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Temporally coordinated assembly and disassembly of replication factories in the absence of DNA synthesis

Nature Cell Biology volume 2, pages 686–694 (2000)Cite this article

Abstract

Here we show that exposure of aphidicolin-arrested Chinese hamster ovary (CHO) cells to the protein-kinase inhibitors 2-aminopurine or caffeine results in initiation of replication at successively later-replicating chromosomal domains, loss of the capacity to synthesize DNA at earlier-replicating sites, release of Mcm2 proteins from chromatin, and redistribution of PCNA and RPA from early- to late-replicating domains in the absence of detectable elongation of replication forks. These results provide evidence that, under conditions of replicational stress, checkpoint controls not only prevent further initiation but may also be required to actively maintain the integrity of stalled replication complexes.

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Figure 1: Treatment of aphidicolin-arrested cells with 2-AP results in the appearance of late-replication patterns.
Figure 2: Treatment of aphidicolin-arrested cells with 2-AP reveals the temporal program for replication.
Figure 3: Temporally coordinated firing of replicons in the absence of DNA synthesis.
Figure 4: 2-AP and caffeine do not directly inhibit DNA synthesis.
Figure 5: Inhibition of the S-phase checkpoint is accompanied by redistribution of replication proteins.
Figure 6: Initiation at late-firing origins is accompanied by abandonment of primers at early origins.
Figure 7: ATM is not involved in the replication checkpoint.

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Acknowledgements

We thank J. Chen for technical assistance with flow cytometry, and P. Hahn, A. McNairn and B. Knox for critical reading of the manuscript. This work was supported by NIH grant GM57233-01 (to D.M.G.).

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  1. Daniela S. Dimitrova

    Present address: Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York, 14260, USA

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  1. Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, 13210, New York, USA

    Daniela S. Dimitrova & David M. Gilbert

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Correspondence to David M. Gilbert.

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Correspondence and requests for materials should be addressed to D.M.G.

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Dimitrova, D., Gilbert, D. Temporally coordinated assembly and disassembly of replication factories in the absence of DNA synthesis. Nat Cell Biol 2, 686–694 (2000). https://doi.org/10.1038/35036309

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