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

Abstract

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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Acknowledgements

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 (http://www.ncbi.nlm.nih.gov/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). https://doi.org/10.1038/nature01900

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