Cells detect and respond to changes in their environment in a number of ways. Perhaps the best studied of these are changes in gene transcription1
, protein abundance2, 3
, and protein modification4, 5
, all of which have been subjected to genome-scale analysis. Cells also regulate the intracellular localization of proteins to accommodate different environmental conditions, but this form of regulation has not been analyzed systematically.
The DNA damage response consists of transcriptional, translational and post-translational facets, and several lines of evidence suggest that post-translational regulation is particularly important. At the single gene level, there is little if any correlation between transcriptional regulation in response to DNA damage and requirement for drug resistance6-8
. Likewise, blocking mRNA translation does not prevent cells from completing S-phase when challenged with the replication inhibitor hydroxyurea (HU), nor does it affect cell viability after HU treatment9, 10
. Critical roles of phosphorylation-, ubiquitylation-, and sumoylation-dependent signaling in the DNA damage response have been well characterized11-13
. Together, these data suggest that post-translational regulation of existing proteins play a paramount role in the DNA damage response.
Regulated protein re-localization is a hallmark of the cellular response to genotoxic drugs that cause DNA damage or DNA replication stress. In yeast, DNA damage response proteins including the single stranded DNA binding complex RPA, the double-strand DNA break processing complex MRX, the DNA damage sensor Ddc2, and proteins involved in homologous recombination relocalize from a diffuse nuclear distribution to form subnuclear foci in cells treated with genotoxic drugs14, 15
. In the case of the recombination protein Rad52, these foci co-localize with induced double-stranded breaks suggesting that they represent centers for DNA repair15
. Other localization changes occur including the re-localization of the small ribonucleotide reductase (RNR) subunits to the cytoplasm16
. Some aspects of the regulated localization of DNA repair proteins to subnuclear foci are conserved, as RPA, the Ddc2 homologue ATRIP, and recombination proteins form foci in response to DNA damage in both yeast and human cells15
. Mutations that disrupt phosphorylation of H2AX, or delete the ubiquitin interacting domains of Rad18 or Polη specifically disrupt the accumulation of repair proteins at nuclear foci and render cells sensitive to DNA damaging agents17-20
highlighting the importance of this post-translational regulation.
Despite the frequent occurrence, conservation, and importance of protein localization changes in response to DNA damage, they have not been examined systematically in any organism. We used high-throughput microscopic analysis of the GFP-tagged yeast ORF collection to define the total proteome localization and abundance changes that occur in response to drug-induced DNA replication stress, and to identify DNA damage response modules. When combined with high-throughput genetic interaction methods the approach identifies and orders DNA damage response pathways. This method is readily applicable to any chemical or genetic stress in which the re-localization of proteins is suspected to play a role.