During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.
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We wish to thank the Fangman–Brewer laboratory members for support and helpful discussions. We also acknowledge G. Findlay for helping with the construction of the rad53 exo1 double mutant and for critically reading the manuscript. We are grateful to M. Foiani for providing the pCH8 plasmid containing the rad53K227A mutation and G. D'Urso for S. pombe strains and helpful discussions. We also thank the staff at the Center for Expression Arrays. Seattle for their service of microarray slide hybridizations and scanning. We extend our gratitude to M. Thornquist, J. Haessler and U. Khan for helpful advice. This work was supported by National Institute of General Medical Sciences (NIGMS) grant 18926 to W.L.F., B.J.B. and M.K.R. W.F. was supported by a Ruth L. Kirschstein Postdoctoral Fellowship from the National Institutes of Health (NIH).
The authors declare no competing financial interests.
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