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S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex


The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome1,2,3,4,5. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards3,6,7. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks6,7,8,9. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.

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Figure 1: Locations of replication and checkpoint proteins on chromosome VI.
Figure 2: Interactions of Pol1 and Tof1 with chromosome VI during normal S-phase progression.
Figure 3: S-phase-specific association of Tof1 and Mrc1 with Cdc45.
Figure 4: Location of Cdc45 and replicated regions in wild type (WT) and mrc1 mutants treated with HU.
Figure 5: A model for the function of Mrc1 and Tof1.

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  1. Zhou, B. S. & Elledge, S. J. The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439 (2000)

    ADS  CAS  Article  Google Scholar 

  2. Melo, J. & Toczyski, D. A unified view of the DNA-damage checkpoint. Curr. Opin. Cell Biol. 14, 237–245 (2002)

    CAS  Article  Google Scholar 

  3. Shirahige, K. et al. Regulation of DNA-replication origins during cell-cycle progression. Nature 395, 618–621 (1998)

    ADS  CAS  Article  Google Scholar 

  4. Kondo, T., Wakayama, T., Naiki, T., Matsumoto, K. & Sugimoto, K. Recruitment of Mec1 and Ddc1 checkpoint proteins to double-strand breaks through distinct mechanisms. Science 294, 867–870 (2001)

    ADS  CAS  Article  Google Scholar 

  5. Melo, J., Cohen, J. & Toczyski, D. Two checkpoint complexes are independently recruited to sites of DNA damage in vivo. Genes Dev. 15, 2809–2821 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Lopes, M. et al. The DNA replication checkpoint response stabilizes stalled replication forks. Nature 412, 557–561 (2001)

    ADS  CAS  Article  Google Scholar 

  7. Tercero, J. A. & Diffley, J. F. Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint. Nature 412, 553–557 (2001)

    ADS  CAS  Article  Google Scholar 

  8. Tercero, J. A., Longhese, M. P. & Diffley, J. F. A central role for DNA replication forks in checkpoint activation and response. Mol. Cell 11, 1323–1336 (2003)

    CAS  Article  Google Scholar 

  9. Sogo, J. M., Lopes, M. & Foiani, M. Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science 297, 599–602 (2002)

    ADS  CAS  Article  Google Scholar 

  10. Shirahige, K., Iwasaki, T., Rashid, M. B., Ogasawara, N. & Yoshikawa, H. Location and characterization of autonomously replicating sequences from chromosome VI of Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 5043–5056 (1993)

    CAS  Article  Google Scholar 

  11. Yamashita, M. et al. The efficiency and timing of initiation of replication of multiple replicons of Saccharomyces cerevisiae chromosome VI. Genes Cells 2, 655–665 (1997)

    CAS  Article  Google Scholar 

  12. Ren, B. et al. Genome-wide location and function of DNA binding proteins. Science 290, 2306–2309 (2000)

    ADS  CAS  Article  Google Scholar 

  13. Iyer, V. R. et al. Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Nature 409, 533–538 (2001)

    ADS  CAS  Article  Google Scholar 

  14. Lengronne, A., Pasero, P., Bensimon, A. & Schwob, E. Monitoring S phase progression globally and locally using BrdU incorporation in TK(+ ) yeast strains. Nucleic Acids Res. 29, 1433–1442 (2001)

    CAS  Article  Google Scholar 

  15. Kurdistani, S. K., Robyr, D., Tavazoie, S. & Grunstein, M. Genome-wide binding map of the histone deacetylase Rpd3 in yeast. Nature Genet. 31, 248–254 (2002)

    CAS  Article  Google Scholar 

  16. Alcasabas, A. A. et al. Mrc1 transduces signals of DNA replication stress to activate Rad53. Nature Cell Biol. 3, 958–965 (2001)

    CAS  Article  Google Scholar 

  17. Zhao, X., Muller, E. G. & Rothstein, R. A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. Mol. Cell 2, 329–340 (1998)

    CAS  Article  Google Scholar 

  18. Labib, K., Tercero, J. A. & Diffley, J. F. Uninterrupted MCM2–7 function required for DNA replication fork progression. Science 288, 1643–1647 (2000)

    ADS  CAS  Article  Google Scholar 

  19. Foss, E. J. Tof1p regulates DNA damage responses during S phase in Saccharomyces cerevisiae. Genetics 157, 567–577 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Tanaka, K. & Russell, P. Mrc1 channels the DNA replication arrest signal to checkpoint kinase Cds1. Nature Cell Biol. 3, 966–972 (2001)

    CAS  Article  Google Scholar 

  21. De Antoni, A. & Gallwitz, D. A novel multi-purpose cassette for repeated integrative epitope tagging of genes in Saccharomyces cerevisiae. Gene 246, 179–185 (2000)

    CAS  Article  Google Scholar 

  22. Paulovich, A. G. & Hartwell, L. H. A checkpoint regulates the rate of progression through S phase in S. cerevisiae in response to DNA damage. Cell 82, 841–847 (1995)

    CAS  Article  Google Scholar 

  23. Winzeler, E. A. et al. Direct allelic variation scanning of the yeast genome. Science 281, 1194–1197 (1998)

    ADS  CAS  Article  Google Scholar 

  24. Cimbora, D. M. et al. Long-distance control of origin choice and replication timing in the human β-globin locus are independent of the locus control region. Mol. Cell. Biol. 20, 5581–5591 (2000)

    CAS  Article  Google Scholar 

  25. Schubeler, D. et al. Genome-wide DNA replication profile for Drosophila melanogaster: a link between transcription and replication timing. Nature Genet. 32, 438–442 (2002)

    Article  Google Scholar 

  26. Zou, L. & Stillman, B. Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p kinase. Mol. Cell. Biol. 20, 3086–3096 (2000)

    CAS  Article  Google Scholar 

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We thank A. De Antoni, E. Schwob, S. Bell and J. Hegemann for the plasmids; K. Tamai for the special preparation of BrdU antibody; H. Araki, K. Okumura, E. Winzeler, M Foiani, T. Yada and K. Umezu for their comments; E. Schwob, F. Uhlmann, R. Cha and H. Yoshikawa for critical reading of the manuscript; T. Itoh, Y. Nakao, A. Nakada and C. Kawagoe for technical assistance; and Y. Sakaki and all the members of the Genome Structure and Function team for their support. This work was supported partly by grants-in-aid on priority areas from the Ministry of Education, Culture, Sports, Science and Technology, Japan, to K.S. Y. Katou is a Junior Research Associate of the RIKEN GSC.

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Correspondence to Katsuhiko Shirahige.

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The authors declare that they have no competing financial interests.

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Data presented in this paper can be obtained from GEO ( with accession number GSE486.

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Katou, Y., Kanoh, Y., Bando, M. et al. S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424, 1078–1083 (2003).

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