Centromere Replication Timing Determines Different Forms of Genomic Instability inSaccharomyces cerevisiaeCheckpoint Mutants During Replication Stress

Abstract

Yeast replication checkpoint mutants lose viability following transient exposure to hydroxyurea, a replication-impeding drug. In an effort to understand the basis for this lethality, we discovered that different events are responsible for inviability in checkpoint-deficient cells harboring mutations in the mec1 and rad53 genes. By monitoring genomewide replication dynamics of cells exposed to hydroxyurea, we show that cells with a checkpoint deficient allele of RAD53, rad53K227A, fail to duplicate centromeres. Following removal of the drug, however, rad53K227A cells recover substantial DNA replication, including replication through centromeres. Despite this recovery, the rad53K227A mutant fails to achieve biorientation of sister centromeres during recovery from hydroxyurea, leading to secondary activation of the spindle assembly checkpoint (SAC), aneuploidy, and lethal chromosome segregation errors. We demonstrate that cell lethality from this segregation defect could be partially remedied by reinforcing bipolar attachment. In contrast, cells with the mec1-1 sml1-1 mutations suffer from severely impaired replication resumption upon removal of hydroxyurea. mec1-1 sml1-1 cells can, however, duplicate at least some of their centromeres and achieve bipolar attachment, leading to abortive segregation and fragmentation of incompletely replicated chromosomes. Our results highlight the importance of replicating yeast centromeres early and reveal different mechanisms of cell death due to differences in replication fork progression.

G1/S and G2/M Cyclin-Dependent Kinase Activities Commit Cells to Death in the Absence of the S-Phase Checkpoint

“…In both cases, cells experience defective DNA replication caused by a condition of nucleotide depletion, and cell death can be overcome by providing cells with additional time to accumulate dNTPs and accomplish DNA replication before chromosome segregation takes place. As proposed for HU-treated mec1⌬ sml1⌬ and rad53⌬ sml1⌬ cells (19), partition of incompletely replicated DNA in mec1⌬ SML1 and rad53⌬ SML1 cells could give rise to lethal chromosome breaks. Consistent with this hypothesis, conditional inactivation of Mec1 by temperature-sensitive mec1 al-leles gives rise to the accumulation of chromosome breakage (8) and of Rad52 foci that can be reversed by nocodazole addition (Fig.…”

“…This hypothesis implies that the segregation of incompletely replicated DNA contributes to kill HU-treated mec1 and rad53 mutants. Because HUtreated mec1⌬ sml1⌬ cells can duplicate their centromeres (19) and therefore are proficient for bipolar attachment that generates tension within the spindle, this precocious spindle elongation can lead to lethal chromosome breaks due to disjunction of incompletely replicated chromosomes (19). Although the force exerted by a bipolar spindle might be insufficient to generate chromosome breakage, the presence of ssDNA in HU-treated mec1 and rad53 mutants (20,50) may cause spindle-induced breakage.…”

“…A hypomorphic mec1 mutant (mec1-100) (38), which does not block late origin firing in HU but is much less HU sensitive than mec1⌬ sml1⌬ cells, argues that regulation of late origin firing plays a relatively minor role in maintaining cell viability after exposure to replication stress (56). Cells lacking Mec1 that are kept viable by SML1 dele-tion have been shown to accumulate chromosome breakages during HU treatment as a consequence of not fully replicated chromosomes being under persistent tension exerted by the mitotic spindle (19). However, inhibiting spindle formation via nocodazole treatment does not improve viability of mec1⌬ sml1⌬ and rad53⌬ sml1⌬ cells during exposure to HU (16,55), suggesting that precocious chromosome segregation per se is not the reason for the loss of viability of HU-treated rad53 and mec1 mutants.…”

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex

“…Genomewide percent replication values were normalized, and the data were smoothed as described (Alvino et al 2007;Feng et al 2009). …”

Conservation of replication timing reveals global and local regulation of replication origin activity

“…The late replication of telomeres may be involved in feedback control of telomere length (Bianchi and Shore 2007), and the early replication of centromeres may be important for proper chromosome segregation (Feng et al 2009). Furthermore, the elevated mutation rates observed in late replicating regions might exert a selective pressure for particular regions to replicate at specific times (Stamatoyannopoulos et al 2009;Chen et al 2010;Lang and Murray 2011;Agier and Fischer 2012;Marsolier-Kergoat and Goldar 2012).…”