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.

• PMID: 9367985
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Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Sidorova JM, Breeden LL

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