Rad53-dependent phosphorylation of Swi6 and down-regulation of CLN1 and CLN2 transcription occur in response to DNA damage in Saccharomyces cerevisiae.
Budding yeast possesses a checkpoint-dependent mechanism of delaying G1 progression in response to UV and ionizing radiation DNA damage. We have shown that after a pulse of DNA damage in G1 with the alkylating agent MMS, there is also a MEC1-, RAD53-, and RAD9-dependent delay in G1. This delay occurs at or before Start, as the MMS-treated cells do not bud, remain sensitive to alpha-factor, and have low CLN1 and CLN2 transcript levels for a longer time than untreated cells. We further show that MMS directly and reversibly down-regulates CLN1 and CLN2 transcript levels. The initial drop in CLN transcript levels in MMS is not RAD53 dependent, but the kinetics of reaccumulation of CLN messages as cells recover from the damage is faster in rad53-11 cells than in wild type cells. This is not an indirect effect of faster progression through G1, because CLN transcripts reaccumulate faster in rad53-11 mutants arrested in G1 as well. In addition, the recovery of CLN mRNA levels can be also hastened by a SWI6 deletion or by overexpression of the truncated Swi4 (Swi4-t) that lacks the carboxy-terminal domain through which Swi4 associates with Swi6. This indicates that both Rad53 and Swi6 are negative regulators of CLN expression after DNA damage. Finally, Swi6 undergoes an MMS-inducible, RAD53-dependent phosphorylation in G1 cells, and Rad53, immunoprecipitated from MMS-treated cells, phosphorylates Swi6 in vitro. On the basis of these observations, we suggest that the Rad53-dependent phosphorylation of Swi6 may delay the transition to S phase by inhibiting CLN transcription.
Sidorova
J M
JM
Fred Hutchinson Cancer Research Center (FHCRC), Basic Sciences Division, Seattle, Washington 98109-1024, USA.
Breeden
L L
LL
D000477:Alkylating Agents; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D016213:Cyclins / Q000502:physiology*; D004249:DNA Damage; D004271:DNA, Fungal / Q000235:genetics; D015536:Down-Regulation; D005656:Fungal Proteins / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal; D008741:Methyl Methanesulfonate; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases; D012331:RNA, Fungal / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic
D000477:Alkylating Agents; C082719:CLN1 protein, S cerevisiae; C082721:CLN2 protein, S cerevisiae; D018797:Cell Cycle Proteins; D016213:Cyclins; D004271:DNA, Fungal; D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; C075111:SWI6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D008741:Methyl Methanesulfonate; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae