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

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.

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Figure 1: High-throughput microscopic screening of yeast GFP collection.
Figure 2: Comparison of biological process enrichment for MMS and hydroxyurea (HU) abundance and localization positives.
Figure 3: Abundance and relocalization positives show drug-specific biological process enrichment.
Figure 4: Global analysis of protein relocalization in response to replication stress.
Figure 5: Relocalization change classes are enriched for protein–protein and genetic interactions.
Figure 6: Cmr1 represents a distinct class of DNA damage response foci.
Figure 7: P-body formation in response to hydroxyurea is regulated by ASC1, MEC1 and TEL1.

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Acknowledgements

We thank M. Cox and B. Andrews for assistance with high-throughput microscopy, A. Baryshnikova, and Q. Morris for advice on the data analysis and R. Tsien for providing the mCherry fusion plasmid. This work was supported by grant 020254 from the Canadian Cancer Society Research Institute to G.W.B., by grant 1R01HG005853 from the National Institutes of Health, grants MOP-102629 and MOP-97939 from the Canadian Institutes of Health Research and grant GL2-01-22 from the Ontario Research Fund to C.B., by grants P41 RR11823 (NCRR) and P41 GM103533 (NIGMS) from the National Institutes of Health to T.N.D., by grants from the Canadian Foundation for Innovation (16304) and the Ontario Institute of Cancer Research Equipment Competition (2007) to J.M., and by a grant from the National Human Genome Research Institute to C.N.

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J.M.T. and G.W.B. designed experiments and carried out data analysis. J.M.T. carried out the primary screen and carried out or co-ordinated experiments for Figs 6 and 7. A.Y. carried out experiments for Figs 6 and 7. A.Y.L. and C.N. carried out GSEA and biological enrichment analysis and generated enrichment networks. M.R., D.J. and T.N.D. constructed the database of images available through the Yeast Resource Center. J.M. established the high-throughput microscopy platform and provided advice on the microscopy and analysis. M.C. and C.B. co-ordinated the CMR1 SGA analysis. J.A.H. and J.O. carried out functional analysis of CMR1 and LSM1, respectively. The manuscript was written by J.M.T. and G.W.B. with contributions from C.N.

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Correspondence to Grant W. Brown.

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Tkach, J., Yimit, A., Lee, A. et al. Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress. Nat Cell Biol 14, 966–976 (2012). https://doi.org/10.1038/ncb2549

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