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

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455.
Molecular and Cellular Biology (Impact Factor: 4.78). 11/2012; 33(2). DOI: 10.1128/MCB.05831-11
Source: PubMed


Most DNA alterations occur during DNA replication in S phase of the cell cycle. However, the majority of eukaryotic cells exist in a non-dividing, quiescent state. Little is known about the factors involved in preventing DNA instability within this stationary-phase cell population. Previously, we utilized a unique assay system to identify mutations that increased minisatellite alterations specifically in quiescent cells in Saccharomyces cerevisiae. Here, we conducted a modified version of the Synthetic Genetic Array analysis to determine if checkpoint signaling components play a role in stabilizing minisatellites in stationary phase yeast cells. Our results revealed that a subset of checkpoint components, specifically MRC1, CSM3, TOF1, DDC1, RAD17, MEC3, TEL1, MEC1 and RAD53, prevent stationary-phase minisatellite alterations within the quiescent cell subpopulation of stationary phase cells. Pathway analysis revealed at least three pathways, with MRC1, CSM3 and TOF1 acting in a pathway independent of MEC1 and RAD53. Overall, our data indicate that some well-characterized checkpoint components maintain minisatellite stability in stationary-phase cells, but are regulated differently in those cells compared to actively growing cells. For the MRC1-dependent pathway, the checkpoint itself may not be the important element, but rather loss of the checkpoint proteins' other functions that contribute to DNA instability.

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    • "We investigated the hits of each screen independently to characterize candidate genes associated with each individual minisatellite. Several hits identified in the ade2-min3 screen were genes associated with checkpoint function and were described previously (Alver et al. 2013). Other identified genes involve DNA replication and repair. "
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    ABSTRACT: Repetitive elements comprise a significant portion of most eukaryotic genomes. Minisatellites, a type of repetitive element composed of repeat units 15-100bp in length, are stable in actively dividing cells but change in composition during meiosis and in stationary phase cells. Alterations within minisatellite tracts have been correlated with the onset of a variety of diseases, including diabetes mellitus, myoclonus epilepsy and several types of cancer. However, little is known about the factors preventing minisatellite alterations. Previously, our lab developed a color segregation assay in which a minisatellite was inserted into the ADE2 gene in the yeast Saccharomyces cerevisiae to monitor alteration events. We demonstrated that minisatellite alterations that occur in stationary phase cells give rise to a specific colony morphology phenotype known as blebbing. Here, we performed a modified version of the Synthetic Genetic Array (SGA) analysis to screen for mutants that produce a blebbing phenotype. Screens were conducted using two distinctly different minisatellite tracts: the ade2-min3 construct consisting of three identical 20bp repeats, and the ade2-h7.5 construct, consisting of 7.5 28bp variable repeats. Mutations in 102 and 157 genes affect the stability of the ade2-min3 and ade2-h7.5 alleles, respectively. Only seven hits overlapped both screens, indicating that different factors regulate repeat stability depending upon minisatellite size and composition. Importantly, we demonstrate that mismatch repair influences the stability of the ade2-h7.5 allele, indicating that this type of DNA repair stabilizes complex minisatellites in stationary phase cells. Our work provides insight into the factors regulating minisatellite stability.
    G3-Genes Genomes Genetics 03/2013; 3(4). DOI:10.1534/g3.112.005397 · 3.20 Impact Factor
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    ABSTRACT: The spindle checkpoint is essential to ensure proper chromosome segregation and thereby maintain genomic stability. Mitotic arrest deficiency 2 (Mad2), a critical component of the spindle checkpoint, is overexpressed in many cancer cells. Thus, we hypothesized that Mad2 overexpression could specifically make cancer cells susceptible to death by inducing a synthetic dosage lethality defect. Because the spindle checkpoint pathway is highly conserved between yeast and humans, we performed a synthetic genetic array analysis in yeast, which revealed that Mad2 overexpression induced lethality in 13 gene deletions. Among the human homologs of candidate genes, knockdown of PPP2R1A, a gene encoding a constant regulatory subunit of protein phosphatase 2, significantly inhibited the growth of Mad2-overexpressing tumor cells. PPP2R1A inhibition induced Mad2 phosphorylation and suppressed Mad2 protein levels. Depletion of PPP2R1A inhibited colony formation of Mad2-overexpressing HeLa cells but not of unphosphorylated Mad2 mutant-overexpressing cells, suggesting that the lethality induced by PP2A depletion in Mad2-overexpressing cells is dependent on Mad2 phosphorylation. Also, the PP2A inhibitor cantharidin induced Mad2 phosphorylation and inhibited the growth of Mad2-overexpressing cancer cells. Aurora B knockdown inhibited Mad2 phosphorylation in mitosis, resulting in the blocking of PPP2R1A inhibition-induced cell death. Taken together, our results strongly suggest that PP2A is a good therapeutic target in Mad2-overexpressing tumors.
    Proceedings of the National Academy of Sciences 01/2014; 111(4). DOI:10.1073/pnas.1315588111 · 9.67 Impact Factor


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