Precocious S-phase entry in budding yeast prolongs replicative state and increases dependence upon Rad53 for viability.
Precocious entry into S phase due to overproduction of G1 regulators can cause genomic instability. The mechanisms of this phenomenon are largely unknown. We explored the consequences of precocious S phase in yeast by overproducing a deregulated form of Swi4 (Swi4-t). Swi4 is a late G1-specific transcriptional activator that, in complex with Swi6, binds to SCB elements and activates late G1-specific genes, including G1 cyclins. We find that wild-type cells tolerate Swi4-t, whereas checkpoint-deficient rad53-11 cells lose viability within several divisions when Swi4-t is overproduced. Rad53 kinase activity is increased in cells overproducing Swi4-t, indicating activation of the checkpoint. We monitored the transition from G1 to S in cells with Swi4-t and found that there is precocious S-phase entry and that the length of S phase is extended. Moreover, there were more replication intermediates, and firing of at least a subset of origins may have been more extensive in the cells expressing Swi4-t. Our working hypothesis is that Rad53 modulates origin firing based upon growth conditions to optimize the rate of S-phase progression without adversely affecting fidelity. This regulation becomes essential when S phase is influenced by Swi4-t.
Sidorova
Julia M
JM
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. jsidorov@fred.fhcrc.org
Breeden
Linda L
LL
D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D016213:Cyclins / Q000502:physiology; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis*; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D016193:G1 Phase / Q000502:physiology; D018816:Genes, cdc / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D016196:S Phase / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*
C082719:CLN1 protein, S cerevisiae; C082721:CLN2 protein, S cerevisiae; D018797:Cell Cycle Proteins; D016213:Cyclins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; C062214:SWI4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C069930:rad9 protein; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae