Phosphorylation-mediated signaling is essential for maintenance of the eukaryotic genome. The evolutionarily conserved kinases ATR and ATM sense specific DNA structures generated upon DNA damage or replication stress and mediate an extensive signaling network that impinges upon most nuclear processes. ATR/ATM signaling is highly regulated and can function in a context-dependent manner. Thus, the ability to quantitatively monitor most, if not all, signaling events in this network is essential to investigate the mechanisms by which kinases maintain genome integrity. Here we describe a method for the Quantitative Mass-Spectrometry Analysis of Phospho-Substrates (QMAPS) to monitor in vivo DNA damage signaling in a systematic, unbiased, and quantitative manner. Using the model organism Saccharomyces cerevisiae, we provide an example for how QMAPS can be applied to define the effect of genotoxins, illustrating the importance of quantitatively monitoring multiple kinase substrates to comprehensively understanding kinase action. QMAPS can be easily extended to other organisms or signaling pathways where kinases can be deleted or inhibited.
DNA damage checkpoint DNA damage signaling Phosphorylation Quantitative mass spectrometry Saccharomyces cerevisiae
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We thank Beatriz S. Almeida for technical support. M.B.S. is supported by grants from the National Institutes of Health (R01-GM097272), F.M.B.d.O. is supported by grants from FAPERJ No E-26/010.002831/2014 and No E-26/010.003001/2014 and from CNPq No 446143/2014 and D.K. is supported by Cornell Vertebrate Genomic Scholarship.
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