acid; MS medium, Murashige and Skoog medium; PFGE, pulsed-field-gel electrophoresis; Topoll, topoisomerase II; TRAP, telomere repeat amplifcation protocol.
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Abstract |
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It has been shown previously that apoptosis of tobacco cells induced by cadmium ions shows a relatively long lag period between exposure and cell death. This lag phase lasts for 3 d in TBY-2 cell cultures and is characterized by the maintenance of full cell viability despite extensive fragmentation of DNA into pieces of chromatin loop size. Experiments reported here demonstrate that cell death can be prevented if 50 µM CdSO4 is removed from the growth medium during the lag phase, suggesting that an irreversible apoptotic trigger is delivered within 24 h, between the third and fourth days of cadmium treatment. The post-cadmium recovery phase was characterized by DNA repair at the level of 50–200 kb and increased telomerase activity. Analysis of high-molecular-weight DNA by pulsed-field-gel electrophoresis revealed that the majority of DNA strand breaks was repaired within 48 h after cadmium withdrawal. Telomerase activity increased 2.5-fold in the recovery phase, but elevated levels were also found in cell extracts from apoptotic cells suggesting that telomerase might be associated with DNA repair, but it is not capable of inhibiting ongoing apoptosis. Limited exposure of TBY-2 cells to cadmium elicits non-random DNA damage of relatively high magnitude that can be repaired. It is proposed that plants might have developed a highly efficient DNA repair system to cope with transient genotoxic stress.
Key words: Key words: Apoptosis, cadmium, DNA domain fragmentation, telomerase, tobacco BY-2 cells.
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Introduction |
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Cadmium is a known carcinogen and represents a serious environmental problem for both humans and animals (Freedman et al., 1988). Although cadmium is a genotoxic metal, the molecular basis of cadmium genotoxicity is not well defined. In mammalian cells, cadmium enhances the mutagenicity of UV light, suggesting its interference with DNA repair processes and the activity of detoxifying enzymes (reviewed in Beyersmann and Hechtenberg, 1997; Hartwig, 1994). In plants, cadmium induces a number of genome-related changes including chromosomal aberrations (Zhang and Xiao, 1998), decrease of mitotic index in root cells (Zhang and Yang, 1994), and abnormalities in nucleolar structure (Jiang et al., 1994; Zhang and Yang, 1994). At the biochemical level, the accumulation of oxidized proteins and lipid peroxides was observed in pea upon cadmium stress (Sandalio et al., 2001).
Apoptosis in plants occurs in response to pathogens (Keen, 1990) and to changes in environmental conditions. Typical DNA fragmentation and changes in the morphology of nuclei were observed during temperature stress (Koukalová et al., 1997; McCabe and Leaver, 2000), after UV-irradiation (Danon and Gallois, 1998) and after exposure to chemicals and toxins (Wang et al., 1996). Apoptosis of tobacco cells, manifested by chromatin condensation and DNA fragmentation after exposure to chemicals (salicylic acid, okadaic acid, hydrogen peroxide, campthotecin), can be reversible during the early stages following the removal of the inducing agent (O’Brien et al., 1998). DNA lesions induced by ongoing apoptosis may represent an abundant substrate for healing by telomerase, and a number of reports from the animal kingdom are consistent with this theory (Leteurtre et al., 1997; Hande et al., 1998). To date, the up-regulation of telomerase in response to DNA damaging agents has not been reported in plants.
In a previous paper on cadmium sulphate-triggered apoptosis in tobacco BY-2 cells (Fojtová and Kovaík, 2000), the most striking observation was the relatively late onset of cell death upon exposure to 50 µM CdSO4. During the first 3 d of cadmium treatment, cell proliferation and morphology were similar to non-treated cells. On the third day, DNA cleavage into units 50–200 kb in length, termed as domain fragmentation, was observed and prolonged exposure (4–7 d) led to a rapid decrease of cell viability and further degradation of DNA to oligonucleosomal units. Thus, in TBY-2 cells, the domain and oligonucleosomal DNA fragmentation are separated by at least 24 h and this experimental system can be used to study the kinetics of domain DNA fragmentation in plant apoptosis.
Here the focus is on the reversibility of cadmium-induced DNA fragmentation during the first 3 d of cadmium treatment and on DNA repair processes taking place after the removal of Cd2+ ions. A relationship between recovery of genome integrity and telomerase activity is demonstrated.
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Materials and methods |
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Chemicals
CdSO4 and fluorescein diacetate were obtained from Sigma (USA), [14C]thymidine (specific activity 4 MBq ml–1) from UVVVR (Czech Republic).
Cell culture and cadmium treatment
The tobacco bright yellow (TBY-2) cells (Kato et al., 1972) were grown in standard liquid Murashige and Skoog (MS) medium supplemented with sucrose (3%, w/v), thiamine (1 mg l–1), KH2PO4 (200 mg l–1), myoinositol (100 mg l–1) and 2,4-dichlorophenoxyacetic acid (0.2 mg l–1) in 100 ml Erlenmeyer flasks at 27 °C with shaking at 120 rev min–1. The cells were regularly subcultured twice a week.
For cadmium stress studies, the cells at a density of about 3 millions cells ml–1 were subcultured at dilution of 1:10 into MS medium with CdSO4 (final concentration 50 µM). After 3 d and 4 d of cultivation, respectively, cells were extensively washed with MS medium and further cultivated without cadmium sulphate for 1–4 d, defined as the post-cadmium phase.
In double strand break induction/rejoining experiments, the DNA was metabolically pre-labelled with [14C] thymidine as follows: about 3x106 of TBY-2 cells were cultivated in 10 ml of MS medium containing 0.4 MBq of [14C]thymidine for 3 d. The incorporated [14C]thymidine was measured in a ß-scintillation counter Wallac 1410 (LKB, Pharmacia) after DNA precipitation with trichloroacetic acid. Typically, incorporated radioactivity reached a plateau (0.2–1.0 dpm per cell) after 24 h when about 50% of the total had been incorporated.
Analysis of high-molecular-weight DNA
Cells from about 3 ml of TBY-2 cell suspension culture were lyophilized at –20 °C, then homogenized in liquid nitrogen and immediately resuspended in a buffer containing 10 mM 2[N-morpholino]ethanesulphonic acid (MES), pH 5.6, 10 mM NaCl, and 5 mM EDTA. The suspension was mixed with an equal volume of molten 2% (w/v) low melting temperature agarose in 0.4 M mannitol, 20 mM MES, pH 5.6 and transferred to a mould. Agarose blocks were incubated in a lysis buffer (0.5 M EDTA, pH 8.0, 1% (w/v) N-lauroylsarcosine, 0.1 mg ml–1 proteinase K) at 55 °C for 2x24 h and stored in 0.5 M EDTA. The DNA was analysed within 1 week to avoid diffusion of low molecular weight fragments.
Electrophoresis was performed on the Gene Navigator System (Pharmacia Biotech, Sweden) using 1% agarose gel in 45 mM Tris-borate, 1 mM EDTA, pH 8.0. The running conditions (pulse ramping time from 5 s to 50 s, voltage 200 V, temperature 10 °C, time 24 h) enabled separation of fragments between 50–1000 kb in size. After electrophoresis, gels were stained by ethidium bromide and photographed. Gels containing [14C]thymidine-labelled DNA were blotted onto a nylon membrane, exposed to a screen and scanned using a PhosphorImager STORM 860 (Molecular Dynamics, USA).
Southern blot hybridization
DNA separated on pulsed-field-gel electrophoresis (PFGE) was blotted onto nylon membranes (Hybond XL, Amersham Pharmacia Biotech, UK) and hybridized with the 360 bp dimer of the subtelomeric tandem repeated sequence HRS60 (Koukalová et al., 1989) labelled with 32P-dCTP by random priming (DNA Labelling Kit, MBI Fermentas, Lithuania) according to a standard protocol (Sambrook et al., 1988). The radioactive signals were visualized using a PhosphorImager and analysed by ImageQuant software (Molecular Dynamics, USA).
Estimation of total and viable cell count, studies of nuclei morphology
Total cell counts were determined manually in Burker chamber using phase-contrast light microscope (Carl Zeiss, Jena, Germany). Counts of viable TBY-2 cells were determined by fluorescein diacetate staining followed by evaluation of fluorescence of living cells using the blue-fluorescence mode of a PhosphorImager STORM (Kovaík and Fojtová, 1999). Briefly, diluted cell suspensions were mixed with equal volumes of FDA reagent, prepared by diluting stock solution (3 mg FDA ml–1 of acetone) with MS medium at 1:200. After 5 min incubation, 20 µl drops were transferred onto a nylon membrane (Hybond XL) and dried. Fluorescence signals were scanned with a blue fluorescence laser channel (excitation wavelength 450 nm) at a high resolution (100 pixels) using a PhosphorImager. Signals were evaluated with an ImageQuant program using an ellipse integration method, viable cell counts were calculated from the calibration curve (a linear plot of fluorescence units and the number of viable cells determined manually by fluorescence microscopy).
The morphology of nuclei was studied by fluorescence microscopy using cells fixed in a Carnoy’s fixative (methanol:acetic acid, 3:1), transferred onto microscope slides, stained with Hoechst 33258 (1 µg ml–1) for 10 min, and destained in distilled water. Blue fluorescence was visualized using an epifluorescence microscope Olympus AX 70 with image capture and processing; image analysis was performed by the ISIS program (Metasystems, Germany).
Preparation of cell extract for telomerase assay
Cell extracts from TBY-2 cells was prepared as described previously (Fitzgerald et al., 1996). Approximately 0.25 g of lyophilized cells were ground in liquid nitrogen, suspended in 1 ml of buffer W (50 mM Tris-acetate pH 7.5, 5 mM MgCl2, 100 mM potassium glutamate, 20 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 0.6 mM vanadyl ribonucleoside complex (NEB), 1.5% (w/v) polyvinylpyrrolidone, 10% glycerol) and centrifuged at 16 000 g for 15 min at 4 °C. The supernatant was supplemented with PEG 8000 (Sigma) to a final concentration of 10%, stirred for 30 min at 4 °C, and centrifuged at 20 000 g for 5 min at 4 °C. The pellet was resuspended in 250 µl of buffer W for 30 min on ice and centrifuged at 20 000 g for 2 min at 4 °C. The supernatant was stored at –70 °C until use. The concentration of total protein in cell extracts was determined according to Bradford (Bradford, 1976).
Telomerase assay
Telomerase was assayed using a modified plant version of the telomere repeat amplification protocol (TRAP) (Fitzgerald et al., 1996; Fajkus et al., 1998). In the first step of the assay, telomerase adds a number of telomeric repeats (GGTTTAG) onto the 3' end of a substrate oligonucleotide. In the second step, the extended products are amplified by PCR using the substrate and reverse primers, generating a ladder of products with seven base increments. Assay buffer contained 50 mM Tris-acetate, pH 8.3, 50 mM potassium glutamate, 0.1% Triton-X-100, 1 mM spermidine, 1 mM DTT, 50 µM of each dNTP, 5 mM MgCl2, 10 mM EGTA, and 100 µg ml–1 BSA. The primer 47F (5'-CGCGGTAGTGATGTGGTTGTGTT-3') was denatured for 5 min at 95 °C and cooled on ice prior to addition to reactions. The reaction mixture, composed of telomerase assay buffer (45.5 µl), 10 pmol of primer 47F and cell extract, was incubated at 26 °C for 45 min in a thermocycler. Controls for false-positive results were run in parallel using heat-treated extracts (94 °C, 10 min). Elongation was terminated by heating the reaction mixture at 94 °C for 10 min and 10 pmol of TP primer (5'-CCGAATTCAACCCTAAACCCTAAACCCTAAACCC-3') and 2 units of DyNAzyme II DNA polymerase (Finnzymes, Finland) were added immediately to each reaction at 80 °C. The extension products were amplified by 35 cycles of PCR (94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s), followed by a terminal extension step (72 °C for 5 min). The products were separated on a 12.5% polyacrylamide gel which was stained with SYBR Green I (Molecular Probes) and scanned on a PhosphorImager STORM in a blue fluorescence mode and the resulting product ladder bands (see above) were quantified using ImageQuant software in each sample. The activity of each sample was then expressed as the percentage telomerase activity with respect to the activity found at the corresponding protein concentration in control cells (cultured in the absence of cadmium).
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Results |
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Early cadmium removal may prevent apoptosis
Previous reports indicated that the death of TBY-2 cells occurs only after 4 d of exposure to 50 µM cadmium sulphate (Fojtová and Kova
ík, 2000). Here the aim was to find out whether the removal of cadmium after different intervals of treatment would prevent apoptosis. In order to determine the point of reversibility, defined as the maximum time interval beyond which cell damage reaches a critical threshold inevitably leading to death, cells were treated with 50 µM CdSO4 for 3 d or 4 d, respectively, then washed and subcultured for up to 4 d in cadmium-free medium. Viable (Fig. 1) and total cell counts were determined and expressed as a ratio (Table 1). TBY-2 cells growing in the presence of the cadmium sulphate for up to 3 d displayed full viability (Table 1). Immediately after transfer to cadmium-free medium, the growth slowed slightly, but then recovered to the control rate (Fig. 1). On the other hand, cells exposed to cadmium for 4 d showed only 
30% viability and viability fell to zero after transfer to cadmium-free medium. This indicated that the changes during the first 3 d of cadmium treatment were fully reversible, while more extended exposure led inevitably to cell death even after the removal of the stress factor. Similar results were obtained in three independent experiments.
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Repair of double strand DNA breaks after cadmium removal
The preapoptotic phase of cadmium treatment was accompanied by DNA fragmentation into chromatin loops 50–200 kb in length (Fojtová and Kova
ík, 2000). Next it was determined whether this initial DNA damage was repaired during the recovery phase described above. Pulsed-field-gel electrophoresis was used to analyse high-molecular-weight DNA from cells treated with 50 µM CdSO4 for 3 d and from a post-cadmium culture (Fig. 2A). On day 3 of the cadmium treatment (lane 2), and 24 h after cadmium removal (lane 3), DNA migrated at the gel front as a smear of unresolved fragments 50–200 kb in length indicating the severe breakdown of genomic DNA. Starting from day 2 following cadmium removal, the signals were shifted to higher molecular weight fractions (Fig. 2A, lanes 4, 5) and on the 4th day of the post-cadmium period, most DNA migrated in the compression zone (about 1 Mb in length) (lane 5). Subsequent Southern hybridization with the subtelomeric probe HRS60 (Fig. 2B) or with telomeric probe (not shown) revealed similar patterns.
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The extensive domain fragmentation of DNA and the virtual absence of dead cells in preapoptotic and post-cadmium phases suggest that restoration of DNA integrity during the post-cadmium period was caused by a repair process, rather than by selection of a rare subpopulation of cells with non-fragmented DNA. In order to demonstrate that double strand breaks are rejoined, cellular DNA was prelabelled with [14C]thymidine during cadmium treatment, then the cells were washed and incubated in cadmium-free medium for 1, 2 or 3 d. An increase of incorporated label was not observed during the post-cadmium period showing the effective removal of free [14C]thymidine (not shown). DNA from cadmium-treated and non-treated cells was separated by PFGE and transferred to a nylon membrane which was exposed to a PhosphorImager screen for 60 d. DNA from non-treated cells migrated to the high-molecular-weight region (Fig. 3, lane 5). As expected, after 3 d with 50 µM CdSO4 most DNA was in the 50–200 kb region at the gel front (lane 1). The removal of cadmium resulted in a shift of the radioactive DNA towards a higher molecular weight after 2 d, when a band in the compression zone (>1 Mb) became visible (lane 3) and its intensity increased after 3 d (lane 4). Thus, recovery of TBY-2 cells from cadmium-induced genotoxic stress is accompanied by the repair of double strand DNA breaks.
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Recovery of genome integrity is accompanied by an increase in telomerase activity
Broken chromosomes become highly unstable and fuse with other broken chromosome ends which leads to overall genomic instability. This may be prevented by ‘healing’ which involves the addition of telomere sequences at the breakpoints by telomerase (reviewed in Gill and Friebe, 1998). To resolve whether telomerase participates in the recovery from the genotoxic stress imposed by cadmium, telomerase activity was monitored in the course of cadmium sulphate treatment and the post-cadmium phase; serial dilutions of nuclear extracts from each phase of the experiment were used for TRAP assays. The final values of relative telomerase activity (see Materials and methods) were then calculated as an average of 5–7 values obtained at individual extract concentrations for each time-point. A remarkable increase in telomerase activity was detected during the phase of recovery from cadmium sulphate treatment (Fig. 4C, D, E), reaching a maximum (234% of the activity in control cells) on the second day of the recovery phase (Fig. 4D; and day 5 of the experiment II in Fig. 5), and falling slightly below the normal level on the fourth day of the recovery phase (see Fig. 4F and day 7 of the experiment II). A small increase in telomerase activity could be observed in cells treated with cadmium for 3 d (Fig. 4B) or 4 d (beyond the ‘point of reversibility’)—see Fig. 5, days 3 and 4 of experiment I. The addition of 50 µM CdSO4 to the TRAP reaction mixtures did not affect the assay, suggesting that the presence of cadmium itself does not directly influence telomerase activity either positively or negatively (not shown). These observations suggest that telomerase participates in the genotoxic-stress-response and, together with DNA polymerases, is involved in the re-establishment of genome integrity.
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Discussion |
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A previous report indicated that apoptosis induced by cadmium ions in TBY-2 cell cultures can be dissected into a relatively long initial phase lasting for 3 d, followed by cell death associated with oligonucleosomal fragmentation of DNA (Fojtová and Kova
ík, 2000). It is shown here that death is prevented if cadmium is removed from the medium during the initial phase. Since on day 4, viable cells could not be recovered even after removal of 50 µM cadmium, the critical phase of this type of apoptosis is limited to about 24 h between days 3 and 4 of cadmium treatment, when a cell death signal is irreversibly triggered and DNA degradation is manifested. The progressive breakdown of high-molecular-weight DNA has been regularly observed during the initial phase. In these experiments, it was not possible to detect significant amounts of intact DNA of >1 Mb after 3 d of cadmium treatment suggesting that most cells possessed severely damaged DNA. During the first 24 h of the post-cadmium phase, the DNA was still significantly fragmented, but after 48 h it showed a dramatic shift towards higher molecular weight and after 4 d most DNA was of >1 Mb in length. This suggests that DNA repair mechanisms were activated in the post-cadmium phase. To demonstrate directly that ligation of breaks had indeed occurred in vivo, pulsed-field-gel-electrophoresis was used to show that, in the post-cadmium phase, the average size of the 14C-thymidine-labelled DNA molecules increased markedly compared to the initial apoptotic phase. Gorbunova and Levy (1997) recently reported end-joining of transfected plasmid DNA in tobacco cells. Thus, end-joining of double strand breaks may significantly contribute to the recovery of plant cells from the temporal genotoxic stress. The DNA cleavage patterns accompanying cadmium stress clearly differed from those observed after gamma irradiation of cells (Hall et al., 1992). While in the latter study the sizes of the DNA fragments were randomly distributed, these results are most consistent with targeted fragmentation into chromatin loops of 50–200 kb. This conclusion is supported by the similarity between the size of ‘preapoptotic’ DNA fragments and that of fragments obtained after treatment of maize protoplasts with VM-26, an inhibitor of topoisomerase II (TopoII) (Espinas and Carballo, 1993). TopoII sites frequently (but not always) co-localize with matrix attachment regions (Gromova et al., 1995). Hence, TopoII and/or other nuclease sensitive sites in chromatin could be primary targets of the cadmium genotoxicity.
Telomerase could be considered as a key enzyme in maintaining chromosomal integrity (reviewed in Blackburn, 2000). In this study’s experiments, the maximum increase of activity of telomerase was found in the 48 h interval of a post-cadmium phase. Interestingly, over this interval the intensive repair of double strand breaks was observed. Correlative evidence for a link between the end-joining of double strand breaks and telomerase was thus obtained. Presumably, the recovery of cells from cadmium stress requires multiple enzyme activities involved in the re-establishment of genome integrity. It will be interesting to examine whether the activation of telomerase would also occur in other systems that involve increased DNA repair, for example, after UV irradiation. The weakly elevated levels of telomerase activity were found in extracts from cells in the apoptotic phase (day 4 in cadmium). Since about 70% of cells are already dead at this stage, this activity may originate from the remaining viable cells that might contain high enzyme activity. Nevertheless, activated telomerase cannot inhibit the apoptosis already initiated between days 3 and 4 of cadmium treatment (Fig. 1). Although tobacco contains exceptionally long telomeres (Fajkus et al., 1995), the double strand breaks occurring in telomeric or subtelomeric regions (not shown and Fig. 2B) might result in short telomeres or chromosomes without telomeres. Possibly, active telomerase helps even very short telomeres to be functionally capped.
Perhaps the most interesting aspect of cadmium stress is the relationship between cell viability and the extent of the DNA damage. The initiation stage and the early post-cadmium phase are characterized by extensive DNA cleavage into chromatin loops. In most animal systems, once this type of fragmentation is initiated, cell death and, in some cases, oligonucleosomal fragmentation inevitably occur (Huang et al., 1995). However, TBY-2 cells remained fully viable and oligonucleosomal fragmentation did not appear in the post-cadmium phase (not shown). The morphology of cell nuclei at day 3 of cadmium treatment did not show significant condensation of chromatin; in fact, the nuclear volume was slightly larger than controls (Fig. 6), which could indicate the arrest of cells in the G2 phase or a certain degree of aneuploidy. How can the observed severe DNA fragmentation be explained in the context of full cell viability? A trivial explanation is that the DNA became fragmented in the course of its preparation. But this possibility is considered unlikely. Domain fragmentation of DNA from lymphocytes was only seen under conditions of low EDTA concentration (Szabo and Bacso, 1996), whereas here all buffers contained 0.5 M EDTA. Moreover, DNA from control cells prepared in parallel did not show the fragmentation and elevation of telomerase activity correlated with end-joining of double strand breaks. The interpretation that fragmentation of high-molecular-weight DNA into 50–200 kb pieces represents a reparable DNA damage in transient cadmium stress is favoured. Saturation of DNA repair enzymes with prolonged genotoxic stress could lead to the activation of apoptotic signals sensing the ‘irreparable’ DNA damage. This assumption is strengthened because the addition of 100 µM cadmium shortened the initial preapoptotic period of cadmium tolerance to about 48 h (Fojtová and Kovaík, 2000). As in other studies that use DNA-damaging drugs to induce apoptosis, it is difficult to separate breaks introduced by treatment from those that represent the initial breaks of apoptosis. Possibly, the cleavage of DNA to 50–200 kb fragments could represent a regular phase of the apoptotic pathway (Oberhammer et al., 1993). In this context the reversibility of the early stages of apoptosis induced with various stresses has been described in plant cells (O’Brien et al., 1998).
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An open question remains as to how genome integrity is restored to allow cells to resume the cell cycle after the removal of cadmium. a central role is proposed for matrix-attachment regions in this process. The assembly of an extensively fragmented genome on the nuclear matrix may contribute to the correct repair via DNA recombination and repair machinery, an idea supported because (i) the size of fragments in the ‘reversible phase’ corresponds to the size of chromatin loops, (ii) chromosome breaks and recombination events preferentially occur at nuclear matrix attachment sites where DNA is bound to topoisomerase II (Blasquez et al., 1989) and (iii) the DNA fragmentation induced by TopoII inhibitor was reversible in maize protoplasts (Espinas and Carballo, 1993). Most likely, the contacts between the ends of chromatin loops and the nuclear matrix proteins probably survive the reversible stage of cadmium-induced genome fragmentation and thus DNA–protein bonds would provide a way of conserving the nuclear position and function of individual loop domains. The maintenance of the loop organization of nuclear chromatin may thus be functionally more important than the simple integrity of the sugar–phosphate backbone of genomic DNA. Hence, the reconstruction of the genome could hardly be possible without the maintenance or re-establishment of chromatin loop attachments to the nuclear matrix.
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Acknowledgements |
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We thank Dr B Koukalová (Institute of Biophysics, Brno, Czech Republic) and Dr R Hancock (Laval University Cancer Research Center, Quebec, Canada) for helpful comments and critical reading of the manuscript and Dr A Levy (Weizman Institute of Sciences, Rehovot, Israel) for a useful discussion. The excellent technical assistance of Mrs L Jedli
ková and Miss D Fridrichová is highly appreciated. The work was supported by the Grant Agency of the Czech Republic (grants no. 521/01/P042, Z 5004920 and 204/02/0027) and the Ministry of Education of the Czech Republic (MSM143100008).
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