Article

Analysis of Gross-Chromosomal Rearrangements in Saccharomyces cerevisiae

Ludwig Institute for Cancer Research, University of North Texas, Health Science Center, Fort Worth, USA.
Methods in Enzymology (Impact Factor: 2.19). 02/2006; 409:462-76. DOI: 10.1016/S0076-6879(05)09027-0
Source: PubMed

ABSTRACT Cells utilize numerous DNA metabolic pathways and cell-cycle checkpoints to maintain the integrity of their genome. Failure of these mechanisms can lead to genome instability, abnormal cell proliferation, and cell death. This chapter describes a method for the measurement of the rate of accumulating gross-chromosomal rearrangements (GCRs) in haploid cells of the yeast Saccharomyces cerevisiae. The isolation of cells with GCRs relies on the simultaneous loss of two counterselectable markers, CAN1 and URA3, within a nonessential region on the left arm of chromosome V. Healing of DNA breaks by de novo telomere addition, translocations, large interstitial deletions, and chromosome fusion has been detected using a PCR-based procedure for the mapping and amplification of breakpoint junctions, which is also described in detail here. This GCR analysis provides an effective tool for the assessment of the contribution by multiple cellular mechanisms to the maintenance of genome integrity.

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    • "To test nuclear genome instability in a large number of samples, we designed a quantitative and highly sensitive assay called CINA (Figure 1). It is based on the gross chromosomal rearrangement (GCR) assay (Schmidt et al. 2006) but is 10,000-times more sensitive. To improve the sensitivity, we designed the assay based on diploid cells (Figure 1A) with the following features: as in the original GCR assay, chromosome instability is measured by positive selection of two marker losses (URA3 and CAN1, linked in the distal part of chromosome V) (Chen and Kolodner 1999); it uses a diploid, allowing either breakage or full chromosome loss to occur without loss of viability, in contrast to the original GCR assay in which haploid cells can only break within a limited area around the URA3-CAN1 markers and survive; the addition of a LEU2 marker close to CEN V allows the ability to distinguish between chromosome breakage and chromosome loss (Figure 1B) and CINA is performed starting from single cells grown to colonies on the plate of choice (clonal assay). "
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    ABSTRACT: Mitochondrial dysfunctions are an internal cause of nuclear genome instability. Because mitochondria are key regulators of cellular metabolism, we have investigated a potential link between external growth conditions and nuclear chromosome instability in cells with mitochondrial defects. Using Saccharomyces cerevisiae, we find that cells lacking mitochondrial DNA (rho0 cells) have a unique feature, with nuclear chromosome instability that occurs in non-dividing cells and strongly fluctuates depending on the cellular environment. Calorie restriction, lower growth temperatures, growth at alkaline pH, anti-oxidants or presence of nearby wild type cells all efficiently stabilize nuclear genomes of rho0 cells, while high glucose and ethanol boost instability. In contrast, other respiratory mutants that still possess mitochondrial DNA (RHO+) keep fairly constant instability rates under the same growth conditions, like wild type or other RHO+ controls. Our data identify mitochondrial defects as an important driver of nuclear genome instability influenced by environmental factors.
    G3-Genes Genomes Genetics 12/2013; 4(3). DOI:10.1534/g3.113.010108 · 2.51 Impact Factor
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    • "b The rate of accumulating gross-chromosomal rearrangements (GCRs) is calculated by selecting for cells resistant to canavanine (Canr) and 5-fluoro-orotic acid (5-FOAr) and is expressed as Canr 5-FOAr × 10-10 [77]. "
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    ABSTRACT: Genome instability is associated with human cancers and chromosome breakage syndromes, including Bloom's syndrome, caused by inactivation of BLM helicase. Numerous mutations that lead to genome instability are known, yet how they interact genetically is poorly understood. We show that spontaneous translocations that arise by nonallelic homologous recombination in DNA-damage-checkpoint-defective yeast lacking the BLM-related Sgs1 helicase (sgs1Δ mec3Δ) are inhibited if cells lack Mec1/ATR kinase. Tel1/ATM, in contrast, acts as a suppressor independently of Mec3 and Sgs1. Translocations are also inhibited in cells lacking Dun1 kinase, but not in cells defective in a parallel checkpoint branch defined by Chk1 kinase. While we had previously shown that RAD51 deletion did not inhibit translocation formation, RAD59 deletion led to inhibition comparable to the rad52Δ mutation. A candidate screen of other DNA metabolic factors identified Exo1 as a strong suppressor of chromosomal rearrangements in the sgs1Δ mutant, becoming even more important for chromosomal stability upon MEC3 deletion. We determined that the C-terminal third of Exo1, harboring mismatch repair protein binding sites and phosphorylation sites, is dispensable for Exo1's roles in chromosomal rearrangement suppression, mutation avoidance and resistance to DNA-damaging agents. Our findings suggest that translocations between related genes can form by Rad59-dependent, Rad51-independent homologous recombination, which is independently suppressed by Sgs1, Tel1, Mec3 and Exo1 but promoted by Dun1 and the telomerase-inhibitor Mec1. We propose a model for the functional interaction between mitotic recombination and the DNA-damage checkpoint in the suppression of chromosomal rearrangements in sgs1Δ cells.
    Genome Integrity 10/2011; 2:8. DOI:10.1186/2041-9414-2-8
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    • "This implies essential distinctions in the mechanisms of maintenance of haploid and diploid genomes and justifies the use of yeast as a model for studying these mechanisms. Needless to say, gross chromosomal rearrangements do occur in haploid cells [14]–[16] and their rate can be measured [17]–[19]. The level of gross chromosomal rearrangements (GCR) demonstrated in haploid cells is in the range of 10−9 to 10−10 per cell, per generation [14], indicating that their rate in wild type haploid yeast cells is 105 fold lower than in diploid cells [11], [12], and is even lower than the haploid point mutation frequency, which falls between 10−7 and 10−9 depending on the marker used or mutagenic event considered [11], [20]–[23]. "
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    ABSTRACT: Maintaining a stable genome is one of the most important tasks of every living cell and the mechanisms ensuring it are similar in all of them. The events leading to changes in DNA sequence (mutations) in diploid cells occur one to two orders of magnitude more frequently than in haploid cells. The majority of those events lead to loss of heterozygosity at the mutagenesis marker, thus diploid-specific genome stability mechanisms can be anticipated. In a new global screen for spontaneous loss of function at heterozygous forward mutagenesis marker locus, employing three different mutagenesis markers, we selected genes whose deletion causes genetic instability in diploid Saccharomyces cerevisiae cells. We have found numerous genes connected with DNA replication and repair, remodeling of chromatin, cell cycle control, stress response, and in particular the structural maintenance of chromosome complexes. We have also identified 59 uncharacterized or dubious ORFs, which show the genome instability phenotype when deleted. For one of the strongest mutators revealed in our screen, ctf18Δ/ctf18Δ the genome instability manifests as a tendency to lose the whole set of chromosomes. We postulate that this phenomenon might diminish the devastating effects of DNA rearrangements, thereby increasing the cell's chances of surviving stressful conditions. We believe that numerous new genes implicated in genome maintenance, together with newly discovered phenomenon of ploidy reduction, will help revealing novel molecular processes involved in the genome stability of diploid cells. They also provide the clues in the quest for new therapeutic targets to cure human genome instability-related diseases.
    PLoS ONE 06/2011; 6(6):e21124. DOI:10.1371/journal.pone.0021124 · 3.23 Impact Factor
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