title: Centromere replication timing determines different forms of genomic instability in Saccharomyces cerevisiae checkpoint 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.
authors.affiliation: Department of Genome Sciences, University of Washington, Box 355065 Foege Bldg., Room S041, 1705 NE Pacific St., Seattle, Washington 98195, USA.
authors.forename: Wenyi
authors.initials: W
authors.lastname: Feng
authors.forename: Jeff
authors.initials: J
authors.lastname: Bachant
authors.forename: David
authors.initials: D
authors.lastname: Collingwood
authors.forename: M K
authors.initials: MK
authors.lastname: Raghuraman
authors.forename: Bonita J
authors.initials: BJ
authors.lastname: Brewer
mesh_terms: D000483:Alleles; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002503:Centromere / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D019457:Chromosome Breakage / Q000187:drug effects; D020090:Chromosome Segregation / Q000187:drug effects / Q000235:genetics; D015825:Chromosomes, Fungal / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000235:genetics; D042822:Genomic Instability / Q000187:drug effects; D006918:Hydroxyurea / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D016196:S Phase / Q000187:drug effects / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects / Q000235:genetics*; D013997:Time Factors
chemical_list: D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D047908:Intracellular Signaling Peptides and Proteins; C115052:SML1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D006918:Hydroxyurea