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Science 26 July 2002:
Vol. 297. no. 5581, pp. 599 - 602
DOI: 10.1126/science.1074023
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Reports

Fork Reversal and ssDNA Accumulation at Stalled Replication Forks Owing to Checkpoint Defects

José M. Sogo,1* Massimo Lopes,2* Marco Foiani2dagger

Checkpoint-mediated control of replicating chromosomes is essential for preventing cancer. In yeast, Rad53 kinase protects stalled replication forks from pathological rearrangements. To characterize the mechanisms controlling fork integrity, we analyzed replication intermediates formed in response to replication blocks using electron microscopy. At the forks, wild-type cells accumulate short single-stranded regions, which likely causes checkpoint activation, whereas rad53 mutants exhibit extensive single-stranded gaps and hemi-replicated intermediates, consistent with a lagging-strand synthesis defect. Further, rad53 cells accumulate Holliday junctions through fork reversal. We speculate that, in checkpoint mutants, abnormal replication intermediates begin to form because of uncoordinated replication and are further processed by unscheduled recombination pathways, causing genome instability.

1 Institute of Cell Biology, ETH Hönggerberg, CH-8093 Zürich, Switzerland.
2 Istituto F.I.R.C. di Oncologia Molecolare, Via Adamello 16, 20141, Milano, Italy, and Dipartimento di Genetica e di Biologia dei Microrganismi, Università degli Studi di Milano, Via Celoria 26, 20133, Milano, Italy.
*   These authors contributed equally to this work.

dagger    To whom correspondence should be addressed. E-mail: foiani{at}ifom-firc.it

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