Yeast Yeast 2007; 24: 637–652. Published online 29 May 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/yea.1497 Research Article Genetic side effects accompanying gene targeting in yeast: the inﬂuence of short heterologous termini Ivan-Kreˇ simir Svetec, Anamarija ˇ Stafa and Zoran Zgaga* Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia *Correspondence to: Zoran Zgaga, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia. E-mail: zgazo@pbf.hr Received: 21 November 2006 Accepted: 24 March 2007 Abstract We investigated the inﬂuence of short terminal heterologies on recombination between transforming linear DNA fragments and the yeast Saccharomyces cerevisiae genome. The efﬁciency of plasmid integration to the CYC1 locus (ends-in assay) was decreased more than ﬁve-fold when the size of terminal heterology exceeded 28 nucleotides (nt) and a similar inhibitory effect was also observed in the ends-out assay (replacement of the ura3-52 allele by the URA3 gene). Plasmid integration occurred almost exclusively in the target homology and was accompanied by excessive degradation of the heterologous termini. Illegitimate integrations were much more frequent in the ends-out transformation in both the absence (8.9%) and the presence (23.7%) of 45/46 heterologous nucleotides at the ends of the transforming fragment. Interestingly, only about 60% of transformants arose by simple gene replacement, regardless of the presence of heterologous ends, whereas more complex interactions resulted in gene or whole chromosome duplications. Our results warn that different genetic alterations may be introduced in the host strain during ends-out transformation but also indicate possible mechanisms for formation of duplications in the genome. Copyright  2007 John Wiley & Sons, Ltd. Keywords: gene targeting; gene duplication; aneuploidy; homologous recombina- tion; illegitimate recombination Introduction If unrepaired, double-strand breaks (DSBs) in DNA are lethal lesions. Two distinct cellular processes may be used to join the ends of the broken molecule. In a homology-dependent pathway, an intact homologous DNA sequence present in the cell is used as a template for in-register repair synthesis and religation. Mechanistically simpler is repair by illegitimate recombination, reconnect- ing the two DNA ends in a process known as non-homologous end-joining (NHEJ). In this case, any degradation of the termini preceding the end- joining will result in the loss of genetic material. However, both NHEJ and non-allelic homologous recombination (HR) may result in genetic rear- rangements like, for example, extensive homozy- gosis and expression of recessive mutations. In meiotic cells, DSB repair by HR accompanied by crossing-over is needed to ensure proper chromo- some segregation, whereas in vegetative cells the preferred pathway is largely species-speciﬁc. New insights into the processes inﬂuencing the balance between HR and NHEJ were recently discussed by Sonoda et al. (2006). In the yeast Saccharomyces cerevisiae, DSB repair occurs predominantly by HR that may involve different mechanisms (reviewed in Pˆ aques and Haber, 1999; Symington, 2002; Krogh and Symington, 2004; Aylon and Kupiec, 2004) and DSB-induced homologous recombination with exogenous DNA is routinely used for targeted manipulations of the yeast genome. Integration of the plasmid molecule can be stimulated and directed to the homologous sequence present in the yeast genome by restriction enzyme-induced DSBs Copyright  2007 John Wiley & Sons, Ltd.