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.

Rif1 and Exo1 regulate the genomic instability following telomere losses

“…We observed an Exo1‐dependent loss of genes, as well as gene duplication events, in cells lacking telomeres. Exo1 has been previously documented to facilitate or inhibit chromosomal duplication following other type of insults, through a checkpoint‐dependent (Kaochar et al ., 2010) or homology‐directed repair (HDR) dependent mechanism (Štafa et al ., 2014). However, the palindrome formation in PAL cells is both HDR‐independent (PALs are rad52∆ ) and checkpoint‐independent (in rad9∆ and rad24∆ strains) and therefore occurs by a different mechanism.…”

“…Consistent with this hypothesis, replicative senescence is induced by p53 and other checkpoint proteins inactivated in cancers (Shay et al ., 1991; Sugrue et al ., 1997; Schmitt et al ., 2002). Moreover, cancers may originate from cells with telomere attrition (Meeker et al ., 2004) or sometimes with acute telomere losses, due to chromosome fragmentation (Štafa et al ., 2014). …”

Telomere Dysfunction Triggers Palindrome Formation Independently of Double-Strand Break Repair Mechanisms

“…Telomeres shorten prematurely in certain diseases and genetic syndromes, for example, liver cirrhosis, pulmonary fibrosis, and dyskeratosis congenita ( Alder et al 2011 ; Batista et al 2011 ; El-Chemaly et al 2011 ). Moreover, telomeres can be lost completely, as in progenies of human lymphocytes exposed to heavy ions, or during chromothripsis ( Durante et al 2006 ; Štafa et al 2014 ).…”

Novel Approach in the Construction of Bioethanol-Producing Saccharomyces cerevisiae Hybrids

“…Lithium acetate transformation was done as described previously ( 28 ) but since it was observed that the efficiency of transformation in natural isolates was lower than in commonly used laboratory strains, cells were first allowed to recover for 30 min in rich YPD medium and then plated on selective SC-Ura plates. Isolation of the genomic DNA was performed as described previously ( 29 ) and molecular analysis of transformants was done by Southern blotting ( 30 ) using dioxigenin (DIG)-labelled ADE2 gene (Roche, Darmstadt, Germany).…”