An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint.
Checkpoint controls coordinate entry into mitosis with the completion of DNA replication. Depletion of nucleotide precursors by treatment with the drug hydroxyurea triggers such a checkpoint response. However, it is not clear whether the signal for this hydroxyurea-induced checkpoint pathway is the presence of unreplicated DNA, or rather the persistence of single-stranded or damaged DNA. In a yeast artificial chromosome (YAC) we have engineered an approximately 170 kb region lacking efficient replication origins that allows us to explore the specific effects of unreplicated DNA on cell cycle progression. Replication of this YAC extends the length of S phase and causes cells to engage an S/M checkpoint. In the absence of Rad9 the YAC becomes unstable, undergoing deletions within the origin-free region.
van Brabant
A J
AJ
University of Washington, Department of Genetics, Seattle, WA 98195, USA.
Buchanan
C D
CD
Charboneau
E
E
Fangman
W L
WL
Brewer
B J
BJ
D018797:Cell Cycle Proteins / Q000235:genetics; D064447:Checkpoint Kinase 2; D018244:Chromosomes, Artificial, Yeast / Q000502:physiology*; D004249:DNA Damage / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D017353:Gene Deletion; D005800:Genes, Fungal / Q000502:physiology; D018816:Genes, cdc / Q000502:physiology*; D006918:Hydroxyurea; D008938:Mitosis / Q000235:genetics*; D019384:Nucleic Acid Synthesis Inhibitors; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D018741:Replication Origin / Q000235:genetics*; D016196:S Phase / Q000235:genetics*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
D018797:Cell Cycle Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D029701:Saccharomyces cerevisiae Proteins; C069930:rad9 protein; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D006918:Hydroxyurea