title: Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I-induced DNA damage.
abstract: We have streamlined the process of transferring plasmids into any yeast strain library by developing a novel mating-based, high-throughput method called selective ploidy ablation (SPA). SPA uses a universal plasmid donor strain that contains conditional centromeres on every chromosome. The plasmid-bearing donor is mated to a recipient, followed by removal of all donor-strain chromosomes, producing a haploid strain containing the transferred plasmid. As proof of principle, we used SPA to transfer plasmids containing wild-type and mutant alleles of DNA topoisomerase I (TOP1) into the haploid yeast gene-disruption library. Overexpression of Top1 identified only one sensitive mutation, rpa34, while overexpression of top1-T(722)A allele, a camptothecin mimetic, identified 190 sensitive gene-disruption strains along with rpa34. In addition to known camptothecin-sensitive strains, this set contained mutations in genes involved in the Rpd3 histone deacetylase complex, the kinetochore, and vesicle trafficking. We further show that mutations in several ESCRT vesicle trafficking components increase Top1 levels, which is dependent on SUMO modification. These findings demonstrate the utility of the SPA technique to introduce plasmids into the haploid gene-disruption library to discover new interacting pathways.
authors.affiliation: Columbia University Medical Center, Department of Genetics & Development, New York, NY 10032, USA.
authors.forename: Robert J D
authors.initials: RJ
authors.lastname: Reid
authors.forename: Sergio
authors.initials: S
authors.lastname: González-Barrera
authors.forename: Ivana
authors.initials: I
authors.lastname: Sunjevaric
authors.forename: David
authors.initials: D
authors.lastname: Alvaro
authors.forename: Samantha
authors.initials: S
authors.lastname: Ciccone
authors.forename: Marisa
authors.initials: M
authors.lastname: Wagner
authors.forename: Rodney
authors.initials: R
authors.lastname: Rothstein
mesh_terms: D000483:Alleles; D002166:Camptothecin / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D053263:Gene Regulatory Networks; D015698:Genomic Library; D057166:High-Throughput Screening Assays / Q000379:methods*; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011003:Ploidies; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D025841:Small Ubiquitin-Related Modifier Proteins; D006655:Histone Deacetylases; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin