title: Sgs1 and Exo1 suppress targeted chromosome duplication during ends-in and ends-out gene targeting.
abstract: Gene targeting is extremely efficient in the yeast Saccharomyces cerevisiae. It is performed by transformation with a linear, non-replicative DNA fragment carrying a selectable marker and containing ends homologous to the particular locus in a genome. However, even in S. cerevisiae, transformation can result in unwanted (aberrant) integration events, the frequency and spectra of which are quite different for ends-out and ends-in transformation assays. It has been observed that gene replacement (ends-out gene targeting) can result in illegitimate integration, integration of the transforming DNA fragment next to the target sequence and duplication of a targeted chromosome. By contrast, plasmid integration (ends-in gene targeting) is often associated with multiple targeted integration events but illegitimate integration is extremely rare and a targeted chromosome duplication has not been reported. Here we systematically investigated the influence of design of the ends-out assay on the success of targeted genetic modification. We have determined transformation efficiency, fidelity of gene targeting and spectra of all aberrant events in several ends-out gene targeting assays designed to insert, delete or replace a particular sequence in the targeted region of the yeast genome. Furthermore, we have demonstrated for the first time that targeted chromosome duplications occur even during ends-in gene targeting. Most importantly, the whole chromosome duplication is POL32 dependent pointing to break-induced replication (BIR) as the underlying mechanism. Moreover, the occurrence of duplication of the targeted chromosome was strikingly increased in the exo1Δ sgs1Δ double mutant but not in the respective single mutants demonstrating that the Exo1 and Sgs1 proteins independently suppress whole chromosome duplication during gene targeting.
authors.affiliation: Laboratory for Biology and Microbial Genetics, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb 10000, Croatia; Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032, USA. Electronic address: astafa@pbf.hr.
authors.forename: Anamarija
authors.initials: A
authors.lastname: Štafa
authors.affiliation: Laboratory for Biology and Microbial Genetics, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb 10000, Croatia.
authors.forename: Marina
authors.initials: M
authors.lastname: Miklenić
authors.affiliation: Laboratory for Biology and Microbial Genetics, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb 10000, Croatia.
authors.forename: Bojan
authors.initials: B
authors.lastname: Zunar
authors.affiliation: Laboratory for Biology and Microbial Genetics, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb 10000, Croatia.
authors.forename: Berislav
authors.initials: B
authors.lastname: Lisnić
authors.affiliation: Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032, USA.
authors.forename: Lorraine S
authors.initials: LS
authors.lastname: Symington
authors.affiliation: Laboratory for Biology and Microbial Genetics, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb 10000, Croatia. Electronic address: iksvetec@pbf.hr.
authors.forename: Ivan-Krešimir
authors.initials: IK
authors.lastname: Svetec
mesh_terms: D058674:Chromosome Duplication; D053903:DNA Breaks, Double-Stranded; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting; D059765:Homologous Recombination; D053484:RecQ Helicases / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C522758:Pol32 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; C090472:SGS1 protein, S cerevisiae; D053484:RecQ Helicases
keywords: Break-induced replication; Ends-in; Ends-out; Gene targeting; Homologous recombination; Whole chromosome duplication
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