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

Abstract

Re-localization 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 re-organization following drug-induced DNA replication stress. Changes in protein localization and abundance reveal drug-specific patterns of functional enrichments. Classification of proteins by sub-cellular destination allows the identification of pathways that respond to replication stress. We analyzed pairwise combinations of GFP fusions and gene deletion mutants to define and order two novel 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.

A chemostat array enables the spatio-temporal analysis of the yeast proteome

“…A poor correlation was also observed between protein and mRNA fold changes in our dataset (SI Appendix, Fig. S23) and by Tkach et al (6), but a similar comparison of protein and mRNA levels during a diauxic shift showed better correlations between fold change and timing (16). Posttranscriptional mechanisms therefore seem to play a significant role in determining protein induction timing and abundance levels in response to MMS.…”

“…These 16,731 movies consisted of 21 × 10 6 images, from which we analyzed a total of 1.5 × 10 8 cells, with an average of 199 cells quantified per image. By comparison, the original University of California, San Francisco (UCSF) yeast-GFP collection consisted of 14,562 images (5), the study by Tkach et al collected 74,664 images (6), and the entire University of Washington -Yeast Resource Center (UW-YRC) currently contains ∼1.2 × 10 6 images. Overall we performed the equivalent of more than 10,000 unique experiments.…”

“…S20) (4), a recent microscopy study (R 2 log = 0:72; SI Appendix, Fig. S21) (6), and with two methods for determining absolute protein abundance: tandem affinity purification (TAP) western (R 2 log = 0:78) (15) and mass spectrometry (R 2 log = 0:84) (SI Appendix, Fig. S14) (16).…”

“…This library was recently used to establish the static differences in protein abundance and localization in response to DNA replication stress induced by methyl methanesulfonate (MMS) and hydroxyurea (HU) (6), in response to DTT, H 2 O 2 , and nitrogen starvation (7), and 800 cytoplasmic proteins were analyzed upon entry into stationary phase (8). These three recent large-scale screens all relied on standard microtiter plates for imaging the yeast strains at a single time point before and after perturbation.…”

The ribosome: A hot spot for the identification of new types of protein methyltransferases

“…In a high-throughput study, an Rmt2-green fluorescent protein fusion protein was shown to relocate to the nucleus after DNA damage in yeast cells induced by hydroxyurea or methyl methanesulfonate (88). These results suggest the possibility that Rmt2 has additional protein substrates in yeast, although it is also possible that methylation on the ribosomal protein is in some way coupled to DNA transcription or repair.…”