Targeted deletion of mouse Rad1 leads to deficient cellular DNA damage responses

Abstract

The Rad1 gene is evolutionarily conserved from yeast to human. The fission yeast Schizosaccharomyces pombeRad1 ortholog promotes cell survival against DNA damage and is required for G₂/M checkpoint activation. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad1, the mouse ortholog of this gene, were created to evaluate its function in mammalian cells. Mrad1^-/- ES cells were highly sensitive to ultraviolet-light (UV light), hydroxyurea (HU) and gamma rays, and were defective in G₂/M as well as S/M checkpoints. These data indicated that Mrad1 is required for repairing DNA lesions induced by UV-light, HU and gamma rays, and for mediating G₂/M and S/M checkpoint controls. We further demonstrated that Mrad1 plays an important role in homologous recombination repair (HRR) in ES cells, but a minor HRR role in differentiated mouse cells.

Keywords

Rad1 / DNA damage / checkpoint signaling / DNA repair / homologous recombination repair

References

[1] al-Khodairy, F., and Carr, A.M. (1992). DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe. EMBO J 11, 1343–1350 .1563350
[2] Aladjem, M.I., Spike, B.T., Rodewald, L.W., Hope, T.J., Klemm, M., Jaenisch, R., and Wahl, G.M. (1998). ES cells do not activate p53-dependent stress responses and undergo p53-independent apoptosis in response to DNA damage. Curr Biol 8, 145–155 .9443911
[3] An, L., Wang, Y., Liu, Y., Yang, X., Liu, C., Hu, Z., He, W., Song, W., and Hang, H. (2010). Rad9 is required for B cell proliferation and immunoglobulin class switch recombination. J Biol Chem 285, 35267–35273 .20729201
[4] Bao, S., Lu, T., Wang, X., Zheng, H., Wang, L.E., Wei, Q., Hittelman, W.N., and Li, L. (2004). Disruption of the Rad9/Rad1/Hus1 (9-1-1) complex leads to checkpoint signaling and replication defects. Oncogene 23, 5586–5593 .15184880
[5] Bermudez, V.P., Lindsey-Boltz, L.A., Cesare, A.J., Maniwa, Y., Griffith, J.D., Hurwitz, J., and Sancar, A. (2003). Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro. Proc Natl Acad Sci U S A 100, 1633–1638 .12578958
[6] Burtelow, M.A., Roos-Mattjus, P.M., Rauen, M., Babendure, J.R., and Karnitz, L.M. (2001). Reconstitution and molecular analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA damage responsive checkpoint complex. J Biol Chem 276, 25903–25909 .11340080
[7] Doré, A.S., Kilkenny, M.L., Rzechorzek, N.J., and Pearl, L.H. (2009). Crystal structure of the rad9-rad1-hus1 DNA damage checkpoint complex—implications for clamp loading and regulation. Mol Cell 34, 735–745 .19446481
[8] Ellison, V., and Stillman, B. (2003). Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5′ recessed DNA. PLoS Biol 1, E33.14624239
[9] Enoch, T., Carr, A.M., and Nurse, P. (1992). Fission yeast genes involved in coupling mitosis to completion of DNA replication. Genes Dev 6, 2035–2046 .1427071
[10] Freire, R., Murguía, J.R., Tarsounas, M., Lowndes, N.F., Moens, P.B., and Jackson, S.P. (1998). Human and mouse homologs of Schizosaccharomyces pombe rad1(+) and Saccharomyces cerevisiae RAD17: linkage to checkpoint control and mammalian meiosis. Genes Dev 12, 2560–2573 .9716408
[11] Han, L., Hu, Z., Liu, Y., Wang, X., Hopkins, K.M., Lieberman, H.B., and Hang, H. (2010). Mouse Rad1 deletion enhances susceptibility for skin tumor development. Mol Cancer 9, 67.20334655
[12] Hang, H., and Fox, M.H. (2004). Analysis of the mammalian cell cycle by flow cytometry. Methods Mol Biol 241, 23–35 .14970644
[13] Hang, H., and Lieberman, H.B. (2000). Physical interactions among human checkpoint control proteins HUS1p, RAD1p, and RAD9p, and implications for the regulation of cell cycle progression. Genomics 65, 24–33 .10777662
[14] Hartwell, L.H., and Weinert, T.A. (1989). Checkpoints: controls that ensure the order of cell cycle events. Science 246, 629–634 .2683079
[15] Hopkins, K.M., Auerbach, W., Wang, X.Y., Hande, M.P., Hang, H., Wolgemuth, D.J., Joyner, A.L., and Lieberman, H.B. (2004). Deletion of mouse rad9 causes abnormal cellular responses to DNA damage, genomic instability, and embryonic lethality. Mol Cell Biol 24, 7235–7248 .15282322
[16] Hu, Z., Liu, Y., Zhang, C., Zhao, Y., He, W., Han, L., Yang, L., Hopkins, K.M., Yang, X., Lieberman, H.B., (2008). Targeted deletion of Rad9 in mouse skin keratinocytes enhances genotoxin-induced tumor development. Cancer Res 68, 5552–5561 .18632607
[17] Levitt, P.S., Liu, H., Manning, C., and Weiss, R.S. (2005). Conditional inactivation of the mouse Hus1 cell cycle checkpoint gene. Genomics 86, 212–224 .15919177
[18] Levitt, P.S., Zhu, M., Cassano, A., Yazinski, S.A., Liu, H., Darfler, J., Peters, R.M., and Weiss, R.S. (2007). Genome maintenance defects in cultured cells and mice following partial inactivation of the essential cell cycle checkpoint gene Hus1. Mol Cell Biol 27, 2189–2201 .17220276
[19] Lieberman, H.B., Hopkins, K.M., Laverty, M., and Chu, H.M. (1992). Molecular cloning and analysis of Schizosaccharomyces pombe rad9, a gene involved in DNA repair and mutagenesis. Mol Gen Genet 232, 367–376 .1588907
[20] Lindsey-Boltz, L.A., Bermudez, V.P., Hurwitz, J., and Sancar, A. (2001). Purification and characterization of human DNA damage checkpoint Rad complexes. Proc Natl Acad Sci U S A 98, 11236–11241 .11572977
[21] Longhese, M.P., Paciotti, V., Fraschini, R., Zaccarini, R., Plevani, P., and Lucchini, G. (1997). The novel DNA damage checkpoint protein ddc1p is phosphorylated periodically during the cell cycle and in response to DNA damage in budding yeast. EMBO J 16, 5216–5226 .9311982
[22] Lydall, D., and Weinert, T. (1997). G2/M checkpoint genes of Saccharomyces cerevisiae: further evidence for roles in DNA replication and/or repair. Mol Gen Genet 256, 638–651 .9435789
[23] Maynard, S., Swistowska, A.M., Lee, J.W., Liu, Y., Liu, S.T., Da Cruz, A.B., Rao, M., de Souza-Pinto, N.C., Zeng, X., and Bohr, V.A. (2008). Human embryonic stem cells have enhanced repair of multiple forms of DNA damage. Stem Cells 26, 2266–2274 .18566332
[24] Murray, J.M., Carr, A.M., Lehmann, A.R., and Watts, F.Z. (1991). Cloning and characterisation of the rad9 DNA repair gene from Schizosaccharomyces pombe. Nucleic Acids Res 19, 3525–3531 .1852603
[25] Parker, A.E., Van de Weyer, I., Laus, M.C., Oostveen, I., Yon, J., Verhasselt, P., and Luyten, W.H. (1998). A human homologue of the Schizosaccharomyces pombe rad1+ checkpoint gene encodes an exonuclease. J Biol Chem 273, 18332–18339 .9660799
[26] Parrilla-Castellar, E.R., Arlander, S.J., and Karnitz, L. (2004). Dial 9-1-1 for DNA damage: the Rad9-Hus1-Rad1 (9-1-1) clamp complex. DNA Repair (Amst) 3, 1009–1014 .15279787
[27] Paulovich, A.G., and Hartwell, L.H. (1995). A checkpoint regulates the rate of progression through S phase in S. cerevisiae in response to DNA damage. Cell 82, 841–847 .7671311
[28] Pierce, A.J., Hu, P., Han, M., Ellis, N., and Jasin, M. (2001). Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells. Genes Dev 15, 3237–3242 .11751629
[29] Rauen, M., Burtelow, M.A., Dufault, V.M., and Karnitz, L.M. (2000). The human checkpoint protein hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9. J Biol Chem 275, 29767–29771 .10884395
[30] Roos-Mattjus, P., Vroman, B.T., Burtelow, M.A., Rauen, M., Eapen, A.K., and Karnitz, L.M. (2002). Genotoxin-induced Rad9-Hus1-Rad1 (9-1-1) chromatin association is an early checkpoint signaling event. J Biol Chem 277, 43809–43812 .12228248
[31] Rowley, R., Subramani, S., and Young, P.G. (1992). Checkpoint controls in Schizosaccharomyces pombe: rad1. EMBO J 11, 1335–1342 .1563349
[32] Shiomi, Y., Shinozaki, A., Nakada, D., Sugimoto, K., Usukura, J., Obuse, C., and Tsurimoto, T. (2002). Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9-1-1 and Rad17-RFC. Genes Cells 7, 861–868 .12167163
[33] Sohn, S.Y., and Cho, Y. (2009). Crystal structure of the human rad9-hus1-rad1 clamp. J Mol Biol 390, 490–502 .19464297
[34] Tichy, E.D., and Stambrook, P.J. (2008). DNA repair in murine embryonic stem cells and differentiated cells. Exp Cell Res 314, 1929–1936 .18374918
[35] Udell, C.M., Lee, S.K., and Davey, S. (1998). HRAD1 and MRAD1 encode mammalian homologues of the fission yeast rad1(+) cell cycle checkpoint control gene. Nucleic Acids Res 26, 3971–3976 .9705507
[36] Wang, X., Guan, J., Hu, B., Weiss, R.S., Iliakis, G., and Wang, Y. (2004). Involvement of Hus1 in the chain elongation step of DNA replication after exposure to camptothecin or ionizing radiation. Nucleic Acids Res 32, 767–775 .14762204
[37] Wang, X., Hu, B., Weiss, R.S., and Wang, Y. (2006). The effect of Hus1 on ionizing radiation sensitivity is associated with homologous recombination repair but is independent of nonhomologous end-joining. Oncogene 25, 1980–1983 .16278671
[38] Weiss, R.S., Enoch, T., and Leder, P. (2000). Inactivation of mouse Hus1 results in genomic instability and impaired responses to genotoxic stress. Genes Dev 14, 1886–1898 .10921903
[39] Weiss, R.S., Leder, P., and Vaziri, C. (2003). Critical role for mouse Hus1 in an S-phase DNA damage cell cycle checkpoint. Mol Cell Biol 23, 791–803 .12529385
[40] Xu, M., Bai, L., Gong, Y., Xie, W., Hang, H., and Jiang, T. (2009). Structure and functional implications of the human rad9-hus1-rad1 cell cycle checkpoint complex. J Biol Chem 284, 20457–20461 .19535328
[41] Yazinski, S.A., Westcott, P.M., Ong, K., Pinkas, J., Peters, R.M., and Weiss, R.S. (2009). Dual inactivation of Hus1 and p53 in the mouse mammary gland results in accumulation of damaged cells and impaired tissue regeneration. Proc Natl Acad Sci U S A 106, 21282–21287 .19918068
[42] Zhou, M., Zheng, L., Guo, L., and Ding, Z. (2010). Cell Biological Effect and Mechanism of Ultraviolet Radiation. Acta Biophysica Sinica 26, 950–958 .