Rad53-dependent phosphorylation of Swi6 and down-regulation of CLN1 andCLN2 transcription occur in response to DNA damage inSaccharomyces cerevisiae

Abstract

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 aMEC1-, 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 α-factor, and have lowCLN1 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 notRAD53 dependent, but the kinetics of reaccumulation ofCLN messages as cells recover from the damage is faster inrad53-11 cells than in wild type cells. This is not an indirect effect of faster progression through G₁, because CLNtranscripts 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 ofCLN expression after DNA damage. Finally, Swi6 undergoes an MMS-inducible, RAD53-dependent phosphorylation in G₁cells, 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.

Keywords

• MMS
• CLN1
• CLN2
• RAD53
• SWI6
• cell cycle
• checkpoint