Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication.
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.
Feng
Wenyi
W
Department of Genome Sciences, Box 357730, University of Washington, Seattle, WA 98195-7730, USA.
Collingwood
David
D
Boeck
Max E
ME
Fox
Lindsay A
LA
Alvino
Gina M
GM
Fangman
Walton L
WL
Raghuraman
Mosur K
MK
Brewer
Bonita J
BJ
D018797:Cell Cycle Proteins / Q000235:genetics; D064447:Checkpoint Kinase 2; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics; D004277:DNA, Single-Stranded / Q000032:analysis / Q000235:genetics*; D005090:Exodeoxyribonucleases / Q000235:genetics; D016681:Genome, Fungal; D006918:Hydroxyurea / Q000494:pharmacology*; D009154:Mutation / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D018741:Replication Origin / Q000235:genetics*; D016196:S Phase / Q000187:drug effects / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D016615:Telomere / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000187:drug effects / Q000235:genetics*
D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D064447:Checkpoint Kinase 2; C578636:Cds1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; D006918:Hydroxyurea