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Quantitative Analysis of DNA Damage Signaling Responses to Chemical and Genetic Perturbations

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Genome Instability

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1672))

Abstract

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.

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Acknowledgments

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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Authors and Affiliations

  1. Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Cidade Universitária, Rio de Janeiro, 21941-902, Brazil

    Francisco M. Bastos de Oliveira

  2. Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, 339 Weill Hall, Ithaca, NY, 14853-7202, USA

    Dongsung Kim, Michael Lanz & Marcus B. Smolka

Authors
  1. Francisco M. Bastos de Oliveira
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  2. Dongsung Kim
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  3. Michael Lanz
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  4. Marcus B. Smolka
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Corresponding author

Correspondence to Marcus B. Smolka .

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Editors and Affiliations

  1. Dipartimento di Bioscienze, Università degli Studi di Milano, Milano, Milano, Italy

    Marco Muzi-Falconi

  2. Donnelly Centre and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Grant W Brown

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Bastos de Oliveira, F.M., Kim, D., Lanz, M., Smolka, M.B. (2018). Quantitative Analysis of DNA Damage Signaling Responses to Chemical and Genetic Perturbations. In: Muzi-Falconi, M., Brown, G. (eds) Genome Instability. Methods in Molecular Biology, vol 1672. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7306-4_42

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  • DOI: https://doi.org/10.1007/978-1-4939-7306-4_42

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7305-7

  • Online ISBN: 978-1-4939-7306-4

  • eBook Packages: Springer Protocols

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