Research Article
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Formation of higher-order nuclear Rad51 structures is functionally linked to p21 expression and protection from DNA damage-induced apoptosis Elke Raderschall1, Alex Bazarov2, Jiangping Cao3, Rudi Lurz1, Avril Smith1, Wolfgang Mann1, Hans-Hilger Ropers1, John M. Sedivy4, Efim I. Golub5, Eberhard Fritz6 and Thomas Haaf1 1Max Planck Institute of Molecular Genetics, 14195 Berlin, Germany 2Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94040, USA 3Soochow University, Suzhou 215007, P.R. China 4Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA 5Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520, USA 6Institute of Radiation Biology, GSF, National Research Center for Environment and Health, 85758 Neuherberg,
Germany
Author for correspondence (e-mail:
[email protected])
Accepted 4 October 2001 Journal of Cell Science 115, 153-164 (2002) © The Company of Biologists Ltd
Summary After exposure of mammalian cells to DNA damage, the endogenous Rad51 recombination protein is concentrated in multiple discrete foci, which are thought to represent nuclear domains for recombinational DNA repair. Overexpressed Rad51 protein forms foci and higher-order nuclear structures, even in the absence of DNA damage, in cells that do not undergo DNA replication synthesis. This correlates with increased expression of the cyclindependent kinase (Cdk) inhibitor p21. Following DNA damage, constitutively Rad51-overexpressing cells show reduced numbers of DNA breaks and chromatid-type chromosome aberrations and a greater resistance to apoptosis. In contrast, Rad51 antisense inhibition reduces
Introduction DNA double-strand breaks (DSBs), which may occur spontaneously or arise as a direct effect of ionizing radiation and many DNA-damaging agents, are potentially lethal for the cell. Recombinational repair of DSBs is an essential process for genome integrity that has been evolutionarily conserved from bacteria to man. Eukaryotic cells repair DSBs either by direct non-homologous end-joining of the broken ends or by homologous recombination (Jeggo, 1998). Homologous recombination is also responsible for the generation of genetic diversity during meiosis (Shinohara et al., 1992). Recent studies have shown that Rad51 recombinase plays an essential role in homologous recombination in mammalian cells. Similar to Escherichia coli RecA, both yeast and mammalian Rad51 proteins form nucleoprotein filaments on single-stranded (ss) DNA, mediating homologous pairing and strand-exchange reactions between ssDNA and homologous double-stranded DNA (Sung, 1994; Baumann et al., 1996; Gupta et al., 1997). In normal, cultured mammalian cells, the Rad51 protein is detected in multiple discrete foci in the nucleoplasm of a low number of cells by immunofluorescent antibodies. After DNA damage, the percentage of cells with focally concentrated Rad51 protein increases in a time- and dose-dependent manner.
p21 protein levels and sensitizes cells to etoposide treatment. Downregulation of p21 inhibits Rad51 foci formation in both normal and Rad51-overexpressing cells. Collectively, our results show that Rad51 expression, Rad51 foci formation and p21 expression are interrelated, suggesting a functional link between mammalian Rad51 protein and p21-mediated cell cycle regulation. This mechanism may contribute to a highly effective recombinational DNA repair in cell cycle-arrested cells and protection against DNA damage-induced apoptosis. Key words: Apoptosis, Cell cycle arrest, DNA repair, p21, Rad51
Rad51-foci-positive cells are arrested during the cell cycle and undergo unscheduled DNA repair synthesis (Haaf et al., 1995; Haaf et al., 1999). Nuclear foci are formed at sites of DNAdamage-induced ssDNA (Raderschall et al., 1999) and contain the ssDNA-binding replication protein A (RPA) (Golub et al., 1998), which facilitates homologous pairing and DNA strand exchange, mediated by Rad51 (Baumann et al., 1996; Gupta et al., 1998). In addition, Rad foci may also contain Rad52 (Liu et al., 1999) and Rad54 (Tan et al., 1999), which belong to the same epistasis group as Rad51. It seems plausible that DNAdamage-induced Rad51 foci represent a repairosome-type assembly of Rad51 and other proteins that are essential for recombinational DNA repair. In contrast to E. coli RecA and yeast ScRad51, mammalian Rad51 protein appears to be necessary for cell survival. Disruption of both Rad51 alleles conveys embryonic stem cell and early embryonic lethality in mice (Lim and Hasty, 1996; Tsuzuki et al., 1996). Rad51 is transcribed in dividing cell lines and, in general, its expression level in tissues correlates with the proportion of cycling cells (Shinohara et al., 1993; Yamamoto et al., 1996). This is consistent with a role for Rad51 protein in mammalian cell proliferation and/or DNA metabolism. In order to study the conserved and novel
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functions of mammalian Rad51 protein, we have both overexpressed and downregulated human Rad51 in various cell lines. Our data suggest that, in addition to its classical function in homologous recombination, mammalian Rad51 protein is involved in regulatory aspects of the cell cycle and apoptosis. Materials and Methods Overexpression of HsRad51 protein in mammalian cells Primary human PPL and transformed rat TGR fibroblasts (Prouty et al., 1993) were grown in D-MEM medium supplemented with 10% fetal bovine serum and antibiotics. Plasmid pEG928.1 was made by inserting the whole coding sequence of HsRad51 into the HpaI site of retroviral vector pLXSH, which contains the hygromycin phosphotransferase marker (Miller et al., 1993). This plasmid was electroporated into the ectopic retrovirus Y2 packaging line (Mann et al., 2000). Viral supernatants were used to infect PPL and TGR cultures. Stably infected cells were then subjected to selection with 150 µg/ml hygromycin B. Individual clones were expanded into cell lines and screened for elevated Rad51 protein levels by western blotting. Two clonal lines, PPL928.1-2 and TGR928.1-9, showed significant Rad51 overexpression and were selected for further studies. To synchronize Rad51-overexpressing cells, subconfluent TGR928.1-9 cultures were starved for two days in medium containing 0.5% serum. Transition from G0 to G1 phase occurred several hours after feeding the cells with medium containing 10% serum. Plasmid pEG915, which carries the HsRad51 coding sequence inserted in frame with the 5′-terminal sequence of vector pEBVHisB (Invitrogen), was used for transient expression of HsRad51 protein in mammalian cells (Haaf et al., 1995). Cells from Xeroderma pigmentosum type A (XP-A) patients have defects in the enzyme that is responsible for DNA lesion recognition by nucleotide excision repair and, therefore, accumulate DNA damage. The unexcised DNA lesions stimulate intrachromosomal homologous recombination (Bhattacharyya et al., 1990). Compared with normal PPL fibroblasts, SV40-transformed XP-A fibroblasts exhibit an approximately 2.5-fold elevated Rad51 protein level and an increased number (i.e. 10-20%) of nuclei with Rad51 foci, even without the induction of DNA damage (Raderschall et al., 1999). Induction of DNA damage in cultured cells To induce DSBs in DNA, 4 µg/ml of etoposide was added to fresh culture medium for 24 hours. Topoisomerase II binds covalently to double-stranded DNA, then cleaves both strands and reseals the cleaved complex. Etoposide interferes with this breakage and rejoining cycle, trapping the enzyme in the cleaved complex. This results in irreparable DSBs (Mizumoto et al., 1994). Ionizing radiation, that is, exposure to a 60Co irradiator at a dose rate of 9.13 Gy per minute or to a UV-C irradiator at a dose of 10 J/m2, induces mostly single-strand breaks and oxidized apurinic and apyrimidinic sites. The abasic sites are hydrolyzed by cellular endonucleases, thereby producing DNA strand breaks (Demple and Harrison, 1994). In control experiments, cultures were treated with 1 µg/ml cycloheximide for 24 hours, which kills cells by inhibiting overall protein synthesis (Waring, 1990). To quantify the number of radiation-induced chromosome aberrations, subconfluent TGR and TGR928.1-9 cells in Petri dishes were exposed to 60Co γ ray doses of 1-7 Gy and immediately afterwards treated with 0.2 µg/ml colcemid. Metaphases were prepared at 16 hours after irradiation according to standard procedures. The chromosome number was determined in 150 metaphases each for TGR and TGR928.1-9 and the diploid status (2n=42) confirmed for both cell lines. For aberration analyses, metaphase slides were first coded and then screened in a double-blind manner for the presence of chromosome breaks (deletions and rings)
and exchanges (dicentrics) as well as for chromatid breaks (gaps and fragments) and exchanges (triradials). For each radiation dose and cell line, 200 metaphases were evaluated. Mean values and standard deviations were determined from three (0 Gy, 5 Gy) or two (1 Gy, 3 Gy, 7 Gy) independent experiments. Antisense inhibition of Rad51 and p21 Antisense phosphorothioate oligodeoxynucleotides (ODNs) for Rad51 (Rad51-AS, 5′-GGCTTCACTAATTCC-3′) (368-382) and scrambled Rad51 ODNs (Rad51-SC, 5′-TCGCGATCACCTTAT-3′) (MWG Biotech) were resuspended at 100 µM in 10 mM Tris-HCl, pH 7.5 and 1 mM EDTA (Taki et al., 1996). ODNs for p21 were complementary to the region of the initiation codon (p21-AS, 5′CCCAGCCGGTTCTGACATGGCGCC-3′) (Yu et al., 1998). Scrambled p21 ODNs (p21-SC, 5′-CCGCACGGAGCGCTGCGTTCTACC-3′) were used as controls. Subconfluent monolayer cultures were washed with phosphate-buffered saline (PBS: 136 mM NaCl, 2 mM KCl, 10.6 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.3) and incubated for eight hours with D-MEM containing the indicated ODNs (final concentration 100 nM or 400 nM) and lipofectamine (Gibco BRL). Then the cultures were washed twice with medium and grown overnight at 37°C. Gene expression analysis The custom cDNA microarrays used for expression analysis contain >900 ESTs (selected from public databases, i.e. http://www.ncbi.nlm.nih.gov/dbEST) for the investigation of approximately 300 genes. This chip includes genes involved in cell cycle, apoptosis, DNA recombination and repair, together with 10 housekeeping genes as controls. To bind PCR products covalently onto amino silane-coated glass slides, cDNAs were amplified from plasmids using amino-modified vector primers. Using a 96-well format, PCR products were spotted onto activated slides (Guo et al., 1994) using a commercially available robot (Beecher Instruments) that deposits 5 nl of DNA solution at each spotting site, resulting in spot areas of approximately 200 µm in diameter. Aliquots (25 µg) of poly(A) RNA from PPL and PPL928.1-2 cells were reverse transcribed with the Superscript II kit (Gibco BRL) using an oligo(dT) primer in the presence of either Cy3-dUTP or Cy5-dUTP. The differentially fluorescent-labeled targets were hybridized together on cDNA microarrays, and the fluorescent intensities for the two wavelengths at each spot were read by a laser scanner (GMS 418 array scanner). Image analysis was carried out with a custom-made software program that runs as an extension on IP Lab Spectrum software (Chen et al., 1997). The result file contains ratios of mean intensities per pixel for individual spots consisting of at least 20 pixels each. The background fluorescence was substracted from all spots. Immunoblot analysis HsRad51 protein, expressed in E. coli, was isolated and used for preparation of rabbit polyclonal antibodies (Haaf et al., 1995). Goat polyclonal antibodies against the entire human p53 protein (FL-393) and against the C-terminus of human p21 (C-19) were purchased from Santa Cruz Biotechnology. Cell extracts were resolved by electrophoresis on 12% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. The resulting filters were blocked overnight with 5% nonfat dried milk, incubated with the appropriately diluted primary antibodies for one hour, incubated with horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG (Dianova) and washed. Antibody binding was visualized by chemiluminescence (ECL RPN 2209; Amersham). To compare the protein levels in different cell substrates, all filters were re-incubated with rabbit antibodies against β-actin (Sigma). The intensity of the Rad51 signals
Rad51 overexpression in mammalian cells (and other primary antibody signals) was equilibrated to the intensity of the β-actin signals using PCbas2.0 software. Measurement of DNA breaks Quantification of the relative number of DNA breaks was based on random primer extension of 3′-OH ends by Klenow fragment polymerase (Basnakian and Jill James, 1996). 100,000 cells each of PPL and PPL928.1-2 were seeded in culture flasks and grown for 24 hours in medium containing 4 µg/ml etoposide. For each cell line, two samples, each containing 0.5 µg high-molecular weight DNA in a volume of 12 µl ddH2O, were processed. 3′-OH end-containing DNA fragments generated in vivo through single-strand and double-strand breaks were separated by heat denaturation. After reassociation, these DNA fragments served as a primer and the excess high-molecular weight DNA as a template. In a random primer extension reaction, Klenow enzyme incorporated [γ-32P]dCTP into newly synthesized DNA. Incorporation was linearly proportional to the number of DNA breaks present in the sample. The reaction mixture for 10 samples consisted of 25 µl of cold dNTPs (0.5 mM each of dATP, dGTP, and dTTP), 4.5 µl of 33 µM cold dCTP, 0.5 µl of [γ-32P]dCTP (labeled to a specific activity of 3,000 Ci/mmol), 1 µl (5 Units) Klenow enzyme and 94 µl ddH2O. Fifteen µl of this reaction mixture were added to each DNA sample and incubated at 16°C for 30 minutes. The reaction was stopped by adding 25 µl of 12.5 mM EDTA, pH 8.0. The radioactively labeled DNA fragments were purified with a PCR purification kit (Qiagen). Four 5 µl aliquots of each DNA sample were counted in a Packard liquid scintillation counter. Immunofluorescent staining Harvested cells were washed and resuspended in PBS. Aliquots (105 cells in 0.5 ml PBS) were centrifuged onto clean glass slides using a Shandon Cytospin. Immediately after cytocentrifugation, the preparations were fixed in absolute methanol for 30 minutes at –20°C and then rinsed in ice-cold acetone for a few seconds. Following three washes with PBS, the preparations were incubated at 37°C with rabbit anti-HsRad51 or goat anti-p21 antibodies diluted 1:100 with PBS in a humidified incubator for 30 minutes. The slides were then washed in PBS another three times for 10 minutes each and incubated for 30 minutes with fluorescein-isothiocyanate (FITC)- and/or Cy3-conjugated secondary antibodies, appropriately diluted with PBS. After three further washes with PBS, the preparations were counterstained with 1 µg/ml 4,6-diamidino-2phenylindole (DAPI) in 2×SSC for one minute. The slides were mounted in 90% glycerol, 0.1 M Tris-HCl, pH 8.0, and 2.3% 1,4diazobicyclo-2,2,2-octane. Images were taken with a Zeiss epifluorescence microscope equipped with a thermoelectronically cooled charge coupled device camera (Photometrics CH250), which was controlled by an Apple Macintosh computer. Gray scale images were pseudocolored and merged using Oncor Image and Adobe Photoshop software. Measurement of apoptosis Annexin V binds in a calcium-dependent manner to phosphatidylserine, which is translocated from the interior side of the plasma membrane to the outer leaflet during the early stages of apoptosis (Van Engeland et al., 1996). Fluorescein-conjugated annexin V-Fluos (Boehringer Mannheim) was added to the cell culture medium at a final concentration of 1.5 µg/ml for three minutes. Harvested cells were washed twice with fresh culture medium to remove excess annexin V, resuspended in 10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl and 5 mM CaCl2 at a density of approximately 105 cells/ml and centrifuged onto glass slides. After fixation in methanol for 30 minutes, the preparations were incubated with rabbit anti-Rad51 antiserum and Cy3-conjugated anti-rabbit IgG.
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Measurement of DNA replication synthesis In order to visualize cycling cells in situ, 10 µg/ml of 5bromodeoxyuridine (BrdU) was added to the culture medium either two hours or 24 hours before cell harvesting. In place of thymidine, BrdU is incorporated into the DNA of replicating cells. After Rad51 protein staining, the immunofluorescent preparations were fixed overnight in a 3:1 mixture of methanol and acetic acid at –20oC. Since the anti-BrdU antibody only recognizes its epitope if the BrdUsubstituted chromosomal DNA is in the single-stranded form, the slides were denatured in 70% formamide, 2×SSC for one minute at 80oC and then dehydrated in an alcohol series. BrdU incorporation was visualized by indirect anti-BrdU antibody (Boehringer Mannheim) staining. Only cells with intense BrdU labeling of the entire nucleus were considered BrdU-positive and scored as cycling cells. Immunoelectron microscopy Cells were grown on coverslips, fixed in methanol at –20oC for 30 minutes and then in acetone for five minutes. After drying, the cells were treated with 0.5% Triton X-100 in PBS, washed 3×5 minutes in PBS and incubated for one to four hours at 37oC with rabbit antiRad51 antibodies. After washing for 3×5 minutes with PBS, the cells were incubated overnight at 4°C with anti-rabbit IgG coupled to 12 nm colloidal gold particles. The cells were washed again for 10 minutes with PBS and fixed with 2% glutaraldehyde in 50 mM cacodylate buffer, pH 7.2, on ice, followed by treatment with 2% OsO4 in H2O for two hours at room temperature. Samples were dehydrated in an ethanol series and after propyleneoxide treatment embedded in Araldite (Agar Scientific). Ultrathin sections were stained for 20 minutes in 4% uranyl acetate and for 15 minutes in lead citrate and viewed with a Phillips CM100 electron microscope.
Results Overexpression of Rad51 protein induces higher-order nuclear structures Human PPL fibroblasts and rat TGR fibroblasts were stably infected with plasmid pEG928.1 in which the retrovirus-based vector pLXSH carries the whole coding sequence of HsRad51. Western blotting showed an approximately 1.5-fold increase in total Rad51 protein in the human Rad51-overexpressing cell line, PPL928.1-2 (Fig. 1A) and an approximately 2.5-fold overexpression of Rad51 in rat TGR928.1-9 cells (Fig. 1B) compared with the respective parental controls. The affinity-purified antibodies used for western analysis detected two Rad51 bands in human wild-type PPL and overexpressing PPL928.1-2 cells (Fig. 1A). The upper 39-kDa band had the same mobility as HsRad51 expressed in E. coli (data not shown) and, therefore, corresponds to the full-length Rad51 protein. The lower approximately 37-kDa band most probably represents a cleavage product following proteolysis, a mechanism that has been described for Rad51 protein in apoptotic cells (Flygare et al., 1998). As the net amount of fulllength Rad51 protein decreased in etoposide-treated cells, we conclude that the formation of nuclear Rad51 foci in response to DNA damage is not due to de novo protein synthesis (Haaf et al., 1995; Haaf et al., 1999). By immunofluorescence staining, 1-20% of cells in stably Rad51-overexpressing cell populations, PPL928.1-2 and TGR928.1-9, and in transiently tranfected PPL cultures showed nuclear foci which were indistinguishable from DNAdamage-induced Rad51 foci in wild-type cells. In addition, up
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Journal of Cell Science 115 (1) Fig. 1. Detection of overexpressed Rad51 protein in stably transfected cell lines by western blotting. (A) Human wild-type PPL and Rad51-overexpressing PPL928.1-2 cells. Total cell extracts were prepared from untreated and etoposide (etop.)-treated cultures. Equal amounts of total cellular protein were separated by electrophoresis and subjected sequentially to immunoblot analysis with antibodies to Rad51 and βactin. Antibody binding was quantified by densitometric analysis. The β-actin signals were used to equilibrate the slightly different amounts of cell extract loaded per lane. Measurements from three independent western blot experiments were averaged. (B) Rat TGR and Rad51-overexpressing TGR928.1-9 cells. The amount of Rad51 in untreated wild-type (PPL or TGR) cells was chosen as a reference (100%).
to 20% of nuclei from these untreated cells displayed elongated higher-order structures up to 20-30 µm in length. Some nuclei were filled with a network of linear Rad51 structures (Fig. 2A). In contrast, linear Rad51 structures were only rarely (
