Abstract
We have shown previously that chromosome VI of Saccharomyces cerevisiae contains nine origins of DNA replication that differ in initiation frequency and replicate sequentially during the S phase of the cell cycle1,2. Here we show that there are links between activation of these multiple origins and regulation of S-phase progression. We study the effects of a DNA-damaging agent, methyl methane sulphonate (MMS), and of mutations in checkpoint genes such as rad53 (ref. 3) on the activity of origins, measured by two-dimensional gel analysis, and on cell-cycle progression, measured by fluorescence-activated cell sorting. We find that when MMS slows down S-phase progression it also selectively blocks initiation from late origins. A rad53 mutation enhances late and/or inefficient origins and releases the initiation block by MMS. Mutation of rad53 also results in a late origin becoming early replicating. We conclude that rad53 regulates the timing of initiation of replication from late origins during normal cell growth and blocks initiation from late origins in MMS-treated cells. rad53 is, therefore, involved in the cell's surveillance of S-phase progression4,5. We also find that orc2, which encodes subunit 2 of the origin-recognition complex6,7, is involved in suppression of late origins.
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Acknowledgements
We thank J. Rine and B. Stillman for the orc2-1 mutant and its parental strain; L. H. Hartwell for the rad53-1 mutant, the mec1-1 mutant and their parental strain (7830-2-4A); and J. F. X. Diffley for critical reading of this manuscript and for unpublished results. This work was supported by grants-in-aid for Cooperative Research and for Special Projects Research from the Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds for promoting Science and Technology from the Science and Technology Agency of the Japanese Government.
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Shirahige, K., Hori, Y., Shiraishi, K. et al. Regulation of DNA-replication origins during cell-cycle progression. Nature 395, 618–621 (1998). https://doi.org/10.1038/27007
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DOI: https://doi.org/10.1038/27007
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