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: 2.09).
02/2006; 409:462-76. DOI: 10.1016/S0076-6879(05)09027-0
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.
Available from: Lorraine S Symington
- "To better understand the types of GCRs in the rfa1-t33 and sae2D derivatives, we utilized a previously described method to identify the location of the breakpoint by overlap PCR using Ch V primers and then attempted to sequence across the junction using an arbitrary PCR strategy (Figure S3) (Schmidt et al., 2006). We characterized 12 GCR isolates from WT cells and found that repair occurred primarily by telomere addition (Table 1). "
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ABSTRACT: Foldback priming at DNA double-stranded breaks is one mechanism proposed to initiate palindromic gene amplification, a common feature of cancer cells. Here, we show that small (5-9 bp) inverted repeats drive the formation of large palindromic duplications, the major class of chromosomal rearrangements recovered from yeast cells lacking Sae2 or the Mre11 nuclease. RPA dysfunction increased the frequency of palindromic duplications in Sae2 or Mre11 nuclease-deficient cells by ∼1,000-fold, consistent with intra-strand annealing to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric isochromosome. The palindromic duplications were frequently associated with duplication of a second chromosome region bounded by a repeated sequence and a telomere, suggesting the dicentric chromosome breaks and repairs by recombination between dispersed repeats to acquire a telomere. We propose secondary structures within single-stranded DNA are potent instigators of genome instability, and RPA and Mre11-Sae2 play important roles in preventing their formation and propagation, respectively.
Available from: Kristina H Schmidt
- "Gross-chromosomal rearrangement (GCR) assay GCR rates were determined by fluctuation analysis by taking the median rate of at least 15 cultures from at least two isolates   and are shown with 95% confidence intervals . Cells with GCRs were identified by their resistance to canavanine and 5-fluoro-orotic acid (Can r 5-FOA r ), which is indicative of simultaneous inactivation of CAN1 and URA3 on chromosome V. Selective media for the GCR assay was prepared as previously described  "
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ABSTRACT: Exo1 belongs to the Rad2 family of structure-specific nucleases and possesses 5′–3′ exonuclease activity on double-stranded DNA substrates. Exo1 interacts physically with the DNA mismatch repair (MMR) proteins Msh2 and Mlh1 and is involved in the excision of the mispaired nucleotide. Independent of its role in MMR, Exo1 contributes to long-range resection of DNA double-strand break (DSB) ends to facilitate their repair by homologous recombination (HR), and was recently identified as a component of error-free DNA damage tolerance pathways. Here, we show that Exo1 activity increases the hydroxyurea sensitivity of cells lacking Pol32, a subunit of DNA polymerases δ and ζ. Both, phospho-mimicking and dephospho-mimicking exo1 mutants act as hypermorphs, as evidenced by an increase in HU sensitivity of pol32Δ cells, suggesting that they are trapped in an active form and that phosphorylation of Exo1 at residues S372, S567, S587, S692 is necessary, but insufficient, for the accurate regulation of Exo1 activity at stalled replication forks. In contrast, neither phosphorylation status is important for Exo1's role in MMR or in the suppression of genome instability in cells lacking Sgs1 helicase. This ability of an EXO1 deletion to suppress the HU hypersensitivity of pol32Δ cells is in contrast to the negative genetic interaction between deletions of EXO1 and POL32 in MMS-treated cells as well as the role of EXO1 in DNA-damage treated rad53 and mec1 mutants.
Available from: PubMed Central
- "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.
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