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FEMS Yeast Research 5 (2004) 127–132
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Mitochondria damage checkpoint in apoptosis and genome stability
Keshav K. Singh
*
Department of Cancer Genetics, Cell and Virus Building, Room 247, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA
Received 3 March 2004; received in revised form 16 April 2004; accepted 20 April 2004
First published online 18 May 2004
Abstract
Mitochondria perform multiple cellular functions including energy production, cell proliferation and apoptosis. Studies described in this paper suggest a role for mitochondria in maintaining genomic stability. Genomic stability appears to be dependent on
mitochondrial functions involved in maintenance of proper intracellular redox status, ATP-dependent transcription, DNA replication, DNA repair and DNA recombination. To further elucidate the role of mitochondria in genomic stability, I propose a
mitochondria damage checkpoint (mitocheckpoint) that monitors and responds to damaged mitochondria. Mitocheckpoint can
coordinate and maintain proper balance between apoptotic and anti-apoptotic signals. When mitochondria are damaged, mitocheckpoint can be activated to help cells repair damaged mitochondria, to restore normal mitochondrial function and avoid production of mitochondria-defective cells. If mitochondria are severely damaged, mitocheckpoint may not be able to repair the damage
and protect cells. Such an event triggers apoptosis. If damage to mitochondria is continuous or persistent such as damage to mitochondrial DNA resulting in mutations, mitocheckpoint may fail which can lead to genomic instability and increased cell survival in
yeast. In human it can cause cancer. In support of this proposal we provide evidence that mitochondrial genetic defects in both yeast
and mammalian systems lead to impaired DNA repair, increased genomic instability and increased cell survival. This study reveals
molecular genetic mechanisms underlying a role for mitochondria in carcinogenesis in humans.
Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Mitochondrial damage; Apoptosis; Genomic instability; ATP; DNA damage; DNA repair; Oxidative stress; Oxidative damage
1. Introduction
Mitochondria play a central role in many cellular
functions including energy production, respiration,
heme synthesis, lipids synthesis, metabolism of amino
acids, nucleotides, and iron, and maintenance of intracellular homeostasis of inorganic ions, cell motility, cell
proliferation and apoptosis [1,2]. Mitochondria contain
their own DNA (mtDNA) that amounts on average to
about 15% of the DNA content of Saccharomyces cerevisiae. It means that a haploid cell contains about 50
copies of the 75-kb mitochondrial genome. The mtDNA
occurs in small clusters called nucleoids or chondriolites.
The number of mtDNA molecules in nucleoids varies in
size and numbers in response to physiological condi-
*
Tel.: +1-716-845-8017; fax: +1-716-845-1047.
E-mail address: [email protected] (K.K. Singh).
tions. All of the mtDNA molecules in S. cerevisiae are
comprised of polydisperse linear tandem arrays of the
genome. The linear molecules are accompanied by small
amounts of circular forms [3]. While nuclear DNA encodes the majority of the mitochondrial proteins only a
few of these proteins are encoded by mitochondrial
DNA. A recent mitochondrial proteomic study in S.
cerevisiae identified at least 750 mitochondrial proteins
that perform mitochondrial function [4].
The last decade has witnessed an increased interest in
mitochondria, not only because mitochondria were
recognized to play a central role in apoptosis but also
since mitochondrial genetic defects were found to be
involved in the pathogenesis of a number of human
diseases [2,5–9]. Human mitochondrial diseases are
caused both by mutations in mtDNA and in nuclear
DNA involved in mitochondrial function. In this paper
we describe the importance of mitochondria in yeast
apoptosis and genome stability.
1567-1356/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsyr.2004.04.008
128
K.K. Singh / FEMS Yeast Research 5 (2004) 127–132
2. Mitochondria are the major site of oxidative stress in
the cell
Mitochondria are the major source of endogenous
reactive oxygen species (ROS) in the cell, because they
carry the electron transport chain that during oxidative
phoshorylation reduces oxygen to water by addition of
electrons [1,10]. It is estimated that the endogenous
production of ROS within human mitochondria is about
107 molecules/mitochondrion/day during normal oxidative phosphorylation [1,10]. Unlike nuclear DNA,
human mtDNA contains no protective histones, and is
continuously exposed to ROS generated during oxidative phosphorylation (it is estimated that up to 4% of the
oxygen consumed by cells is converted to ROS under
physiological conditions) [1]. ROS induce more persistent damage to mtDNA than to nuclear DNA [11]. ROS
also produce more than 20 types of mutagenic base
modifications in DNA [12]. These DNA lesions cause
mutations in mtDNA that can lead to impairment of
mitochondrial function [1,13]. Taken together, this
makes clear that mtDNA is extremely susceptible to
mutation by ROS-induced damage.
3. Mitochondrial impairment leads to nuclear genome
instability
Although the mitochondrial and nuclear genomes are
physically distinct, there is a high degree of cross-talk
and functional interdependence between the two genomes. Since mitochondria are the major site of ROS in
the cell, and mtDNA is more frequently damaged, we
were interested in identifying the consequences of mitochondrial dysfunction on nuclear genome stability. To
address this question we isolated an S. cerevisiae clone
that lacked mitochondrial genome entirely (q° strain).
We also isolated a q strain containing a large deletion
in mtDNA. Our studies conducted with these strains
suggest that indeed mitochondrial dysfunction leads to
nuclear genome instability [14]. We identified two
pathways: a ROS-dependent and a ROS-independent
pathway involved in mitochondria which leads to nuclear genome instability. Our studies also suggest that
ROS-independent mitochondria-mediated nuclear genome instability was controlled by an error-prone DNA
repair pathway [14]. However, the error-prone repair
pathway was not involved in ROS-dependent nuclear
mutator phenotype. We carried out similar studies in
mammalian cells that suggest that mutations in nuclear
DNA arise due to reduced DNA repair activity. Given
that mitochondria are the major producer of ATP, it is
also likely that mitochondrial dysfunction leads to the
reduction in ATP level that may affect ATP-dependent
pathways involved in transcription, DNA replication,
DNA repair, and DNA recombination (see Table 1).
Table 1
Putative ATPases that may contribute to genomic instability in q° cells
Cellular function
Genes
Transcription
DBP3, DBP6, DBP7, DBP8, DBP9, DBP10,
and PRP2, PRP5
CDC46, CDC47, DCD54, DNA2, HMI1,
MCM2, MCM3, MCM6, ORC5, RRM3
and SGS1
UNG1 and CDC9, DNL4
RAD5, RAD7 RAD16, RAD18, RAD26
Replication
Base excision repair
Nucleotide excision
repair
Mismatch repair
Recombination
Chromosome
maintenance
Mlh1, Msh2, Msh6, PMS1 Msh1
RAD54, RDH54
SMC1, SMC2, SMC3 and SMC4
Consistent with this notion a study recently reported
chromosomal abnormalities in mouse fibroblast cells
lacking mitochondrial MnSOD activity [15] and in response to mitochondrial inhibitors [16]. Together these
studies suggest that mitochondrial dysfunction leads to
nuclear genome instability.
4. Mitochondrial impairment leads to altered expression
of antioxidant genes
In order to identify genes whose expression may
contribute to increased mutagenesis in nuclear DNA we
did a gene expression analysis using Affymetrix microarrays. Our results demonstrate that CTT1 and GPX1
antioxidant genes were down-regulated while GPX2 was
up-regulated in q° cells. This indicates that an altered
level of antioxidant status may contribute to nuclear
DNA mutation. The list of genes and the fold changes in
their expression in q° cells are described in Table 2.
5. Mitochondrial impairment alters nucleotide metabolism
Although our studies suggest that reduced DNA
repair and changes in expression of antioxidant genes
may contribute to mutation in nuclear genome, it is
likely that other factors are involved. Mitochondria are
intimately involved in deoxyribose nucleoside triphosphate (dNTP) biosynthesis [17,18]. It is conceivable
that mitochondrial impairment contributes to muta-
Table 2
Changes in antioxidant gene expression in q° cells
Gene name
Fold changes
CTT1
GPX1
GPX2
)5.9
)5.3
7.9
K.K. Singh / FEMS Yeast Research 5 (2004) 127–132
genesis of the nuclear genome in part due to impaired
nucleotide biosynthesis. In fact, it is well established
that an imbalance in the dNTP pool is mutagenic to
cells [19]. Studies demonstrate that a dNTP pool imbalance can induce nucleotide insertion, frame-shift
mutation [19], sister chromatid exchange, recombination and double-strand break [20–22].
6. Mitochondrial impairment alters ATP utilization
Mitochondria produce energy in the form of adenosine 50 -triphosphate (ATP) in a process termed oxidative
phosphorylation [23]. Under the condition of high
proton-motive force, the F1 F0 -ATP synthase complex
catalyzes the formation of ATP from adenosine 50 -diphosphate (ADP) in the manner that is coupled to the
transport of protons from the intermembrane space
across the inner membrane to the matrix. A decrease in
the proton-motive force as a result of oxygen deprivation to the cell, or because of the uncoupling of oxidative phosporylation, can cause the reversal of the action
of F1 F0 synthase, resulting in the hydrolysis of ATP to
ADP and phosphate. This hydrolytic activity of F1 F0 ATP synthase is regulated directly by the natural inhibitor protein called Inh1 in yeast [23,24]. In yeast, the
inhibitory activity of Inh1 is enhanced by two stabilizing
factors: STF1 and STF2 (stabilizing factors). Interestingly, microarray analysis showed that INH1, STF1 and
STF2 are all down-regulated in q° cells (see Table 3).
Our large-scale gene expression analysis suggests that
down-regulation of genes involved in preventing the
hydrolysis of ATP may allow essential ATP-dependent
functions so that cellular functions are not compromised
severely. However, this view may be simplistic because
the level of RNA does not give a perfect image of enzyme activity.
Table 3
Expression of genes involved in ATP hydrolysis in q° cells
Gene names
Fold changes
INH1
STF1
STF2
)7.2
)2.2
)4.3
Total RNA was isolated from exponentially growing S. cerevisiae
cells according to manufactures guide lines using RNeasy (QIAGEN).
Total RNA (5 lg) was converted in double-stranded cDNA by GIBCO
BRL’s SuperScript Choice system for cDNA synthesis (Life Technologies) and a T7-(dT)24 oligomer provided by Research genetics
(Huntsville, AL). Double-stranded cDNA was purified by phenol/
chloroform extraction and ethanol precipitation. In vitro transcription
was performed with T7 RNA polymerase following the instructions
from BioArray high-yield RNA transcript Labeling kit from Enzo
(Affymetrix). Gene array analysis was conducted using yeast gene array (from Affymetrix) as described [36].
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7. A mitochondria damage checkpoint (mitocheckpoint)
Checkpoint was defined by Hartwell and Weinert [37]
as control mechanism that ensures the proper order of
cellular events by arresting or delaying progression
through the cell cycle in response to DNA damage [25].
Based on our comparative gene expression analysis between the wild-type yeast S. cerevisiae and the q° derivative cells, I propose that cells contain a mitochondria
damage checkpoint (mitocheckpoint) that avoids production of cells defective in mitochondrial function.
Mitocheckpoint monitors the functional state of mitochondria and responds accordingly when mitochondria
are damaged or become dysfunctional. Mitocheckpoint
can adjust the cell cycle response and gene expression to
help repair-damaged mitochondria to restore normal
mitochondrial function [7]. This hypothesis is consistent
with (i) A study reporting cell cycle arrest of human cells
in response to respiratory inhibitors [26]. Following low
doses there was a significant increase in the proportion
of cells in G1. However, exposure to higher doses of
respiratory inhibitors caused G2-M arrest [26]. (ii) Existence of highly coordinated cross-talk between mitochondria and nucleus in yeast [27]. (iii) Mutations in
CIT1 gene (encoding mitochondrial citrate synthase) of
Podospora anserina show a defect in progression to
meiosis, leading to developmental abnormalities. In this
organism, citrate synthase appears to work as meiotic
checkpoint [28]. And (iv) A link between nuclear DNA
and mtDNA replication in Drosophila [29]. In Drosophila, a transcription factor DREF coordinates nuclear and mitochondrial DNA replication. DREF also
controls the expression of genes encoding mitochondrial
single-stranded DNA-binding protein, polymerase b
and accessory subunit of polymerase c involved in
mtDNA replication [29]. DREF’s role in controlling the
expression of the DNA polymerase c and b genes also
establishes a common regulatory mechanism linking
nuclear and mitochondrial DNA replication with repair.
In S. cerevisiae Rtg1, Rtg2 and Rtg3 proteins monitor
the functional state of mitochondria and coordinate
mitochondria-to-nucleus signaling. Among RTG proteins Rtg2p contains an ATP-binding domain that may
be the sensor of the ATP level in the cell [30]. RTG
proteins are important candidates to serve as mitocheckpoint proteins. Several proteins involved in DNA
replication in yeast are absolutely dependent on ATP for
their function (see Table 1). The mitocheckpoint may
regulate ATPases known to be involved in DNA repair
and recombination. Examples of such ATPases are
UNG1 and CDC9 (base excision) RAD5, RAD7 RAD16,
RAD18, RAD26 (Nucleotide Excision repair), Mlh1,
Msh2, Msh6, PMS1 (DNA mismatch repair in the nucleus), Msh1 (DNA mismatch repair in the mitochondria), RAD54, and RDH54 (recombination). In
addition, mitocheckpoint can also regulate SMC
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K.K. Singh / FEMS Yeast Research 5 (2004) 127–132
(Structural Maintenance of Chromosome) proteins that
contain ATPase activity and maintain chromosomal
integrity ([30], see Table 1). The mitocheckpoint may
coordinate transcription of genes by regulating ATPdependent RNA helicases (see Table 1).
The mitocheckpoint must coordinate and maintain a
proper balance between apoptotic and anti-apoptotic
signals. Thus mitochondria must regulate mechanisms
that promote cell survival. Our studies show that a mitochondrial genetic defect causes high frequency of
mutations in the nuclear genome and promotes cell
survival when exposed to DNA-damaging agents such
as adriamycin (see Fig. 1, Table 4 and [14]). Similar to
studies in yeast, we also find that a variety of mammalian q° cells are resistant to apoptosis induced by oxidative agents [31]. Altogether our studies provide
evidence that mitochondrial dysfunction leads to evasion of apoptosis, increased cell survival and genomic
instability in both yeast and mammalian cells [14,31,32].
Fig. 2 summarizes our studies in a model where mitocheckpoint monitors damage to the mitochondria and
responds to dysfunctional state of mitochondria. When
mitochondria are damaged, mitocheckpoint is activated
which helps cells repair the damage and restore normal
mitochondrial function. If the mitochondria are severely
damaged, mitocheckpoint may not be able to protect the
cells. Such an event will trigger apoptosis [33–35]. If
damage to mitochondria is continuous or persistent
(such as mutations in mtDNA) mitocheckpoint system
can fail which can lead to genomic instability (mutator
phenotype, chromosomal aneuploidy, loss of heterozygosity), and increased cell survival and cancer in
humans.
Table 4
Frequency of mutations in nuclear genome (Frequency of CanR mutant 107 )
Adriamycin (lg ml1 )
Untreated
control
20
(lg ml1 )
40
(lg ml1 )
Wild type
q°
5.0
7.1
6.4
11.5
9.7
195.3
Both wild-type and q° cells were grown to log phase as described
[14]. Cells were centrifuged, resuspended in sterilized distilled water
containing various concentrations of adriamycin. Appropriate dilutions were made and cells were plated on YPD and SD medium containing canavanine as described [14]. The CAN1 gene of S. cerevisiae
encodes a transmembrane amino acid transporter that renders the cell
sensitive to a lethal arginine analogue, canavanine. Any inactivating
mutation in this gene results in a canavanine-resistant phenotype
(CAN1R ). Thus, the frequency of canavanine-resistant colonies measures spontaneous nuclear mutational events. Canavanine-resistant
colonies were counted after five days.
% Cell Survival
10
Fig. 2. Integrating apoptosis, genomic instability and cell survival. A
model which integrates a role for mitocheckpoint in apoptosis, genomic
stability, and cell survival. For details see Section 7 in the text.
10
1
0.1
0
20
40
-1
Adriamycin (µg ml )
Fig. 1. Cell survival in response to DNA-damaging agent. Both wildtype (circles) and q° (squares) cells were grown to log phase in YPD as
described [14]. Cells were centrifuged, resuspended in sterilized distilled
water containing various concentrations of adriamycin. Appropriate
dilutions were made and cells were plated on YPD medium. Colonies
were counted after three to four days.
It is interesting to note how much of our current
understanding of genetic and biochemical activities of
the mitochondria is owed to the unique ability of the
humble brewer’s yeast S. cerevisiae to survive without
respiration [3]. The S. cerevisiae genome was the first
eukaryotic genome that was sequenced. A comprehensive approach to the deletion of and expression of all
Open Reading Frames has been performed [4]. In addition to availability of deletion mutants of all genes,
sophisticated biochemical and genetic analysis tools are
also available to perform functional genomics and genome-wide gene expression. In summary, yeast will
continue to serve as an excellent model to understand
K.K. Singh / FEMS Yeast Research 5 (2004) 127–132
the underlying genetic mechanisms involved in apoptosis and genome instability relevant to human carcinogenesis.
Acknowledgements
This research was supported by a grant from the
National Institutes of Health RO1-097714. I thank
Drs. Lene Rasmussen and Anna Rasmussen for their
contribution to the data presented in Table 4 and
Dr. Kylie Keshav for critical reading of this manuscript.
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