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

  1. Julia M. Sidorova and
  2. Linda L. Breeden1
  3. Fred Hutchinson Cancer Research Center (FHCRC), Basic Sciences Division, Seattle, Washington 98109-1024 USA

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 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 α-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 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.

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