**3. Ogg1 and mitochondrial base excision repair**

Oxidative stress can induce many types of DNA base damage including two of the most abundant lesions, 8-hydroxyguanine (8-oxoG) and thymine glycol (TG) (Demple & Harrison, 1994; Dizdaroglu 1992; Bohr et al., 2002; Gredilla et al., 2010). Further, 8-oxoG is more susceptible to oxidative attack than guanine itself, resulting in the formation of oxidation products such as guanidinohydantoin and spiroiminodihydantoin (Bjelland & Seeberg, 2003; Hailer et al., 2005). The 8-oxoG residue exists predominantly in its keto form at physiological pH, resulting in the normal anti conformation around the *N*-glycosylic bond, and forming a common Watson-Crick base pair with cytosine. 8-oxoG adopts a *syn*  conformation and base pairs with adenine leading to transversion mutations in replicating cells (Shibutani 1991), which may play a role in the development of cancer and the process of aging (Ames 1989; Lindahl 1993). In contrast, TG strongly blocks DNA replication (Ide et al., 1985; Clark & Beardsley, 1987) and transcription (Hatahet et al., 1994; Htun & Johnston, 1992) and must be efficiently removed and repaired to maintain genetic stability. Therefore, inefficient repair of oxidative mtDNA damage augments the accumulation of mtDNA damage and mutations that can lead to mitochondrial dysfunction and apoptosis. In this section we focus attention on repair of 8-oxoG by mitochondrial 8-oxoguanine DNA glycosylase 1 (mt-Ogg1) since it is among the best characterized mitochondrial base excision repair (BER) proteins.

The BER pathway accounts for the repair of the majority of spontaneously formed oxidized bases in mtDNA important for preserving the genome stability required for long-term cell survival (Barnes & Lindahl, 2004; Gredilla et al. 2010). All mitochondrial DNA repair enzymes, including those involved in BER, are encoded in the nucleus and imported into the mitochondria (Gredilla et al. 2010). The BER pathway removes small covalent modifications, which do not distort the DNA helix, such as the base modifications generated by ROS and single-strand breaks. The BER pathway in mitochondria and nucleus is highly conserved in all cellular organisms, from bacteria to man. BER is carried out in four sequential enzymatic steps catalyzed by the enzymes DNA glycosylase, AP-endonuclease, DNA polymerase and DNA ligase (Dianova et al., 2001; Gredilla et al., 2010). The initial

show that asbestos causes both a dose- and time-dependent reduction in △ψm that was associated with release of cytochrome c from the mitochondria to the cytoplasm as well as activation of caspase-9 (Panduri et al., 2003). In this study, both an iron chelator (phytic acid) and a free radical scavenger (sodium benzoate) blocked asbestos-induced reductions in △ψm and caspase-9, implying the importance of both iron-derived ROS and the mitochondrial death pathway. Furthermore, asbestos-induced apoptosis in A549 cells that stably overexpress Bcl-xl, an anti-apoptotic Bcl-2 family member, was significantly attenuated as compared to wild-type cells as evidenced by preservation of the OMM integrity and reduced DNA fragmentation (Panduri et al., 2003). Using confocal microscopy and Western blotting of mitochondrial proteins, we showed that asbestos stimulates mitochondrial translocation of pro-apoptotic Bax and that these effects are blocked by phytic acid (Panduri et al., 2006). Notably, using A549-ρ0 cells that lack mtDNA and a functional electron transport chain necessary for mitochondrial ROS generation, asbestos-induced ROS production, caspase-9 activation, and intrinsic apoptosis were all completely blocked (Panduri et al., 2006). These findings establish an important role for mitochondrial ROS in

Oxidative stress can induce many types of DNA base damage including two of the most abundant lesions, 8-hydroxyguanine (8-oxoG) and thymine glycol (TG) (Demple & Harrison, 1994; Dizdaroglu 1992; Bohr et al., 2002; Gredilla et al., 2010). Further, 8-oxoG is more susceptible to oxidative attack than guanine itself, resulting in the formation of oxidation products such as guanidinohydantoin and spiroiminodihydantoin (Bjelland & Seeberg, 2003; Hailer et al., 2005). The 8-oxoG residue exists predominantly in its keto form at physiological pH, resulting in the normal anti conformation around the *N*-glycosylic bond, and forming a common Watson-Crick base pair with cytosine. 8-oxoG adopts a *syn*  conformation and base pairs with adenine leading to transversion mutations in replicating cells (Shibutani 1991), which may play a role in the development of cancer and the process of aging (Ames 1989; Lindahl 1993). In contrast, TG strongly blocks DNA replication (Ide et al., 1985; Clark & Beardsley, 1987) and transcription (Hatahet et al., 1994; Htun & Johnston, 1992) and must be efficiently removed and repaired to maintain genetic stability. Therefore, inefficient repair of oxidative mtDNA damage augments the accumulation of mtDNA damage and mutations that can lead to mitochondrial dysfunction and apoptosis. In this section we focus attention on repair of 8-oxoG by mitochondrial 8-oxoguanine DNA glycosylase 1 (mt-Ogg1) since it is among the best characterized mitochondrial base excision

The BER pathway accounts for the repair of the majority of spontaneously formed oxidized bases in mtDNA important for preserving the genome stability required for long-term cell survival (Barnes & Lindahl, 2004; Gredilla et al. 2010). All mitochondrial DNA repair enzymes, including those involved in BER, are encoded in the nucleus and imported into the mitochondria (Gredilla et al. 2010). The BER pathway removes small covalent modifications, which do not distort the DNA helix, such as the base modifications generated by ROS and single-strand breaks. The BER pathway in mitochondria and nucleus is highly conserved in all cellular organisms, from bacteria to man. BER is carried out in four sequential enzymatic steps catalyzed by the enzymes DNA glycosylase, AP-endonuclease, DNA polymerase and DNA ligase (Dianova et al., 2001; Gredilla et al., 2010). The initial

mediating asbestos-induced AEC apoptosis.

repair (BER) proteins.

**3. Ogg1 and mitochondrial base excision repair** 

steps in the BER pathway are recognition and removal of the aberrant base by a DNA glycosylase. Most DNA glycosylases remove several structurally different damaged bases, and some of them have overlapping substrate specificities, which may indicate that they serve as back-up systems for each other (Dianovet al., 2001). The mammalian DNA glycosylase, Ogg1, recognizes and removes 8-oxoG that is base-paired with cytosine in DNA (Aburatani et al., 1997; Radicella et al., 1997). Ogg1 is a bifunctional DNA glycosylase, with an associated AP-lyase activity, cleaving DNA at abasic sites through a β-elimination mechanism (Bjoras et al., 1997). The human OGG1 gene is located on chromosome 3p26.2. Studies of mice that are deficient in Ogg1 demonstrate that this enzyme is responsible for most of the BER activity that is initiated at 8-oxoG in mammalian cells (Klungland et al., 1999). Interestingly, using fluorometric techniques to identify the site of Ogg1 DNA repair activity following exposure to oxidative stress, the mitochondria, rather than the nucleus, was primary site of Ogg1 DNA repair activity (Mirbahai et al., 2010). In Ogg1 knockout mice, the mitochondrial genome contains almost nine times more 8-oxoguanine than control animals, whereas in the nuclear DNA the level of 8-oxoguanine is increased only twofold (Souza-Pinto et al., 2001). OGG1 gene mutations or polymorphisms increase the risk of various malignancies including lung, kidney, gastric, and colorectal cancer, as well as leukemia (Chevillard et al., 1998; Shinmura et al., 1998; Audebert et al., 2000; Bohr et al., 2002; Elahi et al., 2002; Fortini et al., 2003; Russo et al., 2004; Mambo et al., 2005). Furthermore, reduced Ogg1 activity is a risk factor in lung and head and neck cancer (Paz-Elizur et al., 2008).

Several groups have demonstrated that overexpression of mitochondria-targeted Ogg1 prevents mtDNA damage and intrinsic apoptosis caused by ROS-exposed vascular endothelial and asbestos-exposed cells (Dobson et al., 2002; Ruchko et al., 2005; Rachek et al., 2006; Harrison et al., 2007; Panduri et al., 2009; Ruchko et al., 2010). This suggests a prominent role of mt-Ogg1 in regulating intrinsic apoptosis in diverse settings of oxidative stress. Alternative splicing of the OGG1 transcript results in two isoforms: α-Ogg1 and β-Ogg1 (Gredilla et al., 2010). β-Ogg1 levels in the mitochondria are 20-fold greater than α-Ogg1 levels yet, curiously, β-Ogg1 lacks 8-oxoG DNA glycosylase activity (Hashiguchi et al., 2004). This finding suggests a role for Ogg1 that is independent of DNA repair. Our group recently reported that overexpression of mitochondrial α-Ogg1 mutants lacking 8-oxoG DNA repair activity were as effective as wild type mt-Ogg1 in preventing oxidant-induced caspase-9 activation and intrinsic apoptosis. Mitochondria-targeted Ogg1 did not alter the levels of mitochondrial ROS produced but, interestingly, preserved mitochondrial aconitase suggesting a novel role for Ogg1 as discussed further below (Panduri et al., 2009).

#### **4. Aconitase and mitochondrial DNA**

Aconitase, an enzyme that is vital for carbohydrate and energy metabolism, is responsible for the interconversion of citrate and isocitrate in the tricarboxcylic acid (TCA) cycle (Emptage et al., 1983). The importance of mitochondrial aconitase is suggested by the observation that citrate levels in the human prostate appear important for promoting oncogenic conditions. Normal citrate-producing prostate epithelial cells can develop into citrate-oxidizing malignant cells that result in a net increase of 22 ATP/mol glucose that affords energy for malignant-associated activities (Costello & Franklin, 1994). It has been suggested that mitochondrial aconitase is a key enzyme associated with this bioenergy transformation since loss of its activity reduces cellular survival (Singh et al., 2006).

Role of Ogg1 and Aconitase, p53 91

p53 functions as the "gatekeeper" of the genome by integrating various signals and initiating appropriate biological responses including cell cycle arrest, differentiation, apoptosis, senescence, and anti-angiogenesis (see for reviews Levine 1997; Vogelstein et al., 2000; Vousden et al., 2009). Previous studies have shown that the functions of p53 are mediated by transcriptional activation that regulates expression of downstream target genes (El-Deiry 1998). Expression of some cellular genes, including WAF1, CIP1, p21, IGF-BP3, mdm2, cyclin G, PCNA, and GADD45, are directly regulated by p53-mediated transactivation (Ko & Prives, 1996). p53 is also a redox-sensitive transcription factor whose function is integrally connected to ROS production as well as mediating the down-stream cellular effects following oxidative stress including the induction of apoptotic cell death (reviewed in Sablina et al., 2005; Janicke et al., 2008; Vaseva et al., 2009; Liu et al., 2010). ROS can induce p53 expression whereas p53 stabilization can augment further ROS production, often via effects on the mitochondria (Janicke et al., 2008; Liu et al., 2010). The mitochondria are an important target of transcription-dependent and -independent actions of p53 required to trigger apoptosis. By regulating thousands of genes, either directly or indirectly, p53 is implicated in numerous key cellular roles, including a recently described role for mtDNA maintenance (El-Deiry et al., 1992; Janicke et al., 2008, Bakhanashvili et al., 2008;

The mechanism by which p53 regulates cellular responses following exposure to oxidative stress generally depends on the levels of ROS. A biphasic response is seen in which low basal p53 expression promotes ROS homeostasis and cell survival by augmenting antioxidant defenses as one of its tumor-suppressing mechanisms while higher levels of ROS induce persistent p53 expression that blocks the cell cycle enabling time for DNA repair and, if repair is insufficient, triggers apoptosis (Bensaad et al., 2005; Janicke et al., 2008; Vousden et al., 2009). Notably, p53 also enhances Ogg1 activity for 8-oxoG removal suggesting a link between Ogg1, p53 and mtDNA (Achanta & Huang , 2004). A recently described role for p53 in mtDNA maintenance following exposure to mitochondrial ROS is evidenced by its involvement in maintaining mtDNA copy number and mtDNA synthesis (Bakhanashvili et al., 2008; Lebedeva et al., 2009). Cells that are p53-depleted exhibit significant disruption of cellular ROS homeostasis that are characterized by reduced mitochondrial biogenesis and increased H2O2 production (Lebedeva et al., 2009). In contrast, thymic lymphomas derived from the p53-/- mouse (a common model of carcinogenesis) have highly significant upregulation of mitochondrial biogenesis, mitochondrial protein translation, mtDNA copy number, ROS levels, anti-oxidant defenses, proton transport, ATP synthesis, hypoxia response, and glycolysis, indicating important mitochondrial bioenergetic profile changes of cells occurs during the process of malignant transformation (Samper et al., 2009). Hypoxia stimulates mitochondrial ROS production, which activates p53 stabilization and localization to the mitochondria where p53 has many effects including inhibiting MnSOD thereby promoting O2•− formation and greater oxidative damage (Ralph et al., 2010) as well as regulating mtDNA repair and replication as noted above. Taken together, the emerging evidence strongly implicate that p53 is a key regulator of mitochondrial function, including ROS production and associated mtDNA repair following oxidative damage, as well as

mtDNA replication and mitochondrial biogenesis (Ralph et al., 2010).

It is known well that most human tumors contain mutations in one or more p53 gene family members (see for reviews Janicke et al., 2008; Vousden & Prives, 2009). In this section we

**5. p53 and mitochondrial DNA repair** 

Lebedeva et al., 2009).

Mitochondrial aconitase is an iron-sulfur protein that is vulnerable to oxidative inactivation and is implicated as a mitochondrial redox-sensor (Gardner et al., 1994; Bulteau et al., 2003). Aconitase inactivation can further promote oxidant generation by releasing redox-active Fe from the (4Fe–4S)2+ center following exposure to oxidants such as O2•− (Gardner et al., 2000) or deficiency of mitochondrial manganese superoxide dismutase (MnSOD) (Williams et al., 1998). Oxidative-inactivation of aconitase is associated with decreased Drosophila lifespan (Yan et al., 1997). Reduced aconitase activity has also been described in a number of neurodegenerative diseases, including progressive supranuclear palsy (Park 2001), Friedreich's ataxia (Bradley 2000), and Huntington's disease (Tabrizi 1999).

Collectively, the above findings suggested a key role for mitochondrial aconitase beyond the TCA cycle. In this regard, a provocative finding in yeast showed that mitochondrial aconitase preserves mtDNA independent of aconitase's catalytic activity (Chen et al., 2005). This was the first suggestion of a dual role for aconitase as a mitochondrial TCA enzyme as well as in mtDNA maintenance, mitochondrial aconitase co-precipitates with frataxin, which is an iron chaperone protein that prevents aconitase oxidative inactivation and/or augments aconitase reactivation (Bulteau et al., 2004). This study suggested that prevention of oxidative inactivation of mitochondrial aconitase may be important for the pathogenesis of a degenerative disease (e.g. Friedrich's ataxia). Further evidence for this possibility was our recent finding that mt-Ogg1 overexpression completely blocks oxidant induced decreases in AEC mitochondrial aconitase activity and protein expression (Panduri et al., 2009). Moreover, using immunoprecipitation to explore the possible interactive effects between mitochondrial Ogg1 and aconitase, mitochondrial aconitase coprecipitated with both wild-type and mutant mt-Ogg1. Notably, overexpression of mitochondrial aconitase eliminated oxidant induced AEC apoptosis whereas Ogg1 underexpression using shRNA techniques reduced basal mitochondrial aconitase levels and augmented oxidant-induced AEC apoptosis (Panduri et al., 2009). These latter findings are in accord with several recent studies showing that Ogg1 deficiency increases oxidant-induced apoptosis (Youn et al., 2007; Bacsi et al., 2007; Xie et al., 2008). Collectively, these results suggest a novel interaction between an mtDNA repair enzyme (mt-Ogg1) and aconitase in preventing intrinsic AEC apoptosis following exposure to oxidative stress (e.g. asbestos or H2O2).

The underlying mechanisms that account for the interactive protective effect of mt-Ogg1 and aconitase require further study but there are at least two possibilities, which are not mutually exclusive. First, mt-Ogg1 may block key oxidative modification sites on mitochondrial aconitase responsible for triggering degradation by mitochondrial Lon protease (Bota & Davies, 2002; Bota et al., 2005). Lon protease selectively degrades oxidatively modified aconitase at a much higher rate than unexposed aconitase; a finding that may be important in defending the mitochondria against the accumulation of oxidized proteins as well as ensuring that such cells will undergo intrinsic apoptosis (Wallace, 1999; Bota et al., 2005; Bota & Davies, 2002; Panduri et al. 2009). Support for this possibility is our finding that MG132, a protease inhibitor that blocks mitochondrial Lon protease (Granot et al., 2007), attenuates asbestos-induced reductions in mitochondrial aconitase activity (Panduri et al., 2009). Second, overexpression of mt-Ogg1 or aconitase may preserve mtDNA levels necessary to prevent activation of intrinsic apoptosis. Future studies are required to clarify these possibilities as well as to determine precisely how mt-hOgg1 interacts with aconitase and whether other mtDNA repair proteins act similarly.

Mitochondrial aconitase is an iron-sulfur protein that is vulnerable to oxidative inactivation and is implicated as a mitochondrial redox-sensor (Gardner et al., 1994; Bulteau et al., 2003). Aconitase inactivation can further promote oxidant generation by releasing redox-active Fe from the (4Fe–4S)2+ center following exposure to oxidants such as O2•− (Gardner et al., 2000) or deficiency of mitochondrial manganese superoxide dismutase (MnSOD) (Williams et al., 1998). Oxidative-inactivation of aconitase is associated with decreased Drosophila lifespan (Yan et al., 1997). Reduced aconitase activity has also been described in a number of neurodegenerative diseases, including progressive supranuclear palsy (Park 2001),

Collectively, the above findings suggested a key role for mitochondrial aconitase beyond the TCA cycle. In this regard, a provocative finding in yeast showed that mitochondrial aconitase preserves mtDNA independent of aconitase's catalytic activity (Chen et al., 2005). This was the first suggestion of a dual role for aconitase as a mitochondrial TCA enzyme as well as in mtDNA maintenance, mitochondrial aconitase co-precipitates with frataxin, which is an iron chaperone protein that prevents aconitase oxidative inactivation and/or augments aconitase reactivation (Bulteau et al., 2004). This study suggested that prevention of oxidative inactivation of mitochondrial aconitase may be important for the pathogenesis of a degenerative disease (e.g. Friedrich's ataxia). Further evidence for this possibility was our recent finding that mt-Ogg1 overexpression completely blocks oxidant induced decreases in AEC mitochondrial aconitase activity and protein expression (Panduri et al., 2009). Moreover, using immunoprecipitation to explore the possible interactive effects between mitochondrial Ogg1 and aconitase, mitochondrial aconitase coprecipitated with both wild-type and mutant mt-Ogg1. Notably, overexpression of mitochondrial aconitase eliminated oxidant induced AEC apoptosis whereas Ogg1 underexpression using shRNA techniques reduced basal mitochondrial aconitase levels and augmented oxidant-induced AEC apoptosis (Panduri et al., 2009). These latter findings are in accord with several recent studies showing that Ogg1 deficiency increases oxidant-induced apoptosis (Youn et al., 2007; Bacsi et al., 2007; Xie et al., 2008). Collectively, these results suggest a novel interaction between an mtDNA repair enzyme (mt-Ogg1) and aconitase in preventing intrinsic AEC apoptosis following exposure to

The underlying mechanisms that account for the interactive protective effect of mt-Ogg1 and aconitase require further study but there are at least two possibilities, which are not mutually exclusive. First, mt-Ogg1 may block key oxidative modification sites on mitochondrial aconitase responsible for triggering degradation by mitochondrial Lon protease (Bota & Davies, 2002; Bota et al., 2005). Lon protease selectively degrades oxidatively modified aconitase at a much higher rate than unexposed aconitase; a finding that may be important in defending the mitochondria against the accumulation of oxidized proteins as well as ensuring that such cells will undergo intrinsic apoptosis (Wallace, 1999; Bota et al., 2005; Bota & Davies, 2002; Panduri et al. 2009). Support for this possibility is our finding that MG132, a protease inhibitor that blocks mitochondrial Lon protease (Granot et al., 2007), attenuates asbestos-induced reductions in mitochondrial aconitase activity (Panduri et al., 2009). Second, overexpression of mt-Ogg1 or aconitase may preserve mtDNA levels necessary to prevent activation of intrinsic apoptosis. Future studies are required to clarify these possibilities as well as to determine precisely how mt-hOgg1 interacts with aconitase and whether other mtDNA repair

Friedreich's ataxia (Bradley 2000), and Huntington's disease (Tabrizi 1999).

oxidative stress (e.g. asbestos or H2O2).

proteins act similarly.

#### **5. p53 and mitochondrial DNA repair**

p53 functions as the "gatekeeper" of the genome by integrating various signals and initiating appropriate biological responses including cell cycle arrest, differentiation, apoptosis, senescence, and anti-angiogenesis (see for reviews Levine 1997; Vogelstein et al., 2000; Vousden et al., 2009). Previous studies have shown that the functions of p53 are mediated by transcriptional activation that regulates expression of downstream target genes (El-Deiry 1998). Expression of some cellular genes, including WAF1, CIP1, p21, IGF-BP3, mdm2, cyclin G, PCNA, and GADD45, are directly regulated by p53-mediated transactivation (Ko & Prives, 1996). p53 is also a redox-sensitive transcription factor whose function is integrally connected to ROS production as well as mediating the down-stream cellular effects following oxidative stress including the induction of apoptotic cell death (reviewed in Sablina et al., 2005; Janicke et al., 2008; Vaseva et al., 2009; Liu et al., 2010). ROS can induce p53 expression whereas p53 stabilization can augment further ROS production, often via effects on the mitochondria (Janicke et al., 2008; Liu et al., 2010). The mitochondria are an important target of transcription-dependent and -independent actions of p53 required to trigger apoptosis. By regulating thousands of genes, either directly or indirectly, p53 is implicated in numerous key cellular roles, including a recently described role for mtDNA maintenance (El-Deiry et al., 1992; Janicke et al., 2008, Bakhanashvili et al., 2008; Lebedeva et al., 2009).

The mechanism by which p53 regulates cellular responses following exposure to oxidative stress generally depends on the levels of ROS. A biphasic response is seen in which low basal p53 expression promotes ROS homeostasis and cell survival by augmenting antioxidant defenses as one of its tumor-suppressing mechanisms while higher levels of ROS induce persistent p53 expression that blocks the cell cycle enabling time for DNA repair and, if repair is insufficient, triggers apoptosis (Bensaad et al., 2005; Janicke et al., 2008; Vousden et al., 2009). Notably, p53 also enhances Ogg1 activity for 8-oxoG removal suggesting a link between Ogg1, p53 and mtDNA (Achanta & Huang , 2004). A recently described role for p53 in mtDNA maintenance following exposure to mitochondrial ROS is evidenced by its involvement in maintaining mtDNA copy number and mtDNA synthesis (Bakhanashvili et al., 2008; Lebedeva et al., 2009). Cells that are p53-depleted exhibit significant disruption of cellular ROS homeostasis that are characterized by reduced mitochondrial biogenesis and increased H2O2 production (Lebedeva et al., 2009). In contrast, thymic lymphomas derived from the p53-/- mouse (a common model of carcinogenesis) have highly significant upregulation of mitochondrial biogenesis, mitochondrial protein translation, mtDNA copy number, ROS levels, anti-oxidant defenses, proton transport, ATP synthesis, hypoxia response, and glycolysis, indicating important mitochondrial bioenergetic profile changes of cells occurs during the process of malignant transformation (Samper et al., 2009). Hypoxia stimulates mitochondrial ROS production, which activates p53 stabilization and localization to the mitochondria where p53 has many effects including inhibiting MnSOD thereby promoting O2•− formation and greater oxidative damage (Ralph et al., 2010) as well as regulating mtDNA repair and replication as noted above. Taken together, the emerging evidence strongly implicate that p53 is a key regulator of mitochondrial function, including ROS production and associated mtDNA repair following oxidative damage, as well as mtDNA replication and mitochondrial biogenesis (Ralph et al., 2010).

It is known well that most human tumors contain mutations in one or more p53 gene family members (see for reviews Janicke et al., 2008; Vousden & Prives, 2009). In this section we

Role of Ogg1 and Aconitase, p53 93

p53 modulates cellular metabolism by enhancing aerobic respiration and blocking glycolysis in most cell types; findings that are likely important in cellular malignant transformation (Bensaad et al., 2006; Bensaad et al., 2007). Interestingly, there is some evidence that p53 impacts mitochondrial aconitase levels since thymoquinone, a p53-dependent antineoplastic drug, reduces aconitase enzyme activity in isolated rat liver mitochondria (Roepke et al., 2007). Also, mitochondrial aconitase gene expression in prostate carcinoma cells is inhibited by both endogenous p53 induction by camptothecin treatment and exogenous p53 induction by transient overexpression of p53 (Tsui et al., 2011). Further, these investigators showed that mitochondrial aconitase is a p53-downregulated gene. Camptothecin did not affect mitochondrial aconitase reporter activity in p53-null PC-3 cells suggesting that the decrease in mitochondrial aconitase gene expression by camptothecin occurs via p53 activation. The relevance of these findings to other cell types as well as the *in vivo* significance requires

In this review we have summarized emerging evidence demonstrating an important interactive effect between mitochondrial Ogg1, mitochondrial aconitase, and p53 in mtDNA repair and oxidant-induced intrinsic apoptosis. Although we focused on the role of oxidative stress caused by exposure to asbestos fibers, it is likely that many of the described interactive effects between mt-Ogg1, aconitase, p53 and intrinsic apoptosis will have broader implications but this awaits future investigations. Additional studies are necessary to further characterize the role of mitochondrial Ogg1 and aconitase in preventing mtDNA damaging (including following asbestos exposure), p53 activation and intrinsic apoptosis. It will also be of considerable interest to better understand the molecular mechanisms by which mitochondrial Ogg1 binds aconitase. Finally, and perhaps most importantly, we reason that the asbestos paradigm will continue to provide insights into the molecular mechanisms underlying the interactive effects between mt-Ogg1, aconitase, p53 and intrinsic apoptosis that should shed light into the pathogenesis of other more common diseases, such as lung cancer and idiopathic pulmonary fibrosis, for which more effective management regimens are urgently required. Strategies aimed at augmenting mtDNA integrity by increasing mt-Ogg1 and/or aconitase levels to mitigate the deleterious effects of oxidative stress may prove useful for developing novel therapeutic treatments for tumors

and degenerative diseases as well as modulating the effects of aging.

mitochondrial human 8-oxoguanine-DNA glycosylase 1 (mt-hOgg1)

further study.

**6. Conclusion** 

**7. Abbreviations** 

Electron transport chain (ETC)

alveolar epithelial cell (AEC) reactive oxygen species (ROS)

alveolar type II (AT2) cells hydrogen peroxide (H2O2), superoxide anion (O2- ) hydroxyl radical (HO•)

outer mitochondrial membrane (OMM)

8-hydroxydeoxyguanosine (8OHdG)

focus on the role of p53 in the lungs exposed to asbestos fibers. Altered p53 expression has been implicated in the pathophysiology of pulmonary fibrosis, including that due to asbestos, as well as asbestos-associated malignancies, especially bronchogenic lung cancer (Nelson et al., 2001; Mishra et al., 1997; Burmeister et al., 2004; Plataki et al., 2005). Asbestos activates p53 and p21 expression in lung epithelial and mesothelial cells that result in cell cycle arrest (Levresse et al., 1997; Matsuoka et al., 2003; Kopnin et al., 2004). Furthermore, increased p53 levels are detected in lung cancers of patients with asbestosis (Nuorva et al., 1994) and p53 point mutations are present in the lung epithelium of smokers and asbestosexposed individuals (Husgafvel-Pursiainen et al., 1997). Crocidolite asbestos promotes p53 gene mutations predominantly in axons 9 through 11 in BALB/c-3 T3 cells (Lin et al., 2000). Finally, studies in lung epithelial and mesothelial cells using gene expression microarray techniques have established that induction of p53 gene expression following asbestos fiber exposure is an important event (Nymark et al., 2007; Hevel et al., 2008). Thus, p53 has a crucial role regulating lung cellular DNA damage response following exposure to oxidative stress as occurs with asbestos and tobacco smoke.

The mechanisms by which p53 regulate apoptosis are complex and incompletely understood. One established pathway involves intrinsic apoptosis via p53 crosstalk with the mitochondria by increasing transcription of pro-apoptotic stimuli (e.g. Bax and BH3-only proteins) while inhibiting gene expression of anti-apoptotic Bcl-2 family members (Miyashita et al., 1995; Oda et al., 2000; Nakano et al., 2001; Janicke et al., 2008; Vousden & Prives, 2009). There is considerable evidence that p53 phosphorylation at the Ser15 position following exposure to DNA damaging agents, including asbestos, is in part responsible for p53 stabilization and its subsequent mitochondrial translocation. Several different proteins have been implicated in the phosphorylation of p53 at Ser15, including members of the phosphatidylinostitol 3-kinase-related kinase (PI3K) family such as DNA-activated protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) kinase, as well as members of the mitogen-activated protein kinase (MAPK). In one study, suppression of DNA-PK coupled with a mutated form of ATM inhibited asbestos-induced Ser15 phosphorylation and accumulation of p53 (Matsuoka et al., 2003). Considerable evidence has established that p53 is a crucial regulator of mitochondrial function, including ROS generation and mtDNA repair following oxidative damage as well as mitochondrial biogenesis and mtDNA replication (see for review Liu et al., 2010). For example, p53 mediates asbestos-induced mitochondria-regulated apoptosis in lung epithelial cells and this is blocked in cells incapable of producing mitochondrial ROS (Panduri et al., 2006). Notably, loss of p53 results in mtDNA depletion, altered mitochondrial function and increased H2O2 production (Lebedeva et al., 2009).

The above data are providing insights into the molecular mechanisms by which p53 regulates the cellular response to DNA damage caused by exposure to oxidative stress that is likely important in the pathogenesis of inflammation-associated cancer (see for review: Kamp et al., 2011). An important link between p53 and Ogg1 is suggested by the finding that Ogg1 is under transcriptional regulation by p53 in colon and renal epithelial cells (Youn et al., 2007). In this study, the expression and activity of Ogg1 were decreased in HCT116p53−/− cells. Further, gel-shift assays showed that p53 binds to the putative ciselements within the OGG1 promoter while supplementing p53 in HCT116p53−/− cells enhanced OGG1 transcription. In renal epithelial cells, tuberin also regulates OGG1 expression since transcriptional activity of the OGG1 promoter is decreased in tuberin-null cells; an effect that in part is mediated by the transcription factor NF-YA (Habib et al., 2008).

focus on the role of p53 in the lungs exposed to asbestos fibers. Altered p53 expression has been implicated in the pathophysiology of pulmonary fibrosis, including that due to asbestos, as well as asbestos-associated malignancies, especially bronchogenic lung cancer (Nelson et al., 2001; Mishra et al., 1997; Burmeister et al., 2004; Plataki et al., 2005). Asbestos activates p53 and p21 expression in lung epithelial and mesothelial cells that result in cell cycle arrest (Levresse et al., 1997; Matsuoka et al., 2003; Kopnin et al., 2004). Furthermore, increased p53 levels are detected in lung cancers of patients with asbestosis (Nuorva et al., 1994) and p53 point mutations are present in the lung epithelium of smokers and asbestosexposed individuals (Husgafvel-Pursiainen et al., 1997). Crocidolite asbestos promotes p53 gene mutations predominantly in axons 9 through 11 in BALB/c-3 T3 cells (Lin et al., 2000). Finally, studies in lung epithelial and mesothelial cells using gene expression microarray techniques have established that induction of p53 gene expression following asbestos fiber exposure is an important event (Nymark et al., 2007; Hevel et al., 2008). Thus, p53 has a crucial role regulating lung cellular DNA damage response following exposure to oxidative

The mechanisms by which p53 regulate apoptosis are complex and incompletely understood. One established pathway involves intrinsic apoptosis via p53 crosstalk with the mitochondria by increasing transcription of pro-apoptotic stimuli (e.g. Bax and BH3-only proteins) while inhibiting gene expression of anti-apoptotic Bcl-2 family members (Miyashita et al., 1995; Oda et al., 2000; Nakano et al., 2001; Janicke et al., 2008; Vousden & Prives, 2009). There is considerable evidence that p53 phosphorylation at the Ser15 position following exposure to DNA damaging agents, including asbestos, is in part responsible for p53 stabilization and its subsequent mitochondrial translocation. Several different proteins have been implicated in the phosphorylation of p53 at Ser15, including members of the phosphatidylinostitol 3-kinase-related kinase (PI3K) family such as DNA-activated protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) kinase, as well as members of the mitogen-activated protein kinase (MAPK). In one study, suppression of DNA-PK coupled with a mutated form of ATM inhibited asbestos-induced Ser15 phosphorylation and accumulation of p53 (Matsuoka et al., 2003). Considerable evidence has established that p53 is a crucial regulator of mitochondrial function, including ROS generation and mtDNA repair following oxidative damage as well as mitochondrial biogenesis and mtDNA replication (see for review Liu et al., 2010). For example, p53 mediates asbestos-induced mitochondria-regulated apoptosis in lung epithelial cells and this is blocked in cells incapable of producing mitochondrial ROS (Panduri et al., 2006). Notably, loss of p53 results in mtDNA depletion, altered mitochondrial function and increased H2O2 production

The above data are providing insights into the molecular mechanisms by which p53 regulates the cellular response to DNA damage caused by exposure to oxidative stress that is likely important in the pathogenesis of inflammation-associated cancer (see for review: Kamp et al., 2011). An important link between p53 and Ogg1 is suggested by the finding that Ogg1 is under transcriptional regulation by p53 in colon and renal epithelial cells (Youn et al., 2007). In this study, the expression and activity of Ogg1 were decreased in HCT116p53−/− cells. Further, gel-shift assays showed that p53 binds to the putative ciselements within the OGG1 promoter while supplementing p53 in HCT116p53−/− cells enhanced OGG1 transcription. In renal epithelial cells, tuberin also regulates OGG1 expression since transcriptional activity of the OGG1 promoter is decreased in tuberin-null cells; an effect that in part is mediated by the transcription factor NF-YA (Habib et al., 2008).

stress as occurs with asbestos and tobacco smoke.

(Lebedeva et al., 2009).

p53 modulates cellular metabolism by enhancing aerobic respiration and blocking glycolysis in most cell types; findings that are likely important in cellular malignant transformation (Bensaad et al., 2006; Bensaad et al., 2007). Interestingly, there is some evidence that p53 impacts mitochondrial aconitase levels since thymoquinone, a p53-dependent antineoplastic drug, reduces aconitase enzyme activity in isolated rat liver mitochondria (Roepke et al., 2007). Also, mitochondrial aconitase gene expression in prostate carcinoma cells is inhibited by both endogenous p53 induction by camptothecin treatment and exogenous p53 induction by transient overexpression of p53 (Tsui et al., 2011). Further, these investigators showed that mitochondrial aconitase is a p53-downregulated gene. Camptothecin did not affect mitochondrial aconitase reporter activity in p53-null PC-3 cells suggesting that the decrease in mitochondrial aconitase gene expression by camptothecin occurs via p53 activation. The relevance of these findings to other cell types as well as the *in vivo* significance requires further study.

### **6. Conclusion**

In this review we have summarized emerging evidence demonstrating an important interactive effect between mitochondrial Ogg1, mitochondrial aconitase, and p53 in mtDNA repair and oxidant-induced intrinsic apoptosis. Although we focused on the role of oxidative stress caused by exposure to asbestos fibers, it is likely that many of the described interactive effects between mt-Ogg1, aconitase, p53 and intrinsic apoptosis will have broader implications but this awaits future investigations. Additional studies are necessary to further characterize the role of mitochondrial Ogg1 and aconitase in preventing mtDNA damaging (including following asbestos exposure), p53 activation and intrinsic apoptosis. It will also be of considerable interest to better understand the molecular mechanisms by which mitochondrial Ogg1 binds aconitase. Finally, and perhaps most importantly, we reason that the asbestos paradigm will continue to provide insights into the molecular mechanisms underlying the interactive effects between mt-Ogg1, aconitase, p53 and intrinsic apoptosis that should shed light into the pathogenesis of other more common diseases, such as lung cancer and idiopathic pulmonary fibrosis, for which more effective management regimens are urgently required. Strategies aimed at augmenting mtDNA integrity by increasing mt-Ogg1 and/or aconitase levels to mitigate the deleterious effects of oxidative stress may prove useful for developing novel therapeutic treatments for tumors and degenerative diseases as well as modulating the effects of aging.

#### **7. Abbreviations**

Electron transport chain (ETC) outer mitochondrial membrane (OMM) alveolar epithelial cell (AEC) reactive oxygen species (ROS) mitochondrial human 8-oxoguanine-DNA glycosylase 1 (mt-hOgg1) alveolar type II (AT2) cells hydrogen peroxide (H2O2), superoxide anion (O2- ) hydroxyl radical (HO•) 8-hydroxydeoxyguanosine (8OHdG)

Role of Ogg1 and Aconitase, p53 95

Bjelland, S.; Seeberg, E. (2003). "Mutagenicity, toxicity and repair of DNA base damage

Bjoras, M.; Luna, L.; Johnsen, B.; Hoff, E.; Haug, T.; Rognes, T.; Seeberg, E. (1997). "Opposite

Bota, D. A.; and Davies, K. J. A. (2002). "Lon protease preferentially degrades oxidized

Bota, D.A.; Ngo, J.K.; Davies, K.J. (2005). "Downregulation of the human Lon protease

Bota, D. A.; Van Remmen, H.; Davies, K. J. (2002). "Modulation of Lon protease activity and

Boveris, A.; Chance, B. (1977). "The mitochondrial generation of hydrogen peroxide." General properties and effect of hyperbaric oxygen. Biochem. J. 134: 707–716. Bradley, J.L.; Blake, J.C.; Chamberlain, S.; Thomas, P.K.; Cooper, J.M.; Schapira,A.H.(2000).

Bulteau, A. L.; Ikeda-Saito, M.; Szweda, L. I. (2003). "Redox-dependent modulation of aconitase activity in intact mitochondria." Biochemistry 42 (50): 14846–14855. Bulteau, A. L.; O'Neill, H. A.; Kennedy, M. C.; Ikeda-Saito, M.; Isaya, G.; Szweda, L. I. (2004).

Burmeister, B.; Schwerdtle, T.; Poser, I.; Hoffmann, E.; Hartwig, A.; Muller, W. U.;

Chance, B.; Sies, H.; Boveris, A. (1979). "Hydroperoxide metabolism in mammalian organs."

Chen, X. J.; Wang, X.; Kaufman, B. A.; Butow, R. A. (2005). "Aconitase couples metabolic regulation to mitochondrial DNA maintenance." Science 307 (5710):714–717. Chevillard, S.; Radicella, J. P.; Levalois, C.; Lebeau, J.; Poupon, M. F.; Oudard, S.; Dutrillaux,

Clark, J. M.; Beardsley, G. P. (1987). "Functional effects of cis-thymine glycol lesions on DNA

Corral-Debrinski, M.; Horton, T.; Lott, M. T.; Shoffner, J. M.; Beal, M. F.; Wallace, D. C.

Costello, L. C.; Franklin, R. B. (1994). "The bioenergetic theory of prostate malignancy."

base-dependent reactions of a human base excision repair enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites." EMBO J. 16: 6314–6322. Bohr, V. A.; Stevnsner, T.; de Souza-Pinto, N. C. (2002). "Mitochondrial DNA repair of

mitochondrial aconitase by an ATP-stimulated mechanism." Nat. Cell Biol. 4 (9):

impairs mitochondrial structure and function and causes cell death." Free Radic

aconitase turnover during aging and oxidative stress." FEBS Lett. 532 (1–2): 103–

"Clinical, biochemical and molecular genetic correlations in Friedreich's ataxia."

"Frataxin acts as an iron chaperone protein to modulate mitochondrial aconitase

Rettenmeier, A. W.; Seemayer, N. H.; Dopp, E. (2004). "Effects of asbestos on initiation of DNA damage, induction of DNA-strand breaks, P53-expression and apoptosis in primary, SV40-transformed and malignanthumanmesothelial cells."

B.; Boiteux, S. (1998). "Mutations in OGG1, a gene involved in the repair of oxidative DNA damage, are found in human lung and kidney tumors." Oncogene

(1992). "Mitochondrial DNA deletions in human brain: regional variability and

induced by oxidation." Mutat. Res. 531: 37–80.

674–680

106.

Biol Med 38(5):665-77.

Hum Mol Genet 9(2):275-82.

Mutat. Res. 558 (1–2): 81–92.

Physiol Rev 59: 527–605.

16:3083–3086.

Prostate 25:162–166.

activity." Science 305 (5681): 242–245.

synthesis in vitro." Biochemistry 26: 5398–5403.

increase with advanced age." Nat Genet 2: 324–329.

oxidative damage in mammalian cells." Gene 286: 127–134.

mitochondrial DNA (mtDNA) tricarboxcylic acid (TCA) mitochondrial membrane potential (△ψm ) titanium dioxide (TiO2) thymine glycol (TG) base excision repair (BER) 8-hydroxyguanine (8-oxoG) manganese superoxide dismutase (MnSOD)

#### **8. References**


Aburatani, H.; Hippo, Y.; Ishida, T.; Takashima, R., Matsuba, C.; Kodama, T. et al. (1997).

Achanta, G.; Huang, P.;(2004). "Role of p53 in sensing oxidative DNA damage in response to reactive oxygen species-generating agents." Cancer Res 64(17):6233-9. Albring, M.; Griffith, J.; Attardi, G. (1977). "Association of a protein structure of probable

Aljandali, A.; Pollack, H.; Yeldandi, A.; Li, Y.; Weitzman, S. A.; Kamp, D. W. (2001).

Ames, B. N. (1989). "Endogenous oxidative DNA damage aging and cancer." Free Radic.

Anderson, S.; Bankier, A. T.; Barrell, B. G.; de Bruijn, M. H.; Coulson, A. R.; Drouin, J.;

Audebert, M.; Chevillard, S.; Levalois, C.; Gyapay, G.; Vieillefond,A.; Klijanienko J et al.

Babcock, D. F.; Herrington, J.; Goodwin, P. C.; Park, Y. B. and Hille, B. (1997).

Bacsi, A.; Chodaczek, G.; Hazra, T. K.; Konkel, D.; Boldogh, I. (2007). "Increased ROS

Bakhanashvili,M.; Grinberg, S.; Bonda, E.; Simon, A.J.; Moshitch-Moshkovitz, S.; Rahav,

Barnes, D. E.; Lindahl, T. (2004). "Repair and genetic consequences of endogenous DNA

Bensaad, K.; Tsuruta, A.; Selak, M. A.; Vidal, M. N. C.; Nakano, K.; Bartrons, R.; Gottlieb, E.;

Bensaad, K.; Vousden, K. H. (2005). "Savior and slayer: the two faces of p53." Nat. Med. 11:

Bensaad, K.; Vousden, K. H. (2007). "p53:New roles in metabolism." Trends Cell Biol 17:

base damage in mammalian cells." Annu. Rev. Genet. 38: 445–476.

replication." Proc Natl Acad Sci U S A 74: 1348–1352.

radicals." J Lab Clin Med 137 (5): 330–339.

mitochondrial genome." Nature 290: 457–465.

membrane derivation with HeLa cell mitochondrial DNA near its origin of

"Asbestos causes apoptosis in alveolar epithelial cells: role of iron-induced free

Eperon, I. C.; Nierlich, D. P.; Roe, B. A.; Sanger, F.; Schreier, P. H.; Smith, A. J.; Staden, R.; Young, I. G. (1981). "Sequence and organization of the human

"Mitochondrial participation in the intracellular Ca2. Network." J Cell Biol 136:

generation in subsets of OGG1 knockout fibroblast cells." Mech. Ageing Dev.

G.(2008). "p53 in mitochondria enhances the accuracy of DNA synthesis." Cell

Vousden, K. H. (2006). "TIGAR, a p53-inducible regulator of glycolysis and

mitochondrial DNA (mtDNA) tricarboxcylic acid (TCA)

titanium dioxide (TiO2) thymine glycol (TG) base excision repair (BER) 8-hydroxyguanine (8-oxoG)

**8. References** 

mitochondrial membrane potential (△ψm )

manganese superoxide dismutase (MnSOD)

Cancer Res 57: 2151–2156.

Res. Commun. 7: 121–128.

833–844.

1278–1279.

286–291.

128(11–12): 637–649.

Death Differ 15(12):1865-74.

apoptosis." Cell 126: 107–120.

(2000). Cancer Res 60: 4740–4744.


Role of Ogg1 and Aconitase, p53 97

Gardner, P. R.; Nguyen, D. D.; White, C. W. (1994). "Aconitase is a sensitive and critical

Gredilla, R.; Bohr, V.A.; Stevnsner, T.(2010)" Mitochondrial DNA repair and association

Habib, S. L.; Riley, D. J.; Mahimainathan, L.; Bhandari, B.; Choudhury, G. G.; Abboud, H. E.

Hailer, M. K.; Slade, P. G.; Martin, B. D.; Rosenquist, T. A.; Sugden, K. D. (2005).

Hardy, J. A.; Aust, A. E. (1995). "The effect of iron binding on the ability of crocidolite asbestos to catalyze DNA single-strand breaks." Carcinogenesis 16 (2): 319–325. Harrison, J. F.; Rinne, M. L.; Kelley, M. R.; Druzhyna, N. M.; Wilson, G. L.; Ledoux, S.P.

Hashiguchi, K.; Stuart, J. A.; de Souza-Pinto, N. C.; Bohr, V. A. (2004). "The C-terminal α-O

Hatahet, Z.; Purmal, A. A.; Wallace, S. S. (1994). "Oxidative DNA lesions as blocks to in vitro transcription by phage T7 RNA polymerase." Ann. NY Acad. Sci. 726: 346–348. Hevel, J.M.; Olson-Buelow, L.C.; Ganesan, B.; Stevens, J.R.; Hardman, J.P.; Aust, A.E.(2008).

Htun, H.; Johnston, B. H. (1992). "Mapping adducts of DNA structural probes using transcription and primer extension approaches." Methods Enzymol. 212: 272–294. Husgafvel-Pursiainen, K.; Kannio, A.; Oksa, P.; Suitiala, T.; Koskinen, H.; Partanen, R.;

Ide, H.; Kow, Y. W.; Wallace, S. S. (1985). "Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro." Nucleic Acids Res. 13: 8035–8052. Ishikawa,K.; Takenaga,K.; Akimoto,M.; Koshikawa,N.; Yamaguchi,A.; Imanishi,H.;

mutations can regulate tumor cell metastasis." Science. 320(5876):661-4.

Book,N.;Eimerl,S.;Bahat,A.;Lu,B.;Braun,S.;Maurizi,M.R.;Suzuki,

proteasome inhibitors." Mol Endocrinol 21(9):2164-77.

with aging--an update." Exp Gerontol. 45(7-8):478-88.

Natl Acad Sci U S A 91(25): 12248-12252.

NEIL1 and NEIL2." DNA Repair 4: 41–50.

chemotherapeutic agents." Glia. 55: 1416–1425.

Nucleic Acids Res. 32: 5596–5608.

functions." BMC Genomics 9:376.

30 (2): 224–230.

Granot,Z.;Kobiler,O.;Melamed-

Physiol. 294 (1): F281–290.

target of oxygen poisoning in cultured mammalian cells and in rat lungs." Proc

C.K.;Oppenheim,A.B.;Orly,J.(2007). "Turnover of mitochondrial steroidogenic acute regulatory (StAR) protein by Lon protease: the unexpected effect of

(2008). "Tuberin regulates the DNA repair enzyme OGG1." Am. J. Physiol. Renal

"Recognition of the oxidized lesions spiroiminodihydantoin and guanidinohydantoin in DNA by the mammalian base excision repair glycosylases

(2007). "Altering DNA base excision repair: use of nuclear and mitochondrialtargeted N-methylpurine DNA glycosylase to sensitize astroglia to

helix of human Ogg1 is essential for 8-oxoguanine DNA glycosylase activity: the mitochondrial β-Ogg1 lacks this domain and does not have glycosylase activity."

"Novel functional view of the crocidolite asbestos-treated A549 human lung epithelial transcriptome reveals an intricate network of pathways with opposing

Hemminki, K.; Smith, S.; Rosenstock-Leibu, R.; Brandt-Rauf, P. W. (1997). "Mutations, tissue accumulations, and serum levels of p53 in patients with occupational cancers from asbestos and silica exposure." Environ. Mol. Mutagen.

Nakada,K.; Honma,Y.; Hayashi,J.(2008). "ROS-generating mitochondrial DNA

Clayton, D. A. (1982). "Replication of animal mitochondrial DNA." Cell 28: 693–705.


Clayton, D. A. (1984). "Transcription of the mammalian mitochondrial genome." Annu Rev

Demple, B.; Harrison, L. (1994). "Repair of oxidative damage to DNA: enzymology and

De Souza-Pinto, N. C.; Eide, L.; Hogue, B. A.; Thybo, T.; Stevnsner, T.; Seeberg, E.;

Dianov, G.L.;Souza-Pinto,N.; Nyaga,S.G.; Thybo,T.; Stevnsner,T.;Bohr,V.A.(2001)."Base

Dianova I.I.; Bohr, V. A.; and Dianov, G. L. (2001). "Interaction of human AP endonuclease 1

Dizdaroglu, M. (1992). "Oxidative damage to DNA in mammalian chromatin." Mutat. Res.

Dobson, A. W.; Grishko, V. S.; LeDoux, P.; Kelley, M. R.; Wilson, G. L.; Gillespie, M. N.

Dobson, A. W.; Xu, Y.; Kelley, M. R.; LeDoux, S. P.; Wilson, G. L. (2000). "Enhanced

Elahi,A.; Zheng, Z.;Park, J.;Eyring, K.; McCaffrey,T.;Lazarus,P.(2002). "The human OGG1

El-Deiry, W. S.; Kern, S. E.; Pietenpol, J. A.; Kinzler, K. W.; Vogelstein, B. (1992). "Definition

El-Deiry, W. S. (1998). "Regulation of p53 downstream genes, Semin." Cancer Biol. 8:345–

Emptage, M. H.; Kent, T. A.; Kennedy, M. C.; Beinert, H.; Munck, E. (1983). "Mossbauer and

Fortini, P.; Pascucci, B.; Parlanti, E.; D'Errico, M.; Simonelli, V.; Dogliotti, E. (2003). "8-

Franco, R.; Sanchez-Olea, R.; Reyes-Reyes, E. M.; Panayiotidis, M. I. (2009). "Environmental

Gardner, P. R. (1997). "Superoxide-driven aconitase Fe–S center cycling." Biosci Rep 17: 33–

of a consensus binding site for p53, Nat." Genet. 1: 45–49.

Klungland, A.; Bohr, V. A. (2001). "Repair of 8-oxodeoxyguanosine lesions inmitochondrial 498 DNA depends on the oxoguanine DNA glycosylase (OGG1) gene and 8-499 oxoguanine accumulates in the mitochondrial DNA of OGG1-

excision repair in nuclear and mitochondrial DNA." Prog Nucleic Acid Res Mol

with flap endonuclease 1 and proliferating cell nuclear antigen involved in long-

(2002) "Enhanced mtDNA repair capacity protects pulmonary artery endothelial cells from oxidant-mediated death." Am. J. Physiol. Lung Cell Mol. Physiol. 283 (1):

mitochondrial DNA repair and cellular survival after oxidative stress by targeting the human 8-oxoguanine glycosylase repair enzyme to mitochondria." J Biol Chem

DNA repair enzyme and its association with orolaryngeal cancer risk."

EPR studies of activated aconitases: Development of a localized valence state at a subsite of the [4Fe-4S] cluster on binding of citrate." Proc Natl Acad Sci U S A 80:

Oxoguanine DNA damage: at the crossroad of alternative repair pathways." Mutat

toxicity, oxidative stress and apoptosis: menage a trois." Mutat. Res. 674 (1–2): 3–22.

Clayton, D. A. (1982). "Replication of animal mitochondrial DNA." Cell 28: 693–705.

Biochem 53:573–594.

Biol 68:285-97.

275: 331–342.

205–210.

357.

42.

4674–4678.

Res 531:127–139.

275: 37518–37523.

Carcinogenesis 23(7):1229-34.

biology."Annu. Rev. Biochem. 63: 915–948.

defective mice." Cancer Res. 61: 5378–5381.

patch base excision repair." Biochem 40: 12639-12644.

Gardner, P. R.; Nguyen, D. D.; White, C. W. (1994). "Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs." Proc Natl Acad Sci U S A 91(25): 12248-12252.

Granot,Z.;Kobiler,O.;Melamed-Book,N.;Eimerl,S.;Bahat,A.;Lu,B.;Braun,S.;Maurizi,M.R.;Suzuki, C.K.;Oppenheim,A.B.;Orly,J.(2007). "Turnover of mitochondrial steroidogenic acute regulatory (StAR) protein by Lon protease: the unexpected effect of proteasome inhibitors." Mol Endocrinol 21(9):2164-77.


Role of Ogg1 and Aconitase, p53 99

Mambo, E.; Chatterjee, A.; de Souza-Pinto, N. C.; Mayard, S.; Hogue, B. A.; Hoque, M. O.;

Lebedeva, M. A.; Eaton, J. S.; Shadel, G. S.(2009). "Loss of p53 causes mitochondrial DNA

Matsuoka, M.; Igisu, H.; Morimoto, Y. (2003). "Phosphorylation of p53 protein in A549

Medikayala, S.; Piteo, B.; Zhao, X. and Edwards, J. G. (2011). "Chronically elevated glucose

Michikawa, Y.; Mazzucchelli, F.; Bresolin, N.; Scarlato, G.; Attardi, G. (1999). "Aging-

Mirbahai,L.; Kershaw,R.M.; Green,R.M.; Hayden,R.E.; Meldrum,R.A.; Hodges,N.J.(2010).

Mishra, A.; Liu, J. Y.; Brody, A. R.; Morris, G. F. (1997). "Inhaled asbestos fibers induce p53 expression in the rat lung." Am. J. Respir. Cell Mol. Biol. 16 (4): 479–485. Miyashita, T.; Reed, J. C. (1995). "Tumor suppressor p53 is a direct transcriptional activator

Nakano, K.; Vousden, K. H. (2001). "PUMA, a novel proapoptotic gene, is induced by p53."

Nass, M. M. (1969). "Mitochondrial DNA. I. Intramitochondrial distribution and structural relations of single- and double-length circular DNA." J. Mol. Biol. 42: 521–528. Nelson, A.; Mendoza, T.; Hoyle, G. W.; Brody, A. R.; Fermin, C.; Morris, G. F. (2001).

Nuorva, K.; Makitaro, R.; Huhti, E.; Kamel, D.; Vahakangas, K.; Bloigu, R.; Soini, Y.; Paakko,

exposed epithelial and mesothelial lung cell lines." BMC Genomics 8:62. Oda, E.; Ohki, R.; Murasawa, H.; Nemoto, J.; Shibue, T.; Yamashita, T.; Tokino, T.;

Panduri, V.; Weitzman, S. A.; Chandel, N.; Kamp, D. W. (2003). "The mitochondria

epithelial cell apoptosis." Free Radic. Biol. Med. 47 (6): 750–759.

apoptosis." Am. J. Respir. Cell Mol. Biol. 28 (2): 241–248.

"Enhancement of fibrogenesis by the p53 tumor suppressor protein in asbestos-

P. (1994). "p53 protein accumulation in lung carcinomas of patients exposed to asbestos and tobacco smoke." Am. J. Respir. Crit. Care Med. 150 (2): 528–533. Nymark, P.; Lindholm, P.M.; Korpela, M.V.; Lahti, L.; Ruosaari, S.; Kaski, S.; Hollmen, J.;

Anttila,S.; Kinnula,V.L.; Knuutila,S. (2007). "Gene expression profiles in asbestos-

Taniguchi, T.; Tanaka, N. (2000). "Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis." Science 288 (5468): 1053–1058. Panduri, V.; Liu, G.; Surapureddi, S.; Kondapalli, J.; Soberanes, S.; de Souza-Pinto, N. C.;

Bohr, V. A.; Budinger, G. R.; Schumacker, P. T.; Weitzman, S. A.; Kamp, D. W. (2009). "Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung

regulated death pathway mediates asbestos-induced alveolar epithelial cell

hOgg1 activity in lung cancer." Oncogene 24: 4496–4508.

Biochimica et Biophysica Acta 1787:328–334.

region for replication." Science 286: 774–779.

of the human bax gene." Cell, 80 (2): 293–299.

exposed rodents." Chest 120 (1 Suppl): 33S–34S.

OGG1 in live cells." DNA Repair (Amst) 9(2):144-52.

Perspect. 111 (4): 509–512.

Mol. Cell 7 (3):683–694.

C348.

Dizdaroglu, M.; Bohr, V. A.; Sidransky, D. (2005). "Oxidized guanine lesions and

depletion and altered mitochondrial reactive oxygen species homeostasis."

human pulmonary epithelial cells exposed to asbestos fibers." Environ. Health

compromises myocardial mitochondrial DNA integrity by alteration of mitochondrial topoisomerase function." Am. J. Physiol. Cell Physiol. 300: C338–

dependent large accumulation of point mutations in the human mtDNA control

"Use of a molecular beacon to track the activity of base excision repair protein


Janicke, R. U.; Sohn, D.; Schulze-Osthoff, K. (2008). "The dark side of a tumor suppressor:

Jaurand, M. C. (1997). "Mechanisms of fiber-induced genotoxicity." Environ Health Perspect

Kamp, D. W.; Graceffa, P.; Pryor, W. A.; Weitzman, S. A. (1992). "The role of free radicals in

Kamp, D. W.; Israbian, V. A.; Preusen, S. E.; Zhang, C. X.; Weitzman, S. A. (1995). "Asbestos

Kamp, D.W., Shacter E, Weitzman SA. Chronic inflammation and cancer: The role of the

Ke-Hung Tsui, Tsui-Hsia Feng, Yu-Fen Lin, Phei-Lang Chang, Horng-Heng Juang. (2011).

Kim, I.; Xu, W.; Reed, J. C. (2008). "Cell death and endoplasmic reticulum stress: disease

Klungland, A.; Rosewell, I.; Hollenbach, S.; Larsen, E.; Daly, G.; Epe, B. et al. (1999). Proc

Kopnin, P. B.; Kravchenko, I. V.; Furalyov, V. A.; Pylev, L. N.; Kopnin, B. P. (2004). "Cell

Kroemer, G.; Galluzzi, L.; Brenner, C. (2007). "Mitochondrial membrane permeabilization in

Kujoth, G. C.; Hiona, A.; Pugh, T. D.; Someya, S.; Panzer, K.; Wohlgemuth, S. E.; Hofer, T.;

oxidative stress, and apoptosis in mammalian aging." Science 309: 481–484. Lebedeva, M. A.; Eaton, J. S.; Shadel, G. S. (2009). "Loss of p53 causes mitochondrial DNA

Levine, A. J. (1997). "p53, the cellular gatekeeper for growth and division." Cell 88: 323–331. Levresse, V.; Renier, A.; Fleury-Feith, J.; Levy, F.; Moritz, S.; Vivo, C.; Pilatte, Y.; Aurand, M.

Lin, F.; Liu, Y.; Keshava, N.; Li, S. (2000). "Crocidolite induces cell transformation and p53 gene mutation in BALB/c-3T3 cells." Teratog. Carcinog. Mutagen. 20 (5): 273–281. Liu, V. W.; Zhang, C.; Nagley, P. (1998). "Mutations in mitochondrial DNA accumulate

Liu, G.; Beri, R.; Mueller, A.; Kamp, D.W. "Molecular mechanisms of asbestos-induced lung epithelial cell apoptosis." Chemico-Biol Interactions 2010; 188:309-18. Loeb, L. A.; Wallace, D. C. and Martin, G. M. (2005). "The mitochondrial theory of aging and

Ko, L. J.; Prives, C. (1996). " p53: puzzle and paradigm." Genes Dev. 10: 1054-1072.

causes DNA strand breaks in cultured pulmonary epithelial cells: role of iron-

"p53 Downregulates the Gene Expression of Mitochondrial Aconitase in Human

relevance and therapeutic opportunities." Nat. Rev. Drug Discov. 7 (12): 1013–1030.

typespecific effects of asbestos on intracellular ROS levels, DNA oxidation and G1

Seo, A. Y.; Sullivan, R.; Jobling, W. A. et al. (2005). "Mitochondrial DNA mutations,

depletion and altered mitochondrial reactive oxygen species homeostasis."

C. (1997). "Analysis of cell cycle disruptions in cultures of rat pleural mesothelial cells exposed to asbestos fibers." Am. J. Respir. Cell Mol. Biol. 17 (6): 660–671. Lindahl, T. (1993). "Instability and decay of the primary structure of DNA." Nature 362:

differentially in three different human tissues during ageing." Nucleic. Acids Res.

its relationship to reactive oxygen species damage and somatic mtDNA mutations."

asbestos-induced diseases". Free Radic. Biol. Med. 12 (4): 293–315.

catalyzed free radicals." Am. J. Physiol. 268 (3 Pt 1): L471–480.

anti-apoptotic p53." Cell Death Differ. 15 (6): 959–976.

mitochondria. Oncology 2011; 25:400-13.

Natl Acad Sci USA 96: 13300–13305.

cell death." Physiol. Rev 87 (1): 99–163.

Biochim. Biophys. Acta. May 1787 (5): 328-34.

Proc. Natl. Acad. Sci. U S A 102: 18769–18770.

709–715.

26: 1268–1275.

Prostate Carcinoma Cells." The Prostate 71: 62-70.

cell cycle checkpoint." Oncogene 23 (54): 8834–8840.

105 (Suppl. 5): 1073–1084.


Role of Ogg1 and Aconitase, p53 101

Sablina, A. A.; Budanov, A.V.; Ilyinskaya, G. V.; Agapova, L. S.; Kravchenko, J. E.;

Samper, E.; Morgado, L.; Estrada, J. C.; Bernad, A.; Hubbard, A.; Cadenas, S.; Melov, S.

Santos, J. H.; Hunakova, L.; Chen,Y.; Bortner,Carl.; Van Houten, B. (2003). "Cell sorting

Shinmura,K.;Kohno,T.;Kasai,H.;Koda,K.;Sugimura,H.;Yokota,J.(1998). "Infrequent

in damaged DNA, in human gastric cancer." Jpn J Cancer Res 89(8):825-8. Shukla, A.; Gulumian, M.; Hei, T.; Kamp, D. W.; Rahman, Q.; Aust, A. E. (2003). Mossman,

Soong, N. W.; Hinton, D. R.; Cortopassi, G.; Arnheim, N. (1992). "Mosaicism for a specific

Shukla, A.; Jung, M.; Stern, M.; Fukagawa, N. K.; Taatjes, D. J.; Sawyer, D. (2003). "Asbestos

Tamar, P. E.; Ziv, S.; Yael, L. D.; Dalia, E.; Laila, C. (2008). "Roisman, Zvi Livneh DNA repair

Taylor, R. W. and Turnbull, D. M. (2005). "Mitochondrial DNA mutations in human disease.

Trifunovic, A.; Wredenberg, A.; Falkenberg, M.; Spelbrink, J. N.; Rovio, A. T.; Bruder, C. E.;

Upadhyay, D.; Kamp, D. W. (2003). "Asbestos-induced pulmonary toxicity: role of DNA

apoptosis." Am. J. Physiol. Lung Cell Mol. Physiol. 285(5): L1018-1025. Singh, K. K.; Desouki, M. M.; Franklin, R. B.; Costello, L. C. (2006). "Mitochondrial aconitase

Tabrizi,S.J.;Cleeter,M.W.;Xuereb,J.;Taanman,J.W.;Cooper,J.M.;Schapira,A.H.(1999).

cancer risk assessment and prevention." Cancer Letters 266: 60–72.

production." Proc Natl Acad Sci U S A 102(50):17993-8.

damage and apoptosis." Exp Biol Med 228 (6): 650–659.

murine lymphomas." Free Radic. Biol. Med. 46: 387–396.

decay in aging." Proc. Natl. Acad. Sci. U S A 91: 10771–10778.

Free Radic Biol Med 34 (9): 1117–1129.

318–323.

Mol. Cancer 5:14.

Neurol 45(1):25-32.

" Nat. Rev. Genet. 6: 389 –402.

Nat. Med. 11:1306–1313.

Chumakov, P. M. (2005). "The antioxidant function of the p53 tumor suppressor."

(2009). "Increase in mitochondrial biogenesis, oxidative stress, and glycolysis in

experiments link persistent mitochondrial DNA damage with loss of mitochondrial membrane potential and apoptotic cell death." J. Biol. Chem 278(3):1728-1734. Shibutani, S.; Takeshita, M.; Grollman, A. P. (1991). "Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG." Nature 349: 431–434. Shigenaga, M. K.; Hagen, T. M.; Ames, B. N. (1994). "Oxidative damages and mitochondrial

mutations of the hOGG1 gene, that is involved in the excision of 8-hydroxyguanine

B.T. "Multiple roles of oxidants in the pathogenesis of asbestos-induced diseases."

somatic mitochondrial DNA mutation in adult human brain." Nature Genet. 2:

induces mitochondrial DNA damage and dysfunction linked to the development of

and citrate metabolism in malignant and nonmalignant human prostate tissues."

"Biochemical abnormalities and excitotoxicity in Huntington's disease brain." Ann

of oxidative DNA damage in human carcinogenesis: Potential application for

Bohlooly-Y, M.; Oldfors, A.; Wibom, R. (2004). "Premature ageing in mice expressing defective mitochondrial DNA polymerase." Nature 429: 417–423. Trifunovic, A.; Hansson, A.; Wredenberg, A.; Rovio,A.T.; Dufour, E.; Khvorostov, I.;

Spelbrink, J.N.; Wibom, R.; Jacobs, H.T.; Larsson, N.G.(2005). "Somatic mtDNA mutations cause aging phenotypes without affecting reactive oxygen species


Panduri, V.; Weitzman, S. A.; Chandel, N. S.; Kamp, D. W. (2004). "Mitochondrial-derived

Panduri, V. S.; Surapureddi, S.; Soberanes, S. A.; Weitzman, N.; Chandel, D.; Kamp, W.

Plataki, M.; Koutsopoulos, A. V.; Darivianaki, K.; Delides, G.; Siafakas, N. M.; Bouros, D.

Powell, C. D.; Quain, D. E. and Smart, K. A. (2000). "he impact of media composition and

Rachek, L. I.; Grishko, V. I.; Ledoux, S. P.; Wilson, G. L. (2006). "Role of nitric oxide-induced

Rachek, L. I.; Thornley, N. P.; Grishko, V. I.; LeDoux, S. P.; Wilson, G. L. (2006). "Protection

Radicella, J. P.; Dherin, C.; Desmaze, C.; Fox, M. S.; Boiteux, S. (1997). Proc. Natl. Acad. Sci.

Raha, S. ; Robinson, B. H. (2000). "Mitochondria, oxygen free radicals, diseases and ageing."

Ralph, S. J.; Rodríguez-Enríquez, S.; Neuzil, J.; Saavedra, E.; Moreno-Sánchez, R. (2010).

Roepke, M.; Diestel, A.; Bajbouj, K.; Walluscheck, D.; Schonfeld, P.; Roessner, A.; Schneider-

Ruchko, M.; Gorodnya, O.; LeDoux, S. P.; Alexeyev, M. F.; Al-Mehdi, A. B.; Gillespie, M. N.

Ruchko, M. V.; Gorodnya, O. M.; Zuleta, A.; Pastukh, V. M.; Gillespie, M. N. (2010). "The

Russo, M. T.; De Luca, G.; Degan, P.; Parlanti, E.; Dogliotti, E.; Barnes, D. E.; Lindahl, T.;

and Ogg1 DNA glycosylases." Cancer Res. 64: 4411–4414.

Physiol. Lung Cell Mol. Physiol. 286 (6): L1220–L1227.

idiopathic pulmonary fibrosis." Chest. 127 (1): 266–274.

1286–1295.

Microbiol. 31: 46–51.

Med. 40 (5): 754–762.

U S A 94: 8010–8015.

6: 160–169.

13.

Park, L. C. (2001). J. Neurosci. Res. 66 : 1028-1034.

mitochondria." Diabetes 55: 1022–1028.

Molecular Aspects of Medicine 31: 145–170.

Trends Biochem. Sci. 25: 502–508.

Mol. Physiol. 288: L530–L535.

free radicals mediate asbestos-induced alveolar epithelial cell apoptosis." Am. J.

(2006). "P53 mediates amosite asbestos-induced alveolar epithelial cell mitochondria-regulated apoptosis." Am. J. Respir. Cell Mol. Biol. 34 (4) : 443–452. Paradies, G.; Petrosillo, G.; Paradies, V.; Ruggiero, F. M. (2010). "Oxidative stress,

mitochondrial bioenergetics, and cardiolipin in aging." Free Radic. Biol. Med. 48:

(2005). "Expression of apoptotic and antiapoptotic markers in epithelial cells in

petite mutation on the longevity of a polyploid brewing yeast strain." Lett. Appl.

mtDNA damage in mitochondrial dysfunction and apoptosis." Free Radic. Biol.

of INS-1 cells from free fatty acid-induced apoptosis by targeting hOGG1 to

"The causes of cancer revisited: ''Mitochondrial malignancy" and ROS-induced oncogenic transformation - Why mitochondria are targets for cancer therapy."

Stock, R.; Gali-Muhtasib, H. (2007). "Lack of p53 augments thymoquinone-induced apoptosis and caspase activation in human osteosarcoma cells." Cancer Biol. Ther.

(2005). "Mitochondrial DNA damage triggers mitochondrial dysfunction and apoptosis in oxidant-challenged lung endothelial cells." Am. J. Physiol. Lung Cell.

DNA glycosylase Ogg1 defends against oxidant-induced mtDNA damage and apoptosis in pulmonary artery endothelial cells." Free Radic Biol Med 50(9):1107-

Yang, H.; Miller, J. H.; Bignami, M. (2004). "Accumulation of the oxidative base lesion 8-hydroxyguanine in DNA of tumor-prone mice defective in both the Myh


**7** 

*1,2,3Greece 4Germany* 

**Structure-Function Relationship of** 

Horst-Werner Stuerzbecher4 and Constantinos E. Vorgias1

 *32nd Propaedeutic Pathology Clinic, Medical School, Athens University, Athens 4Molecular Cancer Biology Group, Institute of Pathology, UK-SH, Luebeck* 

Accurate transfer of genetic information is vital for all living organisms in order to guarantee species survival. DNA damage occurs spontaneously during a cell's life due to either endogenous causes such as Reactive Oxygen Species (ROS) produced during metabolism or due to exogenous insults such as Ionizing Radiation (IR) or genotoxic agents in food / water and environment, to which an organism is exposed. Endogenous damage, due to intrinsic instability of chemical bonds in DNA structure, occurs spontaneously under normal physiologic conditions and is calculated to be approximately 104 events per cell, per day (Lindahl, 1993). Moreover, during DNA replication base adducts can cause collapse of replication forks and DNA double strand breaks (DSBs) are introduced in order to reinitiate

As the genome carries all necessary information for life and evidently preservation of genome integrity is critical for cell survival, a number of mechanisms have evolved over time to ensure the most effective performance of the genome repair procedure. DNA repair mechanisms are capable of repairing practically all different types of chromosomal lesions (single and double strand breaks, base modifications, etc.) ensuring that genetic information is accurately transferred to the next generation. The cell's response to DNA damage (DNA Damage Response, DDR) encompasses a complex network of proteins, consisting of DNA damage recognition, signal transduction, transcriptional regulation, cell cycle control, DNA repair and verification of the repair efficiency, depending on the type of lesion, the replication status of the genome as well as the cell cycle stage. (scheme 1). Many excellent recent reviews as well as other chapters in the current volume extensively cover this topic

Defects in repair efficiency are the consequence of dysfunction of either upstream damage signalling or the central repair process. The current chapter covers topics referring to

(Rogakou, 1999; Lisby & Rothstein 2005; Murphy & Moynahan, 2010).

**1. Introduction** 

genome duplication process.

*Faculty of Biology, School of Sciences Athens University, Athens* 

**DNA Repair Proteins: Lessons** 

Effrossyni Boutou1,2, Vassiliki Pappa3,

*1Department of Biochemistry & Molecular Biology* 

*2Prenatal Diagnosis Lab, Laiko Hospital, Athens* 

**from BRCA1 and RAD51 Studies** 

