Mechanism of Dun1 activation by Rad53 phosphorylation in Saccharomyces cerevisiae
Mechanism of Dun1 activation by Rad53 phosphorylation in Saccharomyces cerevisiae
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DOI：
10.1074/jbc.m609322200
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发表时间：
2007-01-12
影响因子：
4.8
通讯作者：
Zhou, Huilin
中科院分区：
文献类型：
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作者：
Chen, Sheng-hong;Smolka, Marcus B.;Zhou, Huilin
尽管进行了广泛的研究，但DNA损伤检查点激活的分子机制仍然不完全清楚。为了更好地剖析这一机制，我们利用酵母中的下游DNA损伤检查点激酶的生理底物Sml1，开发了一种基于活性的测定Dun1的方法。通过这个实验，我们证实了Dun1激活的遗传基础。Rad53被发现是Dun1激活的直接原因。我们重建了Rad53对Dun1的激活，发现Rad53对Dun1激活环中Thr-380的磷酸化是Dun1激活的原因。有趣的是，在Rad53的激活环中，进化上保守的Thr-354的磷酸化对Rad53活性的调节也很重要。因此，这种保守的激活环磷酸化模式似乎是Chk2家族激酶激活的一般机制。
Despite extensive studies, the molecular mechanism of DNA damage checkpoint activation remains incompletely understood. To better dissect this mechanism, we developed an activity-based assay for Dun1, a downstream DNA damage checkpoint kinase in yeast, using its physiological substrate Sml1. Using this assay, we confirmed the genetic basis of Dun1 activation. Rad53 was found to be directly responsible for Dun1 activation. We reconstituted the activation of Dun1 by Rad53 and found that phosphorylation of Thr-380 in the activation loop of Dun1 by Rad53 is responsible for Dun1 activation. Interestingly, phosphorylation of the evolutionarily conserved Thr-354 in the activation loop of Rad53 is also important for the regulation of Rad53 activity. Thus, this conserved mode of activation loop phosphorylation appears to be a general mechanism for the activation of Chk2 family kinases.