Mammalian Rad9 plays a role in telomere stability, S- and G2-phase-specific cell survival, and homologous recombinational repair

Abstract

The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of gamma-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.

FIG. 1.

Expression of hRad9. (A) hRad9 protein levels were detected by Western analysis with hRad9-specific antibody in human 293 cells. hRad9 levels in cells expressing mutant (Δ) and wild-type hRad9 are shown. Higher levels of the protein were detected in cells engineered to overexpress hRad9, relative to the vector control, but no difference in hRad9 levels was seen in cells ectopically expressing ΔhRad9. (B) hRad9 levels in different phases of the cell cycle as detected in cell extracts of human 293 cells by immunoblotting with anti-hRad9 antibody. Human 293 cells in different phases were enriched by centrifugal elutriation (41). Lane 1 represents an asynchronous population, lane 2 represents a G₁-phase-enriched population, lane 3 represents an S-phase-enriched population, and lane 4 represents a G₂/M-phase-enriched cell population. (a) hRad9 levels in cells in different phases of the cell cycle. (b) hRad9 levels in cells expressing ΔhRad9 in different phases of the cell cycle. No difference in the levels of hRad9 was seen in different phases of the cell cycle. (C) siRNA-mediated reduction of hRad9 protein in 293 and MCF-7 cells. Cells were transfected with siRNA, and cell lysates were made 48 h posttransfection and examined for hRad9 protein by immunoblotting with anti-hRad9 antibody.

FIG. 2.

Telomere analysis by single-strand extensions (G tails) and telomere FISH. (A) G-tail sizes in cells with or without hRad9 inactivation. Detection of sizes of TRFs on DNA derived from 293 cells with or without hRad9 knockdown. In part a, nondenaturing in-gel hybridizations to genomic DNA digested with restriction enzymes HinfI and RsaI and with a telomeric repeat probe of the C-rich strand are shown. This method allows visualizing G-strand overhangs on telomeres. Signals were quantified by PhosphorImager analysis and corrected for DNA loading by using the rehybridized gel shown in part b. Lane M, molecular mass standards; lane 1, DNA from 293 cells with empty vector; lane 2, DNA from 293 cells expressing ΔhRad9; lane 3, DNA from 293 cells with hRad9 siRNA; lane 4, DNA from 293 cells with control siRNA; lane ss, denatured plasmid single-stranded DNA containing telomeric repeats (positive control); lane ds, double-stranded plasmid DNA used as a negative control (detected only once the DNA is denatured, as shown in part b). Part a shows the same gel as in part b after denaturing of the DNA in the gel and rehybridization with the same probe. The arrow in part b indicates an internal restriction fragment carrying telomeric repeats that was used to correct for DNA loading. (B) Telomere FISH analysis showing human metaphase chromosomal spreads. Telomere signals are red, and centromere signals are green. Part a, metaphase of 293 cells with empty vector; part b, metaphase of 293 cells with control siRNA; part c, metaphase section of MCF-7 cells with control siRNA; part d, metaphase of 293 cells with ΔhRad9; part e, metaphase section of 293 cells with hRad9 siRNA; part f, metaphase section of MCF-7 cells with hRad9 siRNA; part g, metaphase section of 293 cells with mutant TRF2^ΔBΔM showing a chain of chromosomes resulting from single and both TAs; part h, TA with the interstitial region of a chromatin seen in 293 cells expressing mutant TRF2^ΔBΔM; part i, metaphase of 293 cells with expression of mutant TRF2^ΔBΔM and knockdown of hRad9 (note that almost all chromosomes undergo telomere fusions); part j, representative dicentric chromosome with fused telomeres seen in 293 cells expressing mutant TRF2 and inactivated hRad9. Note chromosome end associations, loss of telomeric signals, and gaps, as indicated by arrows. (C) Mouse metaphase chromosomes. Part a, representative metaphase of Mrad9^+/+ cells; part b, Mrad9^−/− metaphase showing polyploidy; part c, representative chromosome end association without loss of telomere signal at the association site seen in Mrad9^−/− cells (Robertsonian fusion); part d, telomere fusion and loss of telomere signals as indicated by arrows in Mrad9^−/− cells. Mrad9^−/− ES cells have chromosome end associations, as well as loss of telomeric signals. (D) hRad9 interacts with TRF2 and Rad51. Immunoprecipitation of endogenous hRad9, TRF2, and Rad51 in 293 cells. Cell extracts were immunoprecipitated with anti-hRad9 antibody, followed by immunoblotting with antibodies to TRF2, Rad51, and hRad9. IgG, immunoglobulin G.

FIG. 2.

Telomere analysis by single-strand extensions (G tails) and telomere FISH. (A) G-tail sizes in cells with or without hRad9 inactivation. Detection of sizes of TRFs on DNA derived from 293 cells with or without hRad9 knockdown. In part a, nondenaturing in-gel hybridizations to genomic DNA digested with restriction enzymes HinfI and RsaI and with a telomeric repeat probe of the C-rich strand are shown. This method allows visualizing G-strand overhangs on telomeres. Signals were quantified by PhosphorImager analysis and corrected for DNA loading by using the rehybridized gel shown in part b. Lane M, molecular mass standards; lane 1, DNA from 293 cells with empty vector; lane 2, DNA from 293 cells expressing ΔhRad9; lane 3, DNA from 293 cells with hRad9 siRNA; lane 4, DNA from 293 cells with control siRNA; lane ss, denatured plasmid single-stranded DNA containing telomeric repeats (positive control); lane ds, double-stranded plasmid DNA used as a negative control (detected only once the DNA is denatured, as shown in part b). Part a shows the same gel as in part b after denaturing of the DNA in the gel and rehybridization with the same probe. The arrow in part b indicates an internal restriction fragment carrying telomeric repeats that was used to correct for DNA loading. (B) Telomere FISH analysis showing human metaphase chromosomal spreads. Telomere signals are red, and centromere signals are green. Part a, metaphase of 293 cells with empty vector; part b, metaphase of 293 cells with control siRNA; part c, metaphase section of MCF-7 cells with control siRNA; part d, metaphase of 293 cells with ΔhRad9; part e, metaphase section of 293 cells with hRad9 siRNA; part f, metaphase section of MCF-7 cells with hRad9 siRNA; part g, metaphase section of 293 cells with mutant TRF2^ΔBΔM showing a chain of chromosomes resulting from single and both TAs; part h, TA with the interstitial region of a chromatin seen in 293 cells expressing mutant TRF2^ΔBΔM; part i, metaphase of 293 cells with expression of mutant TRF2^ΔBΔM and knockdown of hRad9 (note that almost all chromosomes undergo telomere fusions); part j, representative dicentric chromosome with fused telomeres seen in 293 cells expressing mutant TRF2 and inactivated hRad9. Note chromosome end associations, loss of telomeric signals, and gaps, as indicated by arrows. (C) Mouse metaphase chromosomes. Part a, representative metaphase of Mrad9^+/+ cells; part b, Mrad9^−/− metaphase showing polyploidy; part c, representative chromosome end association without loss of telomere signal at the association site seen in Mrad9^−/− cells (Robertsonian fusion); part d, telomere fusion and loss of telomere signals as indicated by arrows in Mrad9^−/− cells. Mrad9^−/− ES cells have chromosome end associations, as well as loss of telomeric signals. (D) hRad9 interacts with TRF2 and Rad51. Immunoprecipitation of endogenous hRad9, TRF2, and Rad51 in 293 cells. Cell extracts were immunoprecipitated with anti-hRad9 antibody, followed by immunoblotting with antibodies to TRF2, Rad51, and hRad9. IgG, immunoglobulin G.

FIG. 2.

Telomere analysis by single-strand extensions (G tails) and telomere FISH. (A) G-tail sizes in cells with or without hRad9 inactivation. Detection of sizes of TRFs on DNA derived from 293 cells with or without hRad9 knockdown. In part a, nondenaturing in-gel hybridizations to genomic DNA digested with restriction enzymes HinfI and RsaI and with a telomeric repeat probe of the C-rich strand are shown. This method allows visualizing G-strand overhangs on telomeres. Signals were quantified by PhosphorImager analysis and corrected for DNA loading by using the rehybridized gel shown in part b. Lane M, molecular mass standards; lane 1, DNA from 293 cells with empty vector; lane 2, DNA from 293 cells expressing ΔhRad9; lane 3, DNA from 293 cells with hRad9 siRNA; lane 4, DNA from 293 cells with control siRNA; lane ss, denatured plasmid single-stranded DNA containing telomeric repeats (positive control); lane ds, double-stranded plasmid DNA used as a negative control (detected only once the DNA is denatured, as shown in part b). Part a shows the same gel as in part b after denaturing of the DNA in the gel and rehybridization with the same probe. The arrow in part b indicates an internal restriction fragment carrying telomeric repeats that was used to correct for DNA loading. (B) Telomere FISH analysis showing human metaphase chromosomal spreads. Telomere signals are red, and centromere signals are green. Part a, metaphase of 293 cells with empty vector; part b, metaphase of 293 cells with control siRNA; part c, metaphase section of MCF-7 cells with control siRNA; part d, metaphase of 293 cells with ΔhRad9; part e, metaphase section of 293 cells with hRad9 siRNA; part f, metaphase section of MCF-7 cells with hRad9 siRNA; part g, metaphase section of 293 cells with mutant TRF2^ΔBΔM showing a chain of chromosomes resulting from single and both TAs; part h, TA with the interstitial region of a chromatin seen in 293 cells expressing mutant TRF2^ΔBΔM; part i, metaphase of 293 cells with expression of mutant TRF2^ΔBΔM and knockdown of hRad9 (note that almost all chromosomes undergo telomere fusions); part j, representative dicentric chromosome with fused telomeres seen in 293 cells expressing mutant TRF2 and inactivated hRad9. Note chromosome end associations, loss of telomeric signals, and gaps, as indicated by arrows. (C) Mouse metaphase chromosomes. Part a, representative metaphase of Mrad9^+/+ cells; part b, Mrad9^−/− metaphase showing polyploidy; part c, representative chromosome end association without loss of telomere signal at the association site seen in Mrad9^−/− cells (Robertsonian fusion); part d, telomere fusion and loss of telomere signals as indicated by arrows in Mrad9^−/− cells. Mrad9^−/− ES cells have chromosome end associations, as well as loss of telomeric signals. (D) hRad9 interacts with TRF2 and Rad51. Immunoprecipitation of endogenous hRad9, TRF2, and Rad51 in 293 cells. Cell extracts were immunoprecipitated with anti-hRad9 antibody, followed by immunoblotting with antibodies to TRF2, Rad51, and hRad9. IgG, immunoglobulin G.

FIG. 3.

Comparison of gamma ray sensitivity. Dose-response curves are shown for 293 cells with or without hRad9 knockdown after IR exposure. The A-T cell line GM5823+hTERT was used as a control to indicate radiosensitivity. (A) Survival curves for exponentially growing cells. Note that cells with hRad9 siRNA or expression of ΔhRad9 have lower survival after IR treatment. (B to D) Survival through the cell cycle of cells with or without ΔhRad9. Dose-response survival curves for cells in the G₁ phase (B), the S phase (C), and the G₂ phase (D) are shown. The cells with ΔhRad9 expression have statistically significantly (P < 0.01 as determined by the chi-square test) enhanced cell killing compared to control cells in the S and G₂ phases. The values shown are means from three experiments.

FIG. 4.

Appearance of γ-H2AX in cells with or without hRad9. Cells were irradiated and collected at different times postexposure. (A) Human 293 cells were irradiated, and γ-H2AX foci were observed at various times postirradiation. For each time point, 100 cells were analyzed. Each experiment was repeated three times. The mean number of foci is plotted against time. Cells with reduced levels of hRad9 caused by hRad9 siRNA showed fewer γ-H2Ax foci until 5 min postirradiation, and the foci persisted for a longer time. (B) Mouse cells with or without Mrad9 were grown on coverslips, labeled with BrdU for 30 min, irradiated with 1 Gy of gamma rays, and processed for detection of foci as previously described (24, 46). BrdU labeling and γ-H2AX were detected by anti-BrdU and anti-phospho-histone-H2AX antibodies, respectively (Upstate).

FIG. 4.

Appearance of γ-H2AX in cells with or without hRad9. Cells were irradiated and collected at different times postexposure. (A) Human 293 cells were irradiated, and γ-H2AX foci were observed at various times postirradiation. For each time point, 100 cells were analyzed. Each experiment was repeated three times. The mean number of foci is plotted against time. Cells with reduced levels of hRad9 caused by hRad9 siRNA showed fewer γ-H2Ax foci until 5 min postirradiation, and the foci persisted for a longer time. (B) Mouse cells with or without Mrad9 were grown on coverslips, labeled with BrdU for 30 min, irradiated with 1 Gy of gamma rays, and processed for detection of foci as previously described (24, 46). BrdU labeling and γ-H2AX were detected by anti-BrdU and anti-phospho-histone-H2AX antibodies, respectively (Upstate).

FIG. 5.

Analysis of chromosome damage and repair in 293 cells with or without wild-type levels of hRad9. (A to C) Chromosomal aberrations analyzed at metaphase. (A) Cells in plateau phase were irradiated with 3 Gy, incubated for 24 h postirradiation, and then subcultured, and metaphases were collected. G₁-type aberrations were examined at metaphase. All categories of asymmetric chromosome aberrations were scored: dicentrics, centric rings, interstitial deletions/acentric rings, and terminal deletions. The frequency of chromosomal aberrations was identical for cells with or without wild-type levels of hRad9, indicating normal repair of G₁-phase-specific chromosome damage. (B) Cells in exponential phase were irradiated with 2 Gy. Metaphases were harvested at 4 h after irradiation, and S-phase types of chromosomal aberrations were scored. Cells with hRad9 knockdown showed significant differences in chromosomal aberration frequencies compared to control cells (P < 0.05, Student t test). (C) Cells in exponential phase were irradiated with 1 Gy. Metaphases were harvested following irradiation, and G₂-type chromosomal aberrations were monitored. Cells with hRad9 knockdown showed significantly higher (P < 0.05, Student t test) frequencies of chromosomal aberrations compared to control cells. (D and E) Chromosome break repair in cells in G₁- and G₂-phase cells. (D) Chromosome break repair in G₁-phase cells. Cells were irradiated with 3 Gy of IR, and chromosome damage was analyzed by a PCC technique. Sixty PCCs were scored for each time point, and the data represent means and standard deviations of two sets of experiments. The mean number of chromosome breaks per cell immediately after irradiation was about 12, and this number was reduced after incubation at 37°C. Cells with or without hRad9 knockdown show no difference in the kinetics or residual levels of breaks. (E) Chromatid gap and break repair in G₂-phase cells. Cells were irradiated with 3 Gy of IR, and chromatid gaps and breaks were analyzed by the PCC technique. The mean number of chromosome breaks per cell immediately after irradiation was about 10, and this number was reduced after incubation at 37°C. Forty five PCCs were scored for each time point, and the data are the mean and standard deviation of two sets of triplicate sample experiments. Note that the rate of chromosome repair is slow in cells with Rad9 siRNA, and such cells have higher frequencies of residual chromosome damage. The difference in residual damage in cells with or without wild-type levels of hRad9 is statistically significant, as demonstrated by the Student t test (P < 0.05).

FIG. 5.

Analysis of chromosome damage and repair in 293 cells with or without wild-type levels of hRad9. (A to C) Chromosomal aberrations analyzed at metaphase. (A) Cells in plateau phase were irradiated with 3 Gy, incubated for 24 h postirradiation, and then subcultured, and metaphases were collected. G₁-type aberrations were examined at metaphase. All categories of asymmetric chromosome aberrations were scored: dicentrics, centric rings, interstitial deletions/acentric rings, and terminal deletions. The frequency of chromosomal aberrations was identical for cells with or without wild-type levels of hRad9, indicating normal repair of G₁-phase-specific chromosome damage. (B) Cells in exponential phase were irradiated with 2 Gy. Metaphases were harvested at 4 h after irradiation, and S-phase types of chromosomal aberrations were scored. Cells with hRad9 knockdown showed significant differences in chromosomal aberration frequencies compared to control cells (P < 0.05, Student t test). (C) Cells in exponential phase were irradiated with 1 Gy. Metaphases were harvested following irradiation, and G₂-type chromosomal aberrations were monitored. Cells with hRad9 knockdown showed significantly higher (P < 0.05, Student t test) frequencies of chromosomal aberrations compared to control cells. (D and E) Chromosome break repair in cells in G₁- and G₂-phase cells. (D) Chromosome break repair in G₁-phase cells. Cells were irradiated with 3 Gy of IR, and chromosome damage was analyzed by a PCC technique. Sixty PCCs were scored for each time point, and the data represent means and standard deviations of two sets of experiments. The mean number of chromosome breaks per cell immediately after irradiation was about 12, and this number was reduced after incubation at 37°C. Cells with or without hRad9 knockdown show no difference in the kinetics or residual levels of breaks. (E) Chromatid gap and break repair in G₂-phase cells. Cells were irradiated with 3 Gy of IR, and chromatid gaps and breaks were analyzed by the PCC technique. The mean number of chromosome breaks per cell immediately after irradiation was about 10, and this number was reduced after incubation at 37°C. Forty five PCCs were scored for each time point, and the data are the mean and standard deviation of two sets of triplicate sample experiments. Note that the rate of chromosome repair is slow in cells with Rad9 siRNA, and such cells have higher frequencies of residual chromosome damage. The difference in residual damage in cells with or without wild-type levels of hRad9 is statistically significant, as demonstrated by the Student t test (P < 0.05).

FIG. 6.

Cell cycle checkpoint analysis. (A) Human 293 cells were exposed to 8 Gy of IR. Twelve hours following irradiation, cells were pulse-labeled with BrdU for 30 min. The proportion of S-phase cells was determined. The percentage of S-phase cells in the irradiated culture relative to those in the unirradiated control is shown. The results represent the mean of three experiments. For each independent experiment, 200 cells were examined. Black bars represent unirradiated control cells that enter into S phase, as they are BrdU positive. Dashed bars represent cells that enter into S phase postirradiation, and their percentage is determined by dividing the number of S-phase irradiated cells by the number of S-phase unirradiated cells. (B) S-phase checkpoint was determined as radioresistant DNA synthesis after exposure to IR. 293 cells were irradiated at the doses indicated. The rate of DNA synthesis was determined 1 h postirradiation by pulse-labeling with [³H]thymidine for 30 min. The value of the unirradiated control was set to 100% for each cell type. The mean and standard deviation of triplicate experimental points are shown. (C) Comparison of the mitotic index among cells after treatment with 2 Gy of IR. Populations were examined for the frequency of mitotic cells at different times postirradiation. The mean represents the value from three independent experiments. For each experiment, 200 metaphases were scored. Note that the mitotic index decreased in cells whether or not hRad9 levels were wild type.

FIG. 7.

hRad9 is not required for IR-induced phosphorylation of ATM and Chk2. (A) 293 cells with or without hRad9 knockdown were irradiated with 2 Gy of IR. Cell extracts were immunoblotted with anti-ATM antibodies and anti-p-Ser-1981-ATM antibody. (B) 293 cells with or without hRad9 knockdown were irradiated with 5 Gy of IR. Cell extracts were immunoblotted with anti-Chk2 antibody and anti-p-T68-Chk2 antibodies. No difference in cells with or without hRad9 was observed for IR-induced phosphorylation of ATM or Chk2.

FIG. 8.

hRad9 is not essential for NHEJ. Depletion of hRad9 was achieved by two different approaches: immunodepletion and use of hRad9 siRNA. (A) Immunodepletion of hRad9 from whole 293-cell extracts as determined by immunoblotting. The extracts were precipitated with protein A-Sepharose beads only (lane 1), with preimmune serum (lane 2), and with hRad9-depleted whole-cell extract (depleted with hRad9 antibody) (lane 3). (B) Ethidium bromide-stained agarose gels show the results of end-joining assays. Plasmid puc19 DNA was linearized by digestion with SalI. The positions of monomer and dimer plasmids are indicated to the right of the gel. Lane 1, molecular weight (MW) markers; lane 2, only plasmid DNA; lane 3, only cell lysate; lane 4, cell lysate and DNA; lane 5, cell lysate with wortmannin and DNA; lane 6, immunoglobulin G (IgG) depleted in cell lysate and then added DNA; lane 7, DNA protein kinase (DNA-PK) depleted in cell lysate and then added DNA; lane 8, cell lysates of cells with control siRNA and plasmid DNA; lane 9, cell lysate from cells with hRad9 siRNA and plasmid DNA; lane 10, immunoglobulin G (IgG) depleted in cell lysate and DNA; lane 11, hRad9 depleted in 293 cell lysates and DNA; lane 12, hRad9 depleted in MCF-7 cells and DNA.

FIG. 9.

hRad9 knockdown influences HR. Impaired I-SceI-induced HR in hRad9-deficient MCF-7 cells was found. HR was measured by dual-color flow cytometric detection of GFP-positive cells. HR frequencies are shown with (+) or without (−) I-SceI induction for untreated cells, for cells treated with control siRNA, and for cells treated with hRad9-specific siRNA. hRad9-deficient and BRCA1-deficient cells show similar reductions in HR. The knockdown of BRCA1 by BRCA1 siRNA in MCF-7 cells has been described previously (72). The results presented are the mean and standard error from four independent experiments.