Abstract
Cell cycle checkpoints play a key role in maintaining genome stability by monitoring the order and integrity of cell division events. Checkpoints induced by DNA damage function to limit the propagation of potentially deleterious mutations. The retinoblastoma tumor suppressor (RB) is a critical effector of DNA damage checkpoint function by eliciting G1-phase cell cycle arrest following genotoxic stress. Here, we describe methodologies for evaluation of three facets of RB action in the DNA damage checkpoint response: (1) transcriptional repression of E2F-regulated genes (cyclin A reporter assay); (2) induction of cell cycle arrest (Brd-U incorporation assay); and (3) inhibition of DNA double-strand break accumulation (phosphorylated-histone H2A.X detection). Together, this combination of techniques allows the evaluation of RB action in the coordinated checkpoint response to DNA damage.
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References
Elledge, S. J. (1996) Cell cycle checkpoints: preventing an identity crisis. Science 274, 1664–1672.
Bartek, J. and Lukas, J. (2001) Mammalian G1-and S-phase checkpoints in response to DNA damage. Curr. Opin. Cell Biol. 13, 738–747.
Zhou, B. B. and Elledge, S. J. (2000) The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439.
Nyberg, K. A., Michelson, R. J., Putnam, C. W., and Weinert, T. A. (2002) Toward maintaining the genome: DNA damage and replication checkpoints. Annu. Rev. Genet. 36, 617–656.
Harrington, E. A., Bruce, J. L., Harlow, E., and Dyson, N. (1998) pRB plays an essential role in cell cycle arrest induced by DNA damage. Proc. Natl. Acad. Sci. USA 95, 11,945–11,950.
Brugarolas, J., Moberg, K., Boyd, S. D., Taya, Y., Jacks, T., and Lees, J. A. (1999) Inhibition of cyclin-dependent kinase 2 by p21 is necessary for retinoblastoma protein-mediated G1 arrest after gamma-irradiation. Proc. Natl. Acad. Sci. USA 96, 1002–1007.
Mittnacht, S. (1998) Control of pRB phosphorylation. Curr. Opin. Genet. Dev. 8, 21–27.
Dyson, N. (1998) The regulation of E2F by pRB-family proteins. Genes Dev. 12, 2245–2262.
Harbour, J. W. and Dean, D. C. (2000) Chromatin remodeling and Rb activity. Curr. Opin. Cell Biol. 12, 685–689.
Nakamura, T., Okuyama, S., Okamoto, S., Nakajima, T., Sekiya, S., and Oda, K. (1995) Down-regulation of the cyclin A promoter in differentiating human embryonal carcinoma cells is mediated by depletion of ATF-1 and ATF-2 in the complex at the ATF/CRE site. Exp. Cell Res. 216, 422–430.
Paull, T. T., Rogakou, E. P., Yamazaki, V., Kirchgessner, C. U., Gellert, M., and Bonner, W. M. (2000) A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10, 886–895.
Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S., and Bonner, W. M. (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868.
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© 2004 Humana Press Inc.
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Mayhew, C.N., Bosco, E.E., Solomon, D.A., Knudsen, E.S., Angus, S.P. (2004). Analysis of RB Action in DNA Damage Checkpoint Response. In: Schönthal, A.H. (eds) Checkpoint Controls and Cancer. Methods in Molecular Biology, vol 281. Humana Press. https://doi.org/10.1385/1-59259-811-0:003
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DOI: https://doi.org/10.1385/1-59259-811-0:003
Publisher Name: Humana Press
Print ISBN: 978-1-58829-500-2
Online ISBN: 978-1-59259-811-3
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