Abstract
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 check-point 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.
Highlights
In Saccharomyces cerevisiae, an evolutionarily conserved kinase cascade, consisting of Mec1, Tel1, Chk1, Rad53, and Dun1, is responsible for cellular responses to DNA damage [1]
Transcription of the ribonucleotide reductase (RNR) genes is up-regulated in response to DNA damage, which is regulated by Dun1 [17]
To ask whether DNA damage treatment could activate Dun1, we compared the phosphorylation of Sml1 by Dun1-TAF that is epitope-tagged and integrated into the DUN1 chromosomal locus, purified from either untreated or MMS-treated cells
Summary
In Saccharomyces cerevisiae, an evolutionarily conserved kinase cascade, consisting of Mec1, Tel1, Chk1, Rad53, and Dun1, is responsible for cellular responses to DNA damage [1]. Analysis of the phosphorylation-defective mutants of Dun1 revealed that Thr-380 in the activation loop of the kinase domain of Dun1 is critical for Dun1 activation by Rad53 and DNA damage-induced Sml1 degradation in vivo. Recombinant WT Rad53 activated WT Dun1, purified from rad53⌬ cells, for Sml1 hyperphosphorylation, whereas Dun1-D328A or Dun1KD, failed to be activated by WT Rad53 (Fig. 3B).
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