Academic journal article Genetics

Centromere Replication Timing Determines Different Forms of Genomic Instability in Saccharomyces Cerevisiae Checkpoint Mutants during Replication Stress

Academic journal article Genetics

Centromere Replication Timing Determines Different Forms of Genomic Instability in Saccharomyces Cerevisiae Checkpoint Mutants during Replication Stress

Article excerpt

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.

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CENTROMERES have long been known to be one of the earliest regions of the budding yeast genome to replicate during S phase (McCarroll and Fangman 1988). However, the biological significance of early replication of centromeres remains speculative, partly owing to the lack ofmutants showing altered or delayed timing of centromere replication. During our investigation of chromosome replication dynamics during nucleotide shortage brought upon by the treatment with hydroxyurea (HU), we discovered that problems with centromere replication can lead to fundamentally different forms of genome instability.

The two mutations that exhibit interesting centromere replication phenotypes are in the genes encoding two essential protein kinases, Mec1 and Rad53, which play pivotal roles in the cellular response to DNA damaging agents as well as in cell cycle arrest in response to HU (Branzei and Foiani 2006; Tourriere and Pasero 2007). Mutations in the kinase domains of Mec1 and Rad53 render the proteins checkpoint deficient and cause the cells carrying such mutations to be hypersensitive to HU. Because Mec1 is an upstream effector of Rad53 in the replication checkpoint pathway, checkpoint-deficient alleles of the two genes are thought to lead to similar phenotypes in response to replication impediments. When rad53 cells encounter HU during S phase, they fail to slow the temporal program of origin firing, expose large regions of single-stranded DNA (ssDNA) at effectively all origins, and elongate their spindles, a phenotype that indicates that the cells are attempting premature chromosome partitioning (Allen et al. 1994; Weinert et al. 1994; Desany et al. 1998; Santocanale and Diffley 1998; Sogo et al. 2002; Feng et al. 2006). Similarly, mec1 cells have also been shown to initiate precocious segregation of unreplicated chromosomes upon exposure to HU (Weinert et al. 1994; Sanchez et al. 1996). Even after the removal of HU, both mec1 and rad53 cells show considerable reduction in their ability to produce progeny. However, the reason for inviability after HU exposure is ill defined.We reasoned that understanding the molecular basis of cell death would help elucidate the role of checkpoint control in DNA replication and cell cycle regulation and in the maintenance of genome integrity.

Previous reports indicated that following transient exposure to HU, rad53 checkpoint-deficient cells are unable to complete DNA replication, which in turn was thought to constitute the primary reason for the loss of viability (Desany et al. …

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