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DNA helicase gene interaction network defined using synthetic lethality analyzed by microarray

Nature Genetics volume 35, pages 277–286 (2003)Cite this article

Abstract

We describe a new synthetic lethality analysis by microarray (SLAM) technique that uses ∼4,600 Saccharomyces cerevisiae haploid deletion mutants with molecular 'bar codes' (TAGs). We used SGS1 and SRS2, two 3′→5′ DNA helicase genes, as 'queries' to identify their redundant and unique biological functions. We introduced these 'query mutations' into a haploid deletion pool by integrative transformation to disrupt the query gene in every cell, generating a double mutant pool. Optimization of integrative transformation efficiency was essential to the success of SLAM. Synthetic interactions defined a DNA helicase genetic network and predicted a role for SRS2 in processing damaged replication forks but, unlike SGS1, not in rDNA replication, DNA topology or lagging strand synthesis. SGS1 and SRS2 have synthetic defects with MRC1 but not RAD9, suggesting that SGS1 and SRS2 function in a parallel pathway with MRC1 to transduce the DNA replication stress signal to the general DNA damage checkpoint pathway. Both helicase genes have rad51-reversible synthetic defects with 5′→3′ DNA helicase RRM3, suggesting that RRM3 helps prevent formation of toxic recombination intermediates. SLAM detects synthetic lethality efficiently and ranks candidate genetic interactions, making it an especially useful method.

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Figure 1: Parallel analysis of YKO strains for synthetic lethality with sgs1Δ.
Figure 2: Microarray analysis of mutants.
Figure 3: rnh35Δ, hst3Δ, ydr279wΔ, ylr154cΔ and ygr081cΔ/slx9Δ have fitness defects with sgs1Δ.
Figure 4: Random spore analysis of fitness defects.
Figure 5: Genetic interaction map for SGS1, SRS2 and RAD27.

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Acknowledgements

We thank R. Crouch and H. Jeong for sharing unpublished results; M. Lee and S. Sookhai-Mahadeo for optimizing the long range PCR; S. Sookhai-Mahadeo, A. IJpma and M. Lee for help with deletion pool construction; C. Boone and A. Tong for strains and discussion; G. Ira and R. Rothstein for discussions and reading the manuscripts; R. Irizarry and J. Bader for discussions on statistical aspects and network analysis; and E. Bolton, M. Lee, X. Pan, F. Spencer and D. Yuan for discussions. This work was supported by a grant from the US National Institutes of Health.

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  1. Department of Molecular Biology & Genetics, The Johns Hopkins University School of Medicine, 617 Hunterian Building, 725 North Wolfe Street, Baltimore, 21205, Maryland, USA

    Siew Loon Ooi & Jef D Boeke

  2. Merck & Company Inc., 3535 General Atomics Court, San Diego, 92121, California, USA

    Daniel D Shoemaker

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Correspondence to Jef D Boeke.

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Ooi, S., Shoemaker, D. & Boeke, J. DNA helicase gene interaction network defined using synthetic lethality analyzed by microarray. Nat Genet 35, 277–286 (2003). https://doi.org/10.1038/ng1258

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