Sidorova J M, Breeden L L
Fred Hutchinson Cancer Research Center (FHCRC), Basic Sciences Division, Seattle, Washington 98109-1024, USA.
Genes Dev. 1997 Nov 15;11(22):3032-45. doi: 10.1101/gad.11.22.3032.
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.
出芽酵母具有一种依赖检查点的机制，可响应紫外线和电离辐射导致的DNA损伤而延迟G1期进程。我们已经表明，在用烷化剂MMS在G1期进行DNA损伤脉冲处理后，G1期也存在依赖MEC1、RAD53和RAD9的延迟。这种延迟发生在起始点或起始点之前，因为经MMS处理的细胞不发芽，对α因子仍敏感，并且与未处理的细胞相比，CLN1和CLN2转录本水平在更长时间内保持较低。我们进一步表明，MMS直接且可逆地下调CLN1和CLN2转录本水平。MMS处理后CLN转录本水平的初始下降不依赖RAD53，但随着细胞从损伤中恢复，CLN信息重新积累的动力学在rad53 - 11细胞中比野生型细胞更快。这不是通过G1期更快进程产生的间接效应，因为在停滞于G1期的rad53 - 11突变体中CLN转录本也更快地重新积累。此外，SWI6缺失或缺乏与Swi6结合的羧基末端结构域的截短型Swi4（Swi4 - t）的过表达也可以加速CLN mRNA水平的恢复。这表明Rad53和Swi6都是DNA损伤后CLN表达的负调节因子。最后，Swi6在G1期细胞中经历MMS诱导的、依赖RAD53的磷酸化，并且从经MMS处理的细胞中免疫沉淀的Rad53在体外使Swi6磷酸化。基于这些观察结果，我们认为Swi6的Rad53依赖的磷酸化可能通过抑制CLN转录来延迟向S期的转变。
