Abstract
The replication checkpoint controls the integrity of replicating chromosomes by stabilizing stalled forks, thus preventing the accumulation of abnormal replication and recombination intermediates that contribute to genome instability. Checkpoint-defective cells are susceptible to rearrangements at chromosome fragile sites when replication pauses, and certain human cancer prone diseases suffer checkpoint abnormalities. It is unclear as to how the checkpoint stabilizes stalled forks and how cells sense replication blocks. We have analysed the checkpoint contribution in controlling replisome–fork association when replication pauses. We show that in yeast wild-type cells, stalled forks exhibit stable replisome complexes and the checkpoint sensors Ddc1 and Ddc2, thus activating Rad53 checkpoint kinase. Ddc1/Ddc2 recruitment on stalled forks and Rad53 activation are influenced by the single-strand-binding protein replication factor A (RFA). rad53 forks exhibit a defective association with DNA polymerases α, ɛ and δ. Further, in rad53 mutants, stalled forks progressively generate abnormal structures that turn into checkpoint signals by accumulating RFA, Ddc1 and Ddc2. We suggest that, following replication blocks, checkpoint activation mediated by RFA-ssDNA filaments stabilizes stalled forks by controlling replisome–fork association, thus preventing unscheduled recruitment of recombination enzymes that could otherwise cause the pathological processing of the forks.
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Acknowledgements
We thank H Araki and Y Kamimura for help in the ChIP procedure, M Lopes, J Sogo and all members of our laboratory for helpful discussions, K Shirahige for communicating unpublished results and H Araki for critical reading of the manuscript. This work was supported by Associazione Italiana per la Ricerca sul Cancro and European Union (Grant RTN2-2001-00177). CL and FV were supported by Adriano Buzzati-Traverso fellowships. JEH was supported by NIH Grants GM20056 and GM61766.
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Lucca, C., Vanoli, F., Cotta-Ramusino, C. et al. Checkpoint-mediated control of replisome–fork association and signalling in response to replication pausing. Oncogene 23, 1206–1213 (2004). https://doi.org/10.1038/sj.onc.1207199
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DOI: https://doi.org/10.1038/sj.onc.1207199
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