DNA bridging of yeast chromosomes VIII leads to near-reciprocal translocation and loss of heterozygosity with minor cellular defects

Abstract

Loss of heterozygosity (LOH) of tumor suppressor genes in somatic cells is a major process leading to several types of cancer; however, its underlying molecular mechanism is still poorly understood. In the present work, we demonstrate that a linear DNA molecule bridging two homologous chromosomes in diploid yeast cells via homologous recombination produce LOH-generating regions of hemizygosity by deletion. The result is a near-reciprocal translocation mutant that is characterized by slight cell cycle defects and increased expression of the multidrug-resistant gene VMR1. When the distance between target regions is approximately 40 kb, the specificity of gene targeting becomes less stringent and an ensemble of gross chromosomal rearrangements arises. These heterogeneous genomic events, together with the low frequency of specific translocation, confirm that several pathways contribute to the healing of a broken chromosome and suggest that uncontrolled recombination between parental homologs is actively avoided by the cell. Moreover, this work demonstrates that the common laboratory practice of making targeted gene deletions may result in a low, but not negligible, frequency of LOH due to the recombination events triggered between homologous chromosomes in mitosis.

Different Aneuploidies Arise From the Same Bridge-Induced Chromosomal Translocation Event in Saccharomyces cerevisiae

“…In the same strain, we also found the overexpression of the caspase YCA1 gene and of GSH1, which is also involved in apoptosis and cellular response to oxidative stress. Interestingly, previous authors (Tosato et al 2009;D. Nikitin personal communication) have revealed that VMR1 upregulation was also present in other homologous and heterologous BIT translocants.…”

“…The names of the analyzed genes are reported in Figure 8 and in Table S3. As in previous expression analyses of BIT translocants, HSC82 was chosen as a constitutive control gene (Nikitin et al 2008;Tosato et al 2009). For all the genes analyzed, an expression level higher than twice, or equal to or lower than half, the expression level of the control strain San1, was considered as a significant up-or downregulation of transcription, respectively.…”

“…In fact, as observed by others in mammalian cells, our findings strongly support that aneuploidy arising from an initial nonreciprocal translocation event may generate a heterogeneous population of mutant cells that are phenotypically and genotypically diverse. At the same time, our laboratory recently showed that near-reciprocal translocation between homologous chromosomes does not affect gene expression at the genomic loci flanking the breakpoints (Tosato et al 2009 FIGURE S1.-Growth test in different carbon sources. The control strain San1 and all the 10 SUSU translocants were cultured in YPD medium and OD600 was measured at identical times.…”

“…The genes GSH1 and GLR1 involved in oxidative stress response were also overexpressed in some of the translocant strains together with the multidrug resistance gene VMR1, which is homologous to the ABCC-type multi-drug transport proteins. This gene was also overexpressed in other BIT translocants between both heterologous and homologous chromosomes (Tosato et al 2009;D. Nikitin, personal communication).…”

“…Recently, our group described a method that allows the generation and subsequent selection of chromo-somal translocations between any two chromosomal loci in diploid yeast strains. This was done by transformation of whole yeast cells with a linear DNA construct, carrying the KAN R selectable marker flanked by DNA sequences homologous to two different chromosomal loci (Tosato et al 2005(Tosato et al , 2009Nikitin et al 2008).…”