Minisatellite alterations in ZRT1 mutants occur via RAD52-dependent and RAD52-independent mechanisms in quiescent stationary phase yeast cells

Abstract

Alterations in minisatellite DNA repeat tracts are associated with a variety of human diseases including type 1 diabetes, progressive myoclonus epilepsy, and some types of cancer. However, in spite of their role in human health, the factors required for minisatellite alterations are not well understood. We previously identified a stationary phase specific increase in minisatellite instability caused by mutations in the high affinity zinc transporter ZRT1, using a minisatellite inserted into the ADE2 locus in Saccharomyces cerevisiae. Here, we examined ZRT1-mediated minisatellite instability in yeast strains lacking key recombination genes to determine the mechanisms by which these alterations occur. Our analysis revealed that minisatellite alterations in a Δzrt1 mutant occur by a combination of RAD52-dependent and RAD52-independent mechanisms. In this study, plasmid-based experiments demonstrate that ZRT1-mediated minisatellite alterations occur independently of chromosomal context or adenine auxotrophy, and confirmed the stationary phase timing of the events. To further examine the stationary phase specificity of ZRT1-mediated minisatellite alterations, we deleted ETR1 and POR1, genes that were previously shown to differentially affect the viability of quiescent or nonquiescent cells in stationary phase populations. These experiments revealed that minisatellite alterations in Δzrt1 mutants occur exclusively in quiescent stationary phase cells. Finally, we show that loss of ZRT1 stimulates alterations in a derivative of the human HRAS1 minisatellite. We propose that the mechanism of ZRT1-mediated minisatellite instability during quiescence is relevant to human cells, and thus, human disease.

Novel Checkpoint Pathway Organization Promotes Genome Stability in Stationary-Phase Yeast Cells

“…We subsequently demonstrated that the DNA alterations were occurring specifically within quiescent cells and not within a subpopulation of slowly growing G 0 cells. We also demonstrated that the alterations were occurring independently of the ADE2 assay system, were not affected by genomic location, and were dependent upon an active recombination system (6,7). Thus, our prior work establishes an assay system in which genomic instability can be readily studied within stationary-phase cells.…”

“…We previously described the ade2-min3 reporter assay and its ability to distinguish between minisatellite alterations occurring during log-phase growth and those occurring during stationary phase (6)(7)(8). Minisatellite alterations occurring during growth of the colony result in a white sector integrated into the red colony, while minisatellite alterations that occur specifically during stationary phase result in a novel colony morphology called "blebbing," in which white Ade ϩ microcolonies form on the surface of the red Ade Ϫ colony.…”

“…(a) The ade2-min3 allele consists of three tandem 20-bp repeats plus one additional base inserted into the ADE2 gene at the XbaI site, producing a 5-bp overhang (ACTAG) at the site of insertion and throwing the ADE2 gene out of frame. The deletion of one repeat unit or the addition of two repeat units restores the ADE2 open reading frame (6,7). (b) Strains bearing the ade2-min3 allele display a variety of blebbing phenotypes.…”

“…We previously developed an assay to monitor DNA stability in stationaryphase yeast cells. Since stationary-phase yeast cells are cells that have entered a cellular state called G 0 , which is equivalent to the mammalian quiescent state (4,5), our assay allows us to analyze events in this critical population (6)(7)(8).…”

“…To allow us to study genomic rearrangement events in stationary-phase cells, we developed a novel color segregation assay that assesses the genomic stability of repetitive DNA tracts in stationary-phase yeast cells (6,7). For this assay, we inserted a short minisatellite into the ADE2 gene (the ade2-min3 allele) of S. cerevisiae (Fig.…”