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Systematic pathway analysis using high-resolution fitness profiling of combinatorial gene deletions

Nature Genetics volume 39, pages 199–206 (2007)Cite this article

Abstract

Systematic genetic interaction studies have illuminated many cellular processes. Here we quantitatively examine genetic interactions among 26 Saccharomyces cerevisiae genes conferring resistance to the DNA-damaging agent methyl methanesulfonate (MMS), as determined by chemogenomic fitness profiling of pooled deletion strains. We constructed 650 double-deletion strains, corresponding to all pairings of these 26 deletions. The fitness of single- and double-deletion strains were measured in the presence and absence of MMS. Genetic interactions were defined by combining principles from both statistical and classical genetics. The resulting network predicts that the Mph1 helicase has a role in resolving homologous recombination–derived DNA intermediates that is similar to (but distinct from) that of the Sgs1 helicase. Our results emphasize the utility of small molecules and multifactorial deletion mutants in uncovering functional relationships and pathway order.

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Figure 1: Identification of genes that confer resistance to MMS.
Figure 2: Fitness measurement of single- and double-deletion strains.
Figure 3: Quantitative genetic interactions predict shared function.
Figure 4: Identification of significant genetic interactions.
Figure 5: Subclassification of alleviating interactions.
Figure 6: Predicted role of Mph1 in resolving homologous recombination–generated DNA intermediates.

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References

  1. Phillips, P.C. The language of gene interaction. Genetics 149, 1167–1171 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Phillips, P.C., Otto, S.P. & Whitlock, M.C. Beyond the Average: the Evolutionary Importance of Gene Interactions and Variability of Epistatic Effects in Epistasis and the Evolutionary Process 20–38 (Oxford Univ. Press, New York, 2000).

    Google Scholar 

  3. Kondrashov, A.S. Deleterious mutations and the evolution of sexual reproduction. Nature 336, 435–440 (1988).

    Article  CAS  PubMed  Google Scholar 

  4. Szafraniec, K., Wloch, D.M., Sliwa, P., Borts, R.H. & Korona, R. Small fitness effects and weak genetic interactions between deleterious mutations in heterozygous loci of the yeast Saccharomyces cerevisiae. Genet. Res. 82, 19–31 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Elena, S.F. & Lenski, R.E. Test of synergistic interactions among deleterious mutations in bacteria. Nature 390, 395–398 (1997).

    Article  CAS  PubMed  Google Scholar 

  6. Maisnier-Patin, S. et al. Genomic buffering mitigates the effects of deleterious mutations in bacteria. Nat. Genet. 37, 1376–1379 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Hartman, J.L., IV, Garvik, B. & Hartwell, L. Principles for the buffering of genetic variation. Science 291, 1001–1004 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Davierwala, A.P. et al. The synthetic genetic interaction spectrum of essential genes. Nat. Genet. 37, 1147–1152 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Kelley, R. & Ideker, T. Systematic interpretation of genetic interactions using protein networks. Nat. Biotechnol. 23, 561–566 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ooi, S.L. et al. Global synthetic-lethality analysis and yeast functional profiling. Trends Genet. 22, 56–63 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Ooi, S.L., Shoemaker, D.D. & Boeke, J.D. DNA helicase gene interaction network defined using synthetic lethality analyzed by microarray. Nat. Genet. 35, 277–286 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Pan, X. et al. A DNA Integrity network in the yeast Saccharomyces cerevisiae. Cell 124, 1069–1081 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Schuldiner, M. et al. Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123, 507–519 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Tong, A.H. et al. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294, 2364–2368 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Tong, A.H. et al. Global mapping of the yeast genetic interaction network. Science 303, 808–813 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Wong, S.L., Zhang, L.V. & Roth, F.P. Discovering functional relationships: biochemistry versus genetics. Trends Genet. 21, 424–427 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Wong, S.L. et al. Combining biological networks to predict genetic interactions. Proc. Natl. Acad. Sci. USA 101, 15682–15687 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Drees, B.L. et al. Derivation of genetic interaction networks from quantitative phenotype data. Genome Biol. 6, R38 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Segre, D., Deluna, A., Church, G.M. & Kishony, R. Modular epistasis in yeast metabolism. Nat. Genet. 37, 77–83 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Collins, S.R., Schuldiner, M., Krogan, N.J. & Weissman, J.S. A strategy for extracting and analyzing large-scale quantitative epistatic interaction data. Genome Biol. 7, R63 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Lee, W. et al. Genome-wide requirements for resistance to functionally distinct DNA-damaging agents. PLoS Genet. 1, e24 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002).

    Article  CAS  PubMed  Google Scholar 

  23. Ashburner, M. et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25–29 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ye, P., Peyser, B.D., Spencer, F.A. & Bader, J.S. Commensurate distances and similar motifs in genetic congruence and protein interaction networks in yeast. BMC Bioinformatics 6, 270 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Kaliraman, V., Mullen, J.R., Fricke, W.M., Bastin-Shanower, S.A. & Brill, S.J. Functional overlap between Sgs1-Top3 and the Mms4-Mus81 endonuclease. Genes Dev. 15, 2730–2740 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Shor, E., Weinstein, J. & Rothstein, R. A genetic screen for top3 suppressors in Saccharomyces cerevisiae identifies SHU1, SHU2, PSY3 and CSM2: four genes involved in error-free DNA repair. Genetics 169, 1275–1289 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Friedl, A.A., Liefshitz, B., Steinlauf, R. & Kupiec, M. Deletion of the SRS2 gene suppresses elevated recombination and DNA damage sensitivity in rad5 and rad18 mutants of Saccharomyces cerevisiae. Mutat. Res. 486, 137–146 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Ulrich, H.D. & Jentsch, S. Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J. 19, 3388–3397 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Huang, M.E., Rio, A.G., Nicolas, A. & Kolodner, R.D. A genomewide screen in Saccharomyces cerevisiae for genes that suppress the accumulation of mutations. Proc. Natl. Acad. Sci. USA 100, 11529–11534 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Punnett, R.C. Mendelism (Macmillan, New York, 1913).

    Book  Google Scholar 

  31. Jana, S. Simulation of quantitative characters from qualitatively acting genes. I. Nonallelic gene interactions involving two or three loci. Theor. Appl. Genet. 42, 119–124 (1972).

    Article  CAS  PubMed  Google Scholar 

  32. Ito, T. et al. A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc. Natl. Acad. Sci. USA 98, 4569–4574 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sung, P. Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. Genes Dev. 11, 1111–1121 (1997).

    Article  CAS  PubMed  Google Scholar 

  34. Avery, L. & Wasserman, S. Ordering gene function: the interpretation of epistasis in regulatory hierarchies. Trends Genet. 8, 312–316 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Krogh, B.O. & Symington, L.S. Recombination proteins in yeast. Annu. Rev. Genet. 38, 233–271 (2004).

    Article  CAS  PubMed  Google Scholar 

  36. Lisby, M., Barlow, J.H., Burgess, R.C. & Rothstein, R. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell 118, 699–713 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. Ellis, N.A. et al. The Bloom's syndrome gene product is homologous to RecQ helicases. Cell 83, 655–666 (1995).

    Article  CAS  PubMed  Google Scholar 

  38. Fabre, F., Chan, A., Heyer, W.D. & Gangloff, S. Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication. Proc. Natl. Acad. Sci. USA 99, 16887–16892 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Liberi, G. et al. Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase. Genes Dev. 19, 339–350 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Prakash, R. et al. Saccharomyces cerevisiae MPH1 gene, required for homologous recombination-mediated mutation avoidance, encodes a 3′ to 5′ DNA helicase. J. Biol. Chem. 280, 7854–7860 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Schurer, K.A., Rudolph, C., Ulrich, H.D. & Kramer, W. Yeast MPH1 gene functions in an error-free DNA damage bypass pathway that requires genes from Homologous recombination, but not from postreplicative repair. Genetics 166, 1673–1686 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Meetei, A.R. et al. A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M. Nat. Genet. 37, 958–963 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Bai, Y. & Symington, L.S.A. Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae. Genes Dev. 10, 2025–2037 (1996).

    Article  CAS  PubMed  Google Scholar 

  44. Klein, H.L. Mutations in recombinational repair and in checkpoint control genes suppress the lethal combination of srs2Δ with other DNA repair genes in Saccharomyces cerevisiae. Genetics 157, 557–565 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. McEachern, M.J. & Haber, J.E. Break-induced replication and recombinational telomere elongation in yeast. Annu. Rev. Biochem. 75, 111–135 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Winzeler, E.A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999).

    Article  CAS  PubMed  Google Scholar 

  47. Guthrie, C. & Fink, G.R . (eds.) Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991).

    Google Scholar 

  48. Rose, M.D., Winston, F. & Heiter, P. Methods in Yeast Genetics: a Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1990).

    Google Scholar 

  49. Erdeniz, N., Mortensen, U.H. & Rothstein, R. Cloning-free PCR-based allele replacement methods. Genome Res. 7, 1174–1183 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank M. Evangelista and S. Pierce for critically reading the manuscript; and B. Andrews, C. Boone, J. Greenblatt, N. Krogan and J. Weissman for discussions. R.P.S. was supported by a postdoctoral fellowship from the Canadian Institutes of Health Research. F.P.R. was supported by grant R01 HG003224 from the National Institutes of Health National Human Genome Research Institute (NIH/NHGRI). This work was supported by a grant from the NHGRI awarded to R.W.D. and G.G.

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Authors and Affiliations

  1. Department of Biochemistry, Stanford University, Stanford, 94305, California, USA

    Robert P St Onge, Julia Oh, Michael Proctor, Eula Fung & Ronald W Davis

  2. Biological Chemistry and Molecular Pharmacology Department, Harvard, Boston, 02115, Massachusetts, USA

    Ramamurthy Mani & Frederick P Roth

  3. Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, M5S3E1, Ontario, Canada

    Corey Nislow & Guri Giaever

  4. Center for Cancer Systems Biology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, 02115, Massachusetts, USA

    Frederick P Roth

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Contributions

R.P.St.O. was involved in every aspect of the study, from experimental design and performance to writing the manuscript. R.M. designed and executed algorithms and performed bioinformatic analysis. J.O. performed genetic experiments and growth curves. M.P. designed custom robotics and automation software. E.F. wrote software and performed database management. R.W.D. contributed intellectually throughout to experimental design and execution. C.N. helped design the experiments and write the manuscript. F.P.R. was involved in data analysis and manuscript preparation. G.G. was involved in every aspect of the study, including manuscript preparation.

Corresponding authors

Correspondence to Frederick P Roth or Guri Giaever.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

96-well growth assay for individual yeast deletion strains. (PDF 141 kb)

Supplementary Fig. 2

Prediction of functional links by 'scaled epsilon'. (PDF 261 kb)

Supplementary Fig. 3

Deletions in genes encoding members of the Shu complex suppress the synthetic lethality of rad54Δhpr5Δ. (PDF 204 kb)

Supplementary Fig. 4

Deletions in HR and SHU genes rescue the MMS sensitivity of the mph1Δmus81Δ double deletion strain. (PDF 193 kb)

Supplementary Fig. 5

Deletions in HR and SHU genes suppress the synthetic lethality of sgs1Δmms4Δ and sgs1Δmus81Δ double deletion mutants. (PDF 308 kb)

Supplementary Fig. 6

Data aggregation identifies additional genetic interactions involving the SHU complex. (PDF 29 kb)

Supplementary Table 1

Chemogenic profiling of the homozygous diploid deletion collection with MMS. (XLS 1927 kb)

Supplementary Table 2

Genetic interaction results for 323 unique gene pairs in the presence of MMS. (XLS 68 kb)

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Onge, R., Mani, R., Oh, J. et al. Systematic pathway analysis using high-resolution fitness profiling of combinatorial gene deletions. Nat Genet 39, 199–206 (2007). https://doi.org/10.1038/ng1948

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