Nature Genetics35, 277 - 286 (2003)
Published online: 19 October 2003; | doi:10.1038/ng1258
DNA helicase gene interaction network defined using synthetic lethality analyzed by microarray
Siew Loon Ooi1, Daniel D Shoemaker2
& Jef D Boeke1
1
Department of Molecular Biology & Genetics, The Johns Hopkins University School of Medicine, 617 Hunterian Building, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
2
Merck & Company Inc., 3535 General Atomics Court, San Diego, California 92121, USA.
Correspondence should be addressed to Jef D Boeke jboeke@jhmi.edu
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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NEWS AND VIEWS Lethal combinations Nature Genetics News and Views (01 Nov 2003)
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'
