Robust G1 checkpoint arrest in budding yeast: dependence on DNA damage signaling and repair.
Although most eukaryotes can arrest in G1 after ionizing radiation, the existence or significance of a G1 checkpoint in S. cerevisiae has been challenged. Previous studies of G1 response to chemical mutagens, X-ray or UV irradiation indicate that the delay before replication is transient and may reflect a strong intra-S-phase checkpoint. We examined the yeast response to double-stranded breaks in G1 using gamma irradiation. G1 irradiation induces repair foci on chromosome spreads and a Rad53 band shift characteristic of activation, which suggest an active DNA damage response. Consistent with a G1 arrest, bud emergence, spindle pole duplication and DNA replication are each delayed in a dose-dependent manner. Sensitivity to mating pheromone is prolonged to over 18 hours when G1 cells are lethally gamma or UV irradiated. Strikingly, G1 delay is the predominant response to continuous gamma irradiation at a dose that confers no loss of viability but delays cell division. Like the G2/M checkpoint, G1 delay is completely dependent on both RAD9 and RAD24 epistasis groups but independent of POL(epsilon). Lethally irradiated rad9 mutants rapidly exit G1 but perform a slow S phase, whereas rad17 and rad24 mutants are completely arrest deficient. Distinct from gamma irradiation, G1 arrest after UV is RAD14 dependent, suggesting that DNA damage processing is required for checkpoint activation. Therefore, as in the yeast G2/M checkpoint response, free DNA ends and/or single-stranded DNA are necessary and sufficient to induce a bona fide G1 checkpoint arrest.
Gerald
Jonathan N Fitz
JN
Center for Molecular Oncology, Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA.
Benjamin
Jacqueline M
JM
Kron
Stephen J
SJ
D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004271:DNA, Fungal / Q000378:metabolism / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D016193:G1 Phase / Q000502:physiology*; D005720:Gamma Rays; D018816:Genes, cdc; D015739:Nocodazole / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D005656:Fungal Proteins; C072975:RAD14 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D045643:DNA Repair Enzymes; D015739:Nocodazole