Summary
The DNA replication checkpoint, also known as the intra-S or S-phase checkpoint, plays a central role in ensuring the accuracy of DNA replication. When replication is impeded by DNA damage or other conditions, this checkpoint delays cell cycle progression and coordinates resumption of replication with DNA repair pathways. One of its critical functions is to stabilize stalled replication forks in a replication-competent state, presumably by maintaining proper assembly of replisome components and preserving DNA structures. Here we describe a series of assays used to study the replication checkpoint. These assays allow us to investigate the specific functions of proteins involved in the replication checkpoint in fission yeast.
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References
Boddy, M. N., and Russell, P. (2001) DNA replication checkpoint.Curr. Biol. 11, R953-R956.
Osborn, A. J., Elledge, S. J., and Zou, L. (2002) Checking on the fork: the DNA-replication stress-response pathway.Trends Cell Biol 12, 509–16.
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, 617–56.
McGlynn, P., and Lloyd, R. G. (2002) Recombinational repair and restart of damaged replication forks.Nat Rev Mol Cell Biol 3, 859–70.
Abraham, R. T. (2001) Cell cycle checkpoint signaling through the ATM and ATR kinases.Genes Dev 15, 2177–96.
Kastan, M. B., and Bartek, J. (2004) Cell-cycle checkpoints and cancer.Nature 432, 316–23.
Khanna, K. K., and Jackson, S. P. (2001) DNA double-strand breaks: signaling, repair and the cancer connection.Nat Genet 27, 247–54.
Kolodner, R. D., Putnam, C. D., and Myung, K. (2002) Maintenance of genome stability inSaccharomyces cerevisiae.Science 297, 552–7.
McGowan, C. H. (2002) Checking in on Cds1 (Chk2): A checkpoint kinase and tumor suppressor.Bioessays 24, 502–11.
Paulovich, A. G., Toczyski, D. P., and Hartwell, L. H. (1997) When checkpoints fail.Cell 88, 315–21.
Zhou, B. B., and Elledge, S. J. (2000) The DNA damage response: putting checkpoints in perspective.Nature 408, 433–9.
Rouse, J., and Jackson, S. P. (2002) Interfaces between the detection, signaling, and repair of DNA damage.Science 297, 547–51.
O’Connell, M. J., Walworth, N. C., and Carr, A. M. (2000) The G2-phase DNA-damage checkpoint.Trends Cell Biol 10, 296–303.
Rhind, N., and Russell, P. (2000) Checkpoints: it takes more than time to heal some wounds.Curr Biol 10, R908–11.
Rhind, N., and Russell, P. (2000) Chk1 and Cds1: linchpins of the DNA damage and replication checkpoint pathways.J Cell Sci 113 (Pt 22), 3889–96.
Tanaka, K., and Russell, P. (2001) Mrc1 channels the DNA replication arrest signal to checkpoint kinase Cds1.Nat Cell Biol 3, 966–72.
Zhao, H., Tanaka, K., Noguchi, E., Noguchi, C., and Russell, P. (2003) Replication checkpoint protein Mrc1 is regulated by Rad3 and Tel1 in fission yeast.Mol Cell Biol 23, 8395–403.
Noguchi, E., Noguchi, C., Du, L. L., and Russell, P. (2003) Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1.Mol Cell Biol 23, 7861–74.
Noguchi, E., Noguchi, C., McDonald, W. H., Yates, J. R., III, and Russell, P. (2004) Swi1 and Swi3 are components of a replication fork protection complex in fission yeast.Mol Cell Biol 24, 8342–55.
Matsumoto, S., Ogino, K., Noguchi, E., Russell, P., and Masai, H. (2005) Hsk1-Dfp1/Him1, the Cdc7-Dbf4 kinase inSchizosaccharomyces pombe, associates with Swi1, a component of the replication fork protection complex.J Biol Chem 280, 42536–42.
Takeda, T., Ogino, K., Tatebayashi, K., Ikeda, H., Arai, K., and Masai, H. (2001) Regulation of initiation of S phase, replication checkpoint signaling, and maintenance of mitotic chromosome structures during S phase by Hsk1 kinase in the fission yeast.Mol Biol Cell 12, 1257–74.
Ansbach, A. B., Noguchi, C., Klansek, I. W., Heidlebaugh, M., Nakamura, T. M., and Noguchi, E. (2008) RFCCtf18 and the Swi1–Swi3 complex function in separate and redundant pathways required for the stabilization of replication forks to facilitate sister chromatid cohesion inSchizosaccharomyces pombe.Mol Biol Cell 19, 595–607.
Capasso, H., Palermo, C., Wan, S., Rao, H., John, U. P., O’Connell, M. J., and Walworth, N. C. (2002) Phosphorylation activates Chk1 and is required for checkpoint-mediated cell cycle arrest.J Cell Sci 115, 4555–64.
Lindsay, H. D., Griffiths, D. J., Edwards, R. J., Christensen, P. U., Murray, J. M., Osman, F., Walworth, N., and Carr, A. M. (1998) S-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response inSchizosaccharomyces pombe.Genes Dev 12, 382–95.
Walworth, N., Davey, S., and Beach, D. (1993) Fission yeast chk1 protein kinase links the rad checkpoint pathway to cdc2.Nature 363, 368–71.
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, 821–8.
Sommariva, E., Pellny, T. K., Karahan, N., Kumar, S., Huberman, J. A., and Dalgaard, J. Z. (2005)Schizosaccharomyces pombe Swi1, Swi3, and Hsk1 are components of a novel S-phase response pathway to alkylation damage.Mol Cell Biol 25, 2770–84.
Lopes, M., Cotta-Ramusino, C., Pellicioli, A., Liberi, G., Plevani, P., Muzi-Falconi, M., Newlon, C. S., and Foiani, M. (2001) The DNA replication checkpoint response stabilizes stalled replication forks.Nature 412, 557–61.
Paciotti, V., Clerici, M., Scotti, M., Lucchini, G., and Longhese, M. P. (2001) Characterization of mec1 kinase-deficient mutants and of new hypomorphic mec1 alleles impairing subsets of the DNA damage response pathway.Mol Cell Biol 21, 3913–25.
Sogo, J. M., Lopes, M., and Foiani, M. (2002) Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects.Science 297, 599–602.
Tercero, J. A., and Diffley, J. F. (2001) Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint.Nature 412, 553–7.
Tercero, J. A., Longhese, M. P., and Diffley, J. F. (2003) A central role for DNA replication forks in checkpoint activation and response.Mol Cell 11, 1323–36.
Kim, W. J., Lee, S., Park, M. S., Jang, Y. K., Kim, J. B., and Park, S. D. (2000) Rad22 protein, a rad52 homologue inSchizosaccharomyces pombe, binds to DNA double-strand breaks.J Biol Chem 275, 35607–11.
Ostermann, K., Lorentz, A., and Schmidt, H. (1993) The fission yeast rad22 gene, having a function in mating-type switching and repair of DNA damages, encodes a protein homolog to Rad52 ofSaccharomyces cerevisiae.Nucleic Acids Res 21, 5940–4.
We thank Adam Leman and Jordan Rapp for helpful discussion. This work was supported by a Leukemia Research Foundation grant (E.N.), Drexel University College of Medicine start-up funds (E.N.), and NIH grant GM59447 (P.R.).
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Noguchi, E., Ansbach, A.B., Noguchi, C., Russell, P. (2009). Assays Used to Study the DNA Replication Checkpoint in Fission Yeast. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 521. Humana Press. https://doi.org/10.1007/978-1-60327-815-7_28
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DOI: https://doi.org/10.1007/978-1-60327-815-7_28
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