Morin Isabelle, Ngo Hien-Ping, Greenall Amanda, Zubko Mikhajlo K, Morrice Nick, Lydall David
Institute for Ageing and Health, Henry Wellcome Laboratory for Biogerontology Research, Newcastle University, Newcastle Upon Tyne, UK.
EMBO J. 2008 Sep 17;27(18):2400-10. doi: 10.1038/emboj.2008.171. Epub 2008 Aug 28.
Exo1 is a nuclease involved in mismatch repair, DSB repair, stalled replication fork processing and in the DNA damage response triggered by dysfunctional telomeres. In budding yeast and mice, Exo1 creates single-stranded DNA (ssDNA) at uncapped telomeres. This ssDNA accumulation activates the checkpoint response resulting in cell cycle arrest. Here, we demonstrate that Exo1 is phosphorylated when telomeres are uncapped in cdc13-1 and yku70Delta yeast cells, and in response to the induction of DNA damage. After telomere uncapping, Exo1 phosphorylation depends on components of the checkpoint machinery such as Rad24, Rad17, Rad9, Rad53 and Mec1, but is largely independent of Chk1, Tel1 and Dun1. Serines S372, S567, S587 and S692 of Exo1 were identified as targets for phosphorylation. Furthermore, mutation of these Exo1 residues altered the DNA damage response to uncapped telomeres and camptothecin treatment, in a manner that suggests Exo1 phosphorylation inhibits its activity. We propose that Rad53-dependent Exo1 phosphorylation is involved in a negative feedback loop to limit ssDNA accumulation and DNA damage checkpoint activation.
Exo1是一种核酸酶，参与错配修复、双链断裂修复、停滞复制叉处理以及由功能失调的端粒引发的DNA损伤反应。在芽殖酵母和小鼠中，Exo1在无帽端粒处产生单链DNA（ssDNA）。这种ssDNA积累激活检查点反应，导致细胞周期停滞。在这里，我们证明在cdc13-1和yku70Delta酵母细胞中端粒无帽时以及响应DNA损伤诱导时，Exo1会发生磷酸化。端粒无帽后，Exo1磷酸化依赖于检查点机制的组分，如Rad24、Rad17、Rad9、Rad53和Mec1，但在很大程度上不依赖于Chk1、Tel1和Dun1。Exo1的丝氨酸S372、S567、S587和S692被确定为磷酸化靶点。此外，这些Exo1残基的突变改变了对无帽端粒和喜树碱处理的DNA损伤反应，其方式表明Exo1磷酸化抑制了其活性。我们提出Rad53依赖的Exo1磷酸化参与负反馈回路，以限制ssDNA积累和DNA损伤检查点激活。
