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Originally published as Genetics Published Articles Ahead of Print on October 5, 2009.
Genetics, Vol. 183, 1249-1260, December 2009, Copyright © 2009
doi:10.1534/genetics.109.107508
Centromere Replication Timing Determines Different Forms of Genomic Instability in Saccharomyces cerevisiae Checkpoint Mutants During Replication Stress
Wenyi Feng*,
Jeff Bachant
,
David Collingwood
,
M. K. Raghuraman* and
Bonita J. Brewer*,1
* Department of Genome Sciences and Department of Mathematics, University of Washington, Seattle, Washington 98195 and Department of Cell Biology and Neuroscience, University of California, Riverside, California 92521
1 Corresponding author: Department of Genome Sciences, University of Washington, Box 355065 Foege Bldg., Room S041, 1705 NE Pacific St., Seattle, WA 98195.
E-mail: bbrewer{at}gs.washington.edu
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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Genetics 2009 183: NP.
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