Abstract
Eukaryotic cells regulate progression through the cell cycle in response to DNA damage. Cell cycle checkpoints are the signal transduction pathways that couple the detection of DNA damage to the proteins that control transitions in the cell cycle. The protein kinase Chk1, originally discovered in fission yeast, but conserved in humans, is essential for preventing mitotic entry in the presence of DNA damage or blocks to DNA replication that cannot be reconciled. Chk1 is phosphorylated in response to DNA damage. Phosphorylation depends on the activity of conserved components of the checkpoint pathway including Rad3, a member of the ATM/ ATR family of kinases. Phosphorylation leads to activation of Chk1 kinase activity. In this chapter, we describe an assay for monitoring the activity of Chk1 isolated.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Pines, J. (1995) Cyclins and cyclin-dependent kinases: a biochemical view. Biochem J. 308,697–711.
Nurse, P. (1994) Ordering S phase and M phase in the cell cycle. Cell 79, 547–550.
Hartwell, L. H. and Weinert, T. A. (1989) Checkpoints: controls that ensure the order of cell cycle events. Science 246, 629–634.
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.
Walworth, N., Davey, S., and Beach, D. (1993) Fission yeast chk1 protein kinase links the rad checkpoint pathway to cdc2. Nature 363, 368–371.
O’Connell, M. J., Walworth, N. C., and Carr, A. M. (2000) The G2-phase DNA-damage checkpoint. Trends CellBiol. 10, 296–303.
Manning, G., Plowman, G. D., Hunter, T., and Sudarsanam, S. (2002) Evolution of protein kinase signaling from yeast to man. Trends Biochem. Sci. 27, 514–520.
Walworth, N. C. and Bernards, R. (1996) rad-Dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint. Science 271, 353–356.
Capasso, H., Palermo, C., Wan, S., et al. (2002) Phosphorylation activates Chk1 and is required for checkpoint-mediated cell cycle arrest. J. Cell Sci. 115, 4555–4564.
Wan, S., Capasso, H., and Walworth, N. C. (1999) The topoisomerase I poison camptothecin generates a Chk1-dependent DNA damage checkpoint signal in fission yeast. Yeast 15, 821–828.
Hanks, S. K. and Hunter T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9, 576–596.
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
Palermo, C., Walworth, N.C. (2005). Assaying Cell Cycle Checkpoints. In: Humphrey, T., Brooks, G. (eds) Cell Cycle Control. Methods in Molecular Biology™, vol 296. Humana Press. https://doi.org/10.1385/1-59259-857-9:345
Download citation
DOI: https://doi.org/10.1385/1-59259-857-9:345
Publisher Name: Humana Press
Print ISBN: 978-1-58829-144-8
Online ISBN: 978-1-59259-857-1
eBook Packages: Springer Protocols