Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication

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Abstract

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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Figure 1: Outline of experimental procedures.
Figure 2: Dynamics of ssDNA formation in wild-type (WT) and rad53 cells.
Figure 3: Elevated levels of ssDNA at telomeric regions.
Figure 4: Identification of new origins from the ssDNA profiles of rad53 cells.
Figure 5: ssDNA profiles of S. pombe chromosomes
Figure 6: Comparison of the distributions of inter-origin distances in S. cerevisiae (cross-hatched bars) and S. pombe (solid black bars).

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Acknowledgements

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).

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Correspondence to Bonita J. Brewer.

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Supplementary Figures S1, S2, S3. Tables S1 and S2 (PDF 3577 kb)

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