Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress

Nat Cell Biol. 2012 Sep;14(9):966-76. doi: 10.1038/ncb2549. Epub 2012 Jul 29.

Abstract

Relocalization of proteins is a hallmark of the DNA damage response. We use high-throughput microscopic screening of the yeast GFP fusion collection to develop a systems-level view of protein reorganization following drug-induced DNA replication stress. Changes in protein localization and abundance reveal drug-specific patterns of functional enrichments. Classification of proteins by subcellular destination enables the identification of pathways that respond to replication stress. We analysed pairwise combinations of GFP fusions and gene deletion mutants to define and order two previously unknown DNA damage responses. In the first, Cmr1 forms subnuclear foci that are regulated by the histone deacetylase Hos2 and are distinct from the typical Rad52 repair foci. In a second example, we find that the checkpoint kinases Mec1/Tel1 and the translation regulator Asc1 regulate P-body formation. This method identifies response pathways that were not detected in genetic and protein interaction screens, and can be readily applied to any form of chemical or genetic stress to reveal cellular response pathways.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • DNA Damage*
  • DNA Replication / genetics
  • DNA Replication / physiology*
  • DNA-Binding Proteins / metabolism
  • GTP-Binding Proteins / metabolism
  • Gene Deletion
  • Histone Deacetylases / metabolism
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Protein Transport / genetics
  • Protein Transport / physiology*
  • Protein-Serine-Threonine Kinases / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae / physiology
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Deletion

Substances

  • ASC1 protein, S cerevisiae
  • Adaptor Proteins, Signal Transducing
  • Cmr1 protein, S cerevisiae
  • DNA-Binding Proteins
  • Intracellular Signaling Peptides and Proteins
  • Saccharomyces cerevisiae Proteins
  • MEC1 protein, S cerevisiae
  • Protein-Serine-Threonine Kinases
  • TEL1 protein, S cerevisiae
  • Histone Deacetylases
  • GTP-Binding Proteins