Abstract
Inhibition of DNA replication in eukaryotic cells was one of the earliest effects of radiation to be reported and quantitated. The elucidation of the mechanism of this inhibition has been the focus of research in several laboratories for four decades. A significant development in recent years is the recognition that the mechanism of this inhibition has both direct (cis-acting) and indirect (trans-acting) components (for a review, see 1). Whereas the direct component is thought to derive from radiation-induced DNA damage that alters chromatin structure and inhibits DNA replication in cis, the indirect component is attributed to the activation by DNA damage of regulatory processes that inhibit DNA replication in trans. The latter mechanism of inhibition is equivalent to the activation of a checkpoint in S-phase (2–5).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Iliakis, G. (1997) Cell cycle regulation in irradiated and nonirradiated cells. Semin. Oncol. 24, 602–615.
Elledge, S. J. (1996) Cell cycle checkpoints: Prevent an identity crisis. Science 274, 1664–1672.
Hartwell, L. H. and Kastan, M. B. (1994) Cell cycle control and cancer. Science 266, 1821–1828.
Hartwell, L. H. and Weinert, T. A. (1989) Checkpoints: Controls that ensure the order of cell cycle events. Science 246, 629–634.
Paulovich, A. G., Toczyski, D. P., and Hartwell, L. H. (1997) When checkpoints fail. Cell 88, 315–321.
Savitsky, K., Bar-Shira, A., Gilad, S., Rotman, G., Ziv, Y., Vanagaite, L., et al. (1995) A single ataxia telangiectasia gene with a product simlar to P1–3 kinase. Science 268, 1749–1753.
Lavin, M. F., Khanna, K. K., and Beamish, H. (1995) Relationship of the ataxiatelangiectasia protein ATM to phosphoinositide 3-kinase. Trends Biochem. Sci. 20, 82–383.
Painter, R. B. and Young, B. R. (1980) Radiosensitivity in ataxia-telangiectasia: a new explanation. Proc. Natl. Acad. Sci. USA 77, 7315–7317.
Paulovich, A. G. and Hartwell, L. H. (1995) A checkpoint regulates the rate of progression through S phase in S. cerivisiae in response to DNA damage. Cell 82, 841–847.
Paulovich, A. G., Margulies, R. U., Garvik, B. M., and Hartwell, L. H. (1997) RAD9, RAD17, and RAD24 are required for S phase regulation in Saccharomyces cerevisiae in response to DNA damage. Genetics 145, 45–62.
Wang, Y., Huq, M. S., Cheng, X., and Iliakis, G. (1995) Regulation of DNA replication in irradiated cells by trans-acting factors. Radiat. Res. 142, 169–175.
Wang, Y., Huq, M. S., and Iliakis, G. (1996) Evidence for activities inhibiting in trans initiation of DNA replication in extract prepared from irradiated cells. iRadiat. Res. 145, 408–418.
Wang, Y., Perrault, A. R., and Iliakis, G. (1997) Down-regulation of DNA replication in extracts of camptothecin-treated cells: activation of an S-phase checkpoint? Cancer Res. 57, 1654–1659.
Challberg, M. D. and Kelly, T. J. (1989) Animal virus DNA replication. Annu. Rev. Biochem. 58, 671–717.
Hurwitz, J., Dean, F. B., Kwong, A. D., and Lee, S.-H. (1990) The in vitro replication of DNA containing the SV40 origin. J. Biol. Chem. 265, 18043–18046.
Kelly, T. J. (1988) SV40 DNA replication. J. Biol. Chem. 263, 17889–17892.
Stillman, B. (1989) Initiation of eukaryotic DNA replication in vitro. Annu. Rev. Cell Biol. 5, 197–245.
Harlow, E., Crawford, L. V., Pim, D. C., and Williamson, N. M. (1981) Monoclonal antibodies specific for simian virus 40 tumor antigens. J. Virol. 39, 861–869.
Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Lanford, R. E. (1988) Expression of simian virus 40 T antigen in insect cells using a baculovirus expression vector. Virology 167, 72–81.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K., Albright, L. M., Coen, D. N., and Varki, A. (1987) Current Protoc. Mol. Biol. 1–3.
Wobbe, C. R., Dean, F., Weissbach, L., and Hurwitz, J. (1985) In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site. Proc. Natl. Acad. Sci. USA 82, 5710–5714.
Prelich, G. and Stillman, B. (1988) Coordinated leading and lagging strand synthesis during SV40 DNA replication in vitro requires PCNA. Cell 53, 117–126.
Li, J. J. and Kelly, T. J. (1984) Simian virus 40 DNA replication in vitro. Proc. Natl. Acad. Sci. USA 81, 6973–6977.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, pp. 1–3.
Simanis, V. and Lane, D. P. (1985) An immunoaffinity purification procedure for SV40 large T antigen. Virology 144, 88–100.
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Humana Press Inc.
About this protocol
Cite this protocol
Iliakis, G., Wang, Y., Wang, H.Y. (1999). Analysis of Inhibition of DNA Replication in Irradiated Cells Using the SV40-Based In Vitro Assay of DNA Replication. In: Henderson, D.S. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 113. Humana Press. https://doi.org/10.1385/1-59259-675-4:543
Download citation
DOI: https://doi.org/10.1385/1-59259-675-4:543
Publisher Name: Humana Press
Print ISBN: 978-0-89603-802-8
Online ISBN: 978-1-59259-675-1
eBook Packages: Springer Protocols