Published Ahead of Print 12 November 2012.
2013, 33(2):457. DOI: 10.1128/MCB.05831-11.
Mol. Cell. Biol.
Bonnie Alver, Maire K. Kelly and David T. Kirkpatrick
Stationary-Phase Yeast Cells
Promotes Genome Stability in
Novel Checkpoint Pathway Organization
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Novel Checkpoint Pathway Organization Promotes Genome Stability
in Stationary-Phase Yeast Cells
Bonnie Alver, Maire K. Kelly, David T. Kirkpatrick
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA
alterations specifically in quiescent cells in Saccharomyces cerevisiae. Here we conducted a modified version of synthetic genetic
the accumulation of genomic instability, two of the major hall-
marks of cancer. At present, little is known about the initial mu-
become cancerous. This is due to a lack of assay systems to assess
genomic stability within this specific cell population. We previ-
ously developed an assay to monitor DNA stability in stationary-
phase yeast cells. Since stationary-phase yeast cells are cells that
have entered a cellular state called G0, which is equivalent to the
mammalian quiescent state (4, 5), our assay allows us to analyze
events in this critical population (6–8).
Checkpoints act as guardians of genomic stability, preventing
the accumulation and replication of damaged DNA. In the yeast
Saccharomyces cerevisiae, several checkpoints arrest the cell cycle
when DNA damage, replication, or mitotic errors occur (Fig. 1).
During S phase, the replication checkpoint is activated when rep-
lication forks stall (9, 10). This allows DNA repair to occur and
prevents the formation of single-stranded DNA (ssDNA), which
can be used as a template for chromosomal rearrangements. The
as Mrc1p or Rad9p (11–13). Phosphorylation of Mrc1p or Rad9p
results in the recruitment of Rad53p to the replication fork and
activation of the replication checkpoint (14–16).
Intra-S-phase and DNA damage checkpoints utilize similar
signal transduction pathways to block cell cycle progression (Fig.
1) (10, 17). The DNA damage signal is propagated by the kinases
Mec1p and Tel1p, a protein functionally redundant with Mec1p
(18–20). Mec1p and Rad24p (a component of the RFC-like com-
plex which loads the PCNA-like clamp composed of Ddc1p,
Rad17p, and Mec3p onto DNA [21, 22]) are independently re-
cruited to the site of DNA damage (23, 24). This localization re-
ost eukaryotic cells spend the majority of their life span in a
quiescent or nondividing state. It is thought that mutations
sults in the activation of Mec1p and leads to the activation of
Rad53p through the phosphorylation of Rad9p. Alternatively,
and prevent cell cycle progression (25).
During mitosis, the spindle assembly checkpoint prevents the
onset of anaphase until all chromosomes are properly attached to
Bub1p, and Bub3p localize to unattached kinetochores (27, 28).
This binding prevents the activation of the anaphase-promoting
complex (APC) through inhibition of its substrate Cdc20p (29).
The inhibition of Cdc20p prevents the degradation of APC sub-
matids are properly aligned. These cell cycle surveillance mecha-
nisms ensure the proper replication of DNA and segregation of
In yeast, stationary-phase cells recapitulate the phenotypes of
quiescent cells found in other eukaryotic systems (4, 5). Recent
studies in S. cerevisiae have shown that the checkpoint kinase
Rad53p is phosphorylated in DNA repair mutants that are in sta-
tionary phase (30). Furthermore, the authors found a dramatic
increase in the amount of spontaneous mutability within these
cells. Due to the discovery of Rad53p activation in stationary-
phase cells, we wanted to determine if other well-characterized
checkpoint components play a role in maintaining genomic sta-
bility once cells have exited the cell cycle.
ary-phase cells, we developed a novel color segregation assay that
Received 20 June 2011 Returned for modification 12 July 2012
Accepted 6 November 2012
Published ahead of print 12 November 2012
Address correspondence to David T. Kirkpatrick, firstname.lastname@example.org.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
January 2013 Volume 33 Number 2Molecular and Cellular Biology p. 457–472mcb.asm.org
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assesses the genomic stability of repetitive DNA tracts in station-
ary-phase yeast cells (6, 7). For this assay, we inserted a short
siae (Fig. 2a). Insertion of the minisatellite tract in the ADE2 gene
pushes the gene out of frame, resulting in a red colony color and
making the cells auxotrophic for adenine. Alterations occurring
within the minisatellite are monitored by examining strains bear-
ing the ade2-min3 allele for the presence of a color segregation
phenotype. If the minisatellite tract loses one repeat or gains two
repeats, the ADE2 gene is pushed back into frame, resulting in
colonies that are now white in color and Ade?.
Previously, we performed a UV mutagenesis screen to find
mutants that destabilized the minisatellite tract within the ade2-
min3 allele (7). From this screen, we identified several mutations
a novel color segregation phenotype called blebbing. Blebs are
white Ade?microcolonies that form on the surface of the red
Ade?colony. Further characterization of the blebbing phenotype
revealed that it resulted from the loss or gain of minisatellite re-
peats that occurred once cells entered stationary phase. Restora-
tion of ADE2 function allowed the cell to escape the adenine nu-
and begin to grow again, forming the white microcolonies on the
red colony. We subsequently demonstrated that the DNA altera-
tions were occurring specifically within quiescent cells and not
within a subpopulation of slowly growing G0cells. We also dem-
ADE2 assay system, were not affected by genomic location, and
were dependent upon an active recombination system (6, 7).
Thus, our prior work establishes an assay system in which
In this study, we used a modified version of synthetic genetic
array (SGA) analysis (31, 32) to determine if well-characterized
checkpoints play an active role in stabilizing minisatellites during
stationary phase. In brief, we mated a query strain containing the
an essential gene. We examined the resulting double mutants for
the presence of a blebbing phenotype and then verified this phe-
notype in a separate strain background. Our results reveal that a
FIG1 Summary of checkpoint factors. (a) In Saccharomyces cerevisiae, several checkpoints regulate cell cycle progression to ensure that DNA damage or errors
made during replication or mitosis can be effectively repaired. These checkpoints include the replication checkpoint, intra-S-phase checkpoint, DNA damage
checkpoints, and mitotic checkpoint. Each utilizes several pathways to signal and activate cell cycle arrest. (b) In stationary-phase G0cells, only a subset of the
the strongest phenotype. Question marks designate speculative interactions or pathways. The pathway denoted in gray dotted lines represents a Rad9p-
dependent backup pathway that is activated when Mrc1p function is compromised.
was streaked onto solid YPD medium, incubated at 30°C for 2 days, and then
left at RT for 6 days. (a) The ade2-min3 allele consists of three tandem 20-bp
repeats plus one additional base inserted into the ADE2 gene at the XbaI site,
repeat units restores the ADE2 open reading frame (6, 7). (b) Strains bearing
rad53-1 strains display both blebbing and sectoring phenotypes.
Alver et al.
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