The function of mammalian Rad9, a gene involved in sensing DNA damage, was examined in the context of chromosome stability and DNA repair in mammalian cells. Human cells in which hRad9 was inactivated by expression of ΔhRad9, or levels of the protein were reduced by siRNA, or mouse cells with Mrad9
) were used. A cDNA fragment encoding aa 269 to 391 of hRad9 was expressed in different human cells, which were subsequently examined for various end points. We found that the C-terminal fragment of hRad9 (aa 269 to 391) demonstrated dominant negative activity for almost all of the assays employed. Thus, expression of this fragment might block the interaction of wild-type hRad9 with other proteins and thus has a dominant negative function. This function of the hRad9 fragment is consistent with the previously described observation of Wang and coworkers (67
), who reported that the C-terminal region of hRad9 interacts with other proteins. We found that, indeed, the frequencies of chromosomal abnormalities, such as chromosome end-to-end associations and chromosomal breaks, were higher in cells with inactivated mammalian Rad9. These chromosomal aberrations appear to involve dysfunctional telomeres, as a significant number of cells showed TAs. Thus, the results suggest that abrogating the function of mammalian Rad9, which is implicated in mediating DNA damage-sensing checkpoints, increases the frequency of unrepaired chromosomal aberrations that can lead to an increase in the number of telomere fusions. This mechanism might contribute to the inviability of mouse embryonic fibroblasts when Mrad9 is inactivated (23
). The frequencies of spontaneous, as well as IR-induced, chromosomal aberrations were dramatically higher in cells with inactivated Rad9 relative to those in controls, suggesting that loss of telomere function occurred by breakage near telomeres, rather than by a telomerase-based mechanism. This is supported by our finding that knockdown of hRad9 does not influence telomerase activity (data not shown). hRad9 forms a heterotrimer with Hus1 and Rad1, which is loaded onto DNA by Rad17-mediated mechanisms, and these proteins are evolutionarily conserved. Interestingly, studies with S. pombe
, S. cerevisiae
, and Caenorhabditis elegans
orthologues also indicate roles for these proteins in telomere metabolism (1
It is important to note that in cells with both alleles of Rad9 inactivated, frequent chromosome breakage with complete loss of telomeric repeats in some chromosomes, as well as chromosome end fusions, occurred under normal growth conditions in the absence of exogenous genotoxic challenges (Fig. and Table ). The fact that the G1 checkpoint was operative in cells with reduced levels of Rad9 is supported by the observations that irradiation induced comparable frequencies of cells in G1 and S phase compared to those cells with normal levels of hRad9 (Fig. ). Furthermore, cells with reduced levels of Rad9 did not show any defect in the S-phase-specific cell cycle checkpoint, as such cells did not differ from control cells for radioresistant DNA synthesis (Fig. ). The mitotic index after irradiation was identical in cells with or without functional Rad9, indicating that mammalian Rad9 inactivation does not influence the G2 checkpoint in the cell lines studied (Fig. ). hRad9 knockdown had no influence on IR-induced ATM Ser-1981 phosphorylation or Chk2 Thre-68 phosphorylation, supporting the argument that mammalian Rad9 has a minimum role in cell cycle checkpoint control postirradiation. In contrast, there was a clear and significant difference in the chromosome aberration frequency if log-phase cells were irradiated, and mitoses shortly thereafter were monitored; at 4 h postirradiation, there was a 1.8-fold difference in the frequencies of S-phase aberrations/metaphase between cells with and without Rad9 fully functional (Fig. ). Furthermore, when G2-induced aberrations were monitored after irradiation, cells with reduced levels of Rad9 showed higher frequencies of aberrations in the same time span compared to controls, indicating very inefficient repair (Fig. ). Utilizing the PCC technique, we found that there was no difference in the kinetics of repair or the residual number of chromosome breaks in G1-phase cells with or without reduced levels of hRad9 (Fig. ); however, cells deficient in Rad9 had a higher frequency of chromosome gaps and breaks in the G2 phase (Fig. ). Consistent with these data, survival studies demonstrated that cells in the S and G2 phases were sensitive to irradiation if Rad9 was inactivated (Fig. ). These data, taken together, reinforce the idea that in cells with hRad9 knockdown, chromosomal damage that might occur during the S or G2 phase could be due to defective chromosome repair (Fig. ). In cells lacking Rad9 protein, cell cycle arrest after DNA damage is operative and the cells do not enter mitosis exceptionally early. Our study reveals that as a consequence of this inability to appropriately repair, such cells enter mitosis with a higher frequency of chromosomal aberrations.
hRad9 is rapidly retained at sites of damaged DNA and colocalizes with the phosphorylated form of H2AX (γ-H2AX) (19
). In addition, we found quantitative differences in the kinetics of appearance and disappearance of γ-H2AX foci and kinetics of repair of G2
chromosomes (Fig. and ), dependent upon whether cells do or do not have functional mammalian Rad9. These results provide strong evidence that mammalian Rad9 plays a critical role in repair of DNA damage.
The association of hRad9 with TRF2 provides further evidence that hRad9 may have a role in maintaining the stability of telomeres. Although knockdown of hRad9 has a minimal role in TRF2 association with telomeres (data not shown) and inactivation of hRad9 and TRF2 did not rescue a telomere instability phenotype (Fig. ), it is possible that TRF2 might be mediating DNA damage signaling through Rad9. This notion is further supported by the data indicating that inactivation of hRad9 influences HR (Fig. ) but not NHEJ (Fig. ).
Our results demonstrate that hRad9 can influence telomere stability and HR. However, it is not clear exactly how the protein functions in these processes. It is possible that hRad9 modulates chromatin structure, an early essential step for HR. This is supported by our demonstration that hRad9 inactivation delays the appearance of IR-induced γ-H2AX foci. Although the data support a role for Rad9 in chromatin remodeling, especially at telomeres, it is not clear whether the protein can influence chromatin structure beyond this limited region. Such a general chromatin alteration should influence repair in all phases of the cell cycle, as has been observed in ATM null cells (42
). However, our results do not support this activity for hRad9 since inactivation of the protein resulted in an enhanced defect in chromosome repair confined only to the S and G2
phases of the cell cycle. Alternatively, Rad9 may interact with and influence the activity of proteins that have a role in HR repair, which occurs predominantly in S- and G2
-phase cells. This is supported by our demonstration of a physical interaction between hRad9 and hRad51. In fact, Shinohara and coworkers (58
) demonstrated that S. cerevisiae
orthologues of some of the hRad9 checkpoint pathway-related proteins interact with Rad51 for repair of DSBs in meiosis via recombination. Furthermore, Grushcow and coworkers (20
), as well as Aylon and Kupiec (2
), showed that the S. cerevisiae
equivalents of hRad9, hRad1, hHus1, and hRad17 pathway members are involved in recombination partner choice. In addition, we observed the delayed appearance of γ-H2AX foci in S-phase cells after IR exposure of Mrad9−/−
relative to Mrad9+/+
cells. Moreover, the high frequency of chromatid gaps and breaks in hRad9-inactivated cells supports a role for the protein in HR repair. Inactivation of mammalian Rad9 could thus contribute, at least in part, to the radiosensitivity, genomic instability, and loss of telomeres observed by mediating the abrogation of HR repair.