A Slowed Cell Cycle Stabilizes the Budding Yeast Genome

Abstract

During cell division, aberrant DNA structures are detected by regulators called checkpoints that slow division to allow error correction. In addition to checkpoint-induced delay, it is widely assumed, though rarely shown, that merely slowing the cell cycle might allow more time for error detection and correction, thus resulting in a more stable genome. Fidelity by a slowed cell cycle might be independent of checkpoints. Here we tested the hypothesis that a slowed cell cycle stabilizes the genome, independent of checkpoints, in the budding yeast Saccharomyces cerevisiae We were led to this hypothesis when we identified a gene (ERV14, an ER cargo membrane protein) that when mutated, unexpectedly stabilized the genome, as measured by three different chromosome assays. After extensive studies of pathways rendered dysfunctional in erv14 mutant cells, we are led to the inference that no particular pathway is involved in stabilization, but rather the slowed cell cycle induced by erv14 stabilized the genome. We then demonstrated that, in genetic mutations and chemical treatments unrelated to ERV14, a slowed cell cycle indeed correlates with a more stable genome, even in checkpoint-proficient cells. Data suggest a delay in G2/M may commonly stabilize the genome. We conclude that chromosome errors are more rarely made or are more readily corrected when the cell cycle is slowed (even ∼15 min longer in an ∼100-min cell cycle). And, some chromosome errors may not signal checkpoint-mediated responses, or do not sufficiently signal to allow correction, and their correction benefits from this "time checkpoint."

Keywords: accuracy; chromosome instability; delayed cell cycle; erv14; speed.

Figure 1

The ChrVII and ChrV disome systems. (A) Schematic of the ChrVII disome system. Two homologs are shown; the CAN1 homolog is black and the non-CAN1 homolog is gray. The 403-site is shown in red and ERV14 is shown in green due to their relevance in the discovery of slowed cell cycle stabilization. (B) Colony phenotypes. (Green) Allelic recombination and chromosome loss result in stable round colonies. (Red) A cell with an unstable chromosome generates a sectored colony containing three types of cells: cells inheriting the unstable chromosome, cells that have lost the unstable chromosome, and cells that have undergone allelic recombination to stabilize the unstable chromosome. (C) Schematic of the ChrV disome system. Two homologs are shown: the CAN1 homolog is black and the non-CAN1 homolog is gray. (D) Same as in B.

Figure 2

The 403-site deletions and ERV14 complementation. (A) Explanations of the various 403-site deletions. Deletions/mutations are on both ChrVII homologs. Various rad9Δ 403-site deletions normalized to rad9Δ. (i) Large deletion of the 403-site, includes deletions of ERV14 and TYW3. (ii) Small deletion of the 403-site, ERV14 and TYW3 are left intact. (iii) Right deletion of the 403-site, ERV14 is intact and TYW3 is deleted. (iv) Left deletion of the 403-site, ERV14 is deleted and TYW3 is intact. (v) atg/atg deletion, only ERV14 start codons mutated. (B) ERV14 complementation of rad9Δ erv14. The rad9Δ erv14 ERV14x2 strain is normalized to rad9Δ. Blue and red squares correspond to genome fold stabilization increase or genome fold stabilization decrease (<1.0 = increased instability), respectively.

Figure 3

DNA damage sensitivity assays. (A) Percentage of viability after 6-hr exposure to 0.005% MMS liquid rich media for rad51Δ, rad17Δrad9, rad9Δ, and mad2Δ vs. erv14⁻ counterparts. (B) Percentage of viability after 6-hr exposure to 0.15 M HU liquid rich media for rad51Δ, rad17Δrad9Δ, rad9Δ, and mad2Δ vs. erv14⁻ counterparts. *Statistically significant P ≤ 0.05 using Kruskal–Wallis test.

Figure 4

DNA content FACS analysis and nuclear profiling. (A) DNA content FACS analysis of checkpoint-proficient, WT and cdc13 cells with their erv14⁻counterparts. Also shown are corresponding doubling times and genome fold stabilization. (B) DNA content FACS analysis of checkpoint-deficient cells rad9Δ and mad2Δ with their erv14⁻ counterparts. Also shown are corresponding doubling times and genome fold stabilization. (C) DNA content FACS analysis of WT^GCR with its erv14⁻ counterpart. Also shown are corresponding doubling times and genome fold stabilization. *Statistically significant P < 0.01 using Kruskal–Wallis test.

Figure 5

G2/M phase delay by mih1Δ. Instability table of checkpoint-proficient and -deficient cells and their mih1Δ counterparts. DNA content FACS analysis of checkpoint-deficient and -proficient rad9Δ and cdc13 cells with their mih1⁻ counterparts. Also shown are corresponding doubling times and genome fold stabilization. Blue and red squares correspond to genome fold stabilization increase or genome fold stabilization decrease (<1.0 = increased instability), respectively. *Statistically significant P < 0.01 using Kruskal–Wallis test.

Figure 6

Slowed cell cycle stabilization models of mutant spindle checkpoint, telomere biology, and DDR cells. Green glow indicates WT/WT-like outcome; red glow indicates aberrant/catastrophic outcome. (A, left) WT MAD2. Mad2 activates spindle checkpoint cell cycle delay, spindle attaches, chromosomes segregate properly. (Center) mad2Δ. Defective spindle checkpoint, no cell cycle delay, chromosomes mis-segregate. (Right) mad2Δ erv14. In a defective spindle checkpoint (mad2Δ) cell, the erv14-induced cell cycle delay creates time for spindle attachment; chromosomes segregate properly. (B, left) WT CDC13 Cdc13 protects telomere during replication, and chromosomes replicate properly. (Center) cdc13. Defective Cdc13 is compromised for telomere binding, allowing degradation of exposed chromosome end by exonucleases, resulting in shorter chromosomes with no telomere end protection, resulting in further degradation and instability. (Right) cdc13 erv14. Mutant Cdc13 is compromised for telomere binding, and the erv14-induced cell cycle delay creates time for more mutant Cdc13 to bind to telomere, resulting in chromosomes replicating properly. (C, left) WT DDR⁺. DDR is fully functional, so a cell cycle delay allows DNA lesions to be recognized and repaired efficiently, and thus chromosomes replicate properly. (Center) In a ddr⁻ cell some DNA lesions escape detection or suffer incomplete repair, resulting in DNA lesions/ssDNA gaps, which persist during DNA replication, generating shorter and damaged chromosomes. (Right) In a ddr⁻ erv14 cell, the partially functioning DDR is permitted more time to repair DNA lesions during the erv14-induced cell cycle delay.