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

Abstract

Most DNA alterations occur during DNA replication in the S phase of the cell cycle. However, the majority of eukaryotic cells exist in a nondividing, 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 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 than in actively growing cells. For the MRC1-dependent pathway, the checkpoint itself may not be the important element; rather, it may be loss of the checkpoint proteins' other functions that contributes to DNA instability.

Synthetic genetic array screen identifies PP2A as a therapeutic target in Mad2-overexpressing tumors

“…Because the spindle checkpoint and cell cycle regulation are highly conserved between yeast and humans (15), we used synthetic genetic array (SGA) technology (16,17) to screen the haploid deletion MATa library (4,541 strains) for candidate genes whose deletion kills Mad2-overexpressing yeast cells. Deletion mutant strains carrying pGAL-MAD2 were spotted onto dextrose or galactose plates.…”

Integrating Information in Biological Ontologies and Molecular Networks to Infer Novel Terms

“…6a), Unicorn identified a sub-complex of the replication fork protection complex (GO:0031298) involving three proteins Csm3p, Mrc1p and Tof1p. These three proteins form the replication fork-pausing complex (FPC)2425, which is associated with replication sites and prevents genomic instability through mediating checkpoint signaling in stationary-phase cells26.…”

A Whole Genome Screen for Minisatellite Stability Genes in Stationary-Phase Yeast Cells

“…We constructed the ade2-min3 query strain DTK893 ( MAT a his3-1 ura3-0 can1 :: MFA1pr-spHIS5 ade2-min3 - URA3MX ) using a two-step PCR process as previously described (Alver et al 2013). To summarize, the plasmid pDC369 was used to generate a URA3MX PCR product with flanking sequence using primers 14193004 and 14193005.…”

“…For our study, we performed a modified SGA analysis as described in our previous work (Alver et al 2013; Tong and Boone 2006; Tong et al 2001) (Figure 1C). To summarize, we inoculated YPD liquid media with a single red colony of query strain DTK1189 5a or DTK1624.…”

“…Genes involved in these pathways include END3 , RAD27 , PKC1 , ZRT1 , and ZAP1 . More recently, we used a modified version of the synthetic genetic array (SGA) protocol (Li et al 2011; Tong and Boone 2006; Tong et al 2001) to screen all of the genes annotated for checkpoint function and found that a subset of those genes also influence stationary phase minisatellite stability (Alver et al 2013). Here, we used our modified SGA protocol to screen the entire yeast genome in an effort to: (1) determine what factors are involved in regulating minisatellite stability in stationary phase cells and (2) determine whether minisatellites varying in repeat composition and size are regulated by different factors in stationary phase cells.…”