DNA replication as a target of the DNA damage checkpoint

Faithful inheritance of the genome from mother to daughter cell requires that it is replicated accurately, in its entirety, exactly once. DNA replication not only has to have high fidelity, but also has to cope with exogenous and endogenous agents that damage DNA during the life cycle of a cell. The DNA damage checkpoint, which monitors and responds to defects in the genome, is critical for the completion of replication. The focus of this review is how DNA replication is regulated by the checkpoint response in the presence of DNA damage and fork stalling agents.     

A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA

We have isolated a Saccharomyces cerevisiae mutant that shows an increased tendency to form cytoplasmic petites (respiration-deficient ρ⁻ or ρ⁰ mutants) in response to treatment of cells growing on a solid medium with the DNA-damaging agent methyl methanesulfonate or ultraviolet light. The mutation in this strain, atm1-1, was found to cause a single amino acid substitution in ATM1, a nuclear gene that encodes the mitochondrial ATP-binding cassette (ABC) transporter. When the mutant cells were grown in liquid glucose medium, they accumulated free iron within the mitochondria and at the same time gave rise to spontaneous cytoplasmic petite mutants, as seen previously in cells carrying a mutation in a gene homologous to the human gene responsible for Friedreich's ataxia. Analysis of the effects of free iron and malonic acid (an inhibitor of oxidative respiration in mitochondria) on the incidence of petites among the mutant cells indicated that spontaneous induction of petites was a consequence of oxidative stress rather than a direct effect of either a defect in the ATM1 gene or the accumulation of free iron. We observed an increase in the incidence of strand breaks in the mitochondrial DNA of the atm1-1 mutant cells. Furthermore, we found that rates of induction of petites and accumulation of strand breaks in mitochondrial DNA were enhanced in the atm1-1 mutant by the introduction of another mutation, mhr1-1, which results in a deficiency in mitochondrial DNA repair. These observations indicate that spontaneous induction of petites in the atm1-1 mutant is a consequence of oxidative damage to mitochondrial DNA mediated by enhanced accumulation of mitochondrial iron. Received: 26 March 1999 / Accepted: 29 June 1999     

Mapping oxidative DNA damage using ligation-mediated polymerase chain reaction technology

Reactive oxygen species induce a pharmacopoeia of oxidized bases in DNA. DNA can be cleaved at most of the sites of these modified bases by digestion with a combination of two base excision repair glycosylases from Escherichia coli, Fpg glycosylase, and endonuclease III. The frequency of the resulting glycosylase-dependent 5'-phosphoryl ends can be mapped at nucleotide resolution along a sequencing gel autoradiogram by a genomic sequencing technique, ligation-mediated polymerase chain reaction (LMPCR). In cultured rat cells, the frequency of endogenous oxidized bases in mitochondrial DNA is sufficiently high, about one oxidized base per 100 kb, to be directly mapped from 0.1 microg of total cellular DNA preparations by LMPCR. Nuclear DNA has a lower frequency of endogenous oxidative base damage which cannot be mapped from 1-microg preparations of total cellular DNA. Preparative gel electrophoresis of the PGK1 and p53 genes from 300 microg of restriction endonuclease-digested genomic DNA showed a 25-fold enrichment for the genes and, after endonuclease digestion followed by LMPCR, gave sufficient signal to map the frequency of oxidized bases from human cells treated with 50 microM H2O2.     

Involvement of DNA polymerase beta in protection against the cytotoxicity of oxidative DNA damage

We had shown previously that DNA polymerase beta (beta-pol) null mouse fibroblasts, deficient in base excision repair (BER), are hypersensitive to monofunctional methylating agents but not to hydrogen peroxide (H2O2). This is surprising because beta-pol is thought to be involved in BER of oxidative as well as methylated DNA damage. We confirm these findings here in early-passage cells. However, with time in culture, beta-pol null cells become hypersensitive to H2O2 and other reactive oxygen species-generating agents. Analysis of in vitro BER reveals a strong deficiency in single-nucleotide BER of 8-oxoguanine (8-oxoG) by both early- and late-passage beta-pol null cell extracts. Therefore, in early-passage wild-type and beta-pol null cells, the capacity for single-nucleotide BER of 8-oxoG does not correlate with cellular sensitivity to H2O2. Expression of beta-pol protein in the late-passage null cells almost completely reverses the H2O2-hypersensitivity phenotype. Methoxyamine (MX) treatment sensitizes late-passage wild-type cells to H2O2 as expected for beta-pol-mediated single-nucleotide BER; however in beta-pol null cells, MX has no effect. The data indicate a role(s) of beta-pol-dependent repair in protection against the cytotoxicity of oxidative DNA damage in wild-type cells.     

Detection of oxidative stress-induced mitochondrial DNA damage using fluorescence correlation spectroscopy

Using fluorescence correlation spectroscopy (FCS), we tested the feasibility of rapid detection of oxidative damage of mitochondrial DNA (mtDNA) in a small volume. The complete mtDNA genome was amplified by long polymerase chain reaction (LPCR), and the product was fluorescently labeled with an intercalating dye, YOYO-1. The fluorescence autocorrelation function was analyzed using a simple two-component model with the diffusion time of 0.21 ms for the LPCR primer and 18 ms for the mtDNA LPCR product. When human embryonic kidney 293 (HEK-293) cells were exposed to 0.4 mM H2O2, the fraction of the mtDNA LPCR product decreased significantly. In contrast, the fraction of the nuclear-encoded beta-globin LPCR product remained unchanged. The analysis time of FCS measurement was very short (5 min) compared with that of gel electrophoresis (3 h). Thus, FCS allowed the rapid detection of the vulnerability of mtDNA to oxidative stress within a small volume element at the subfemtoliter level in solution. These results suggest that the LPCR-FCS method can be used for epidemiological studies of diseases caused by mtDNA damage.     

DNA polymerase lambda protects mouse fibroblasts against oxidative DNA damage and is recruited to sites of DNA damage/repair

DNA polymerase lambda (pol lambda) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol lambda null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol lambda in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol lambda co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol lambda protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.     

Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer's disease

Increasing evidence suggests that oxidative stress is associated with normal aging and several neurodegenerative diseases, including Alzheimer's disease (AD). Here we quantified multiple oxidized bases in nuclear and mitochondrial DNA of frontal, parietal, and temporal lobes and cerebellum from short postmortem interval AD brain and age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring (GC/MS-SIM) and stable labeled internal standards. Nuclear and mitochondrial DNA were extracted from eight AD and eight age-matched control subjects. We found that levels of multiple oxidized bases in AD brain specimens were significantly (p < 0.05) higher in frontal, parietal, and temporal lobes compared to control subjects and that mitochondrial DNA had approximately 10-fold higher levels of oxidized bases than nuclear DNA. These data are consistent with higher levels of oxidative stress in mitochondria. Eight-hydroxyguanine, a widely studied biomarker of DNA damage, was approximately 10-fold higher than other oxidized base adducts in both AD and control subjects. DNA from temporal lobe showed the most oxidative damage, whereas cerebellum was only slightly affected in AD brains. These results suggest that oxidative damage to mitochondrial DNA may contribute to the neurodegeneration of AD.     

Increased mitochondrial oxidative damage and oxidative DNA damage contributes to the neurodegenerative process in sporadic amyotrophic lateral sclerosis

To investigate the possibility that mitochondrial oxidative damage or oxidative DNA damage or both contribute to the neurodegenerative process of sporadic amyotrophic lateral sclerosis (sALS), this study used high-performance liquid chromatography with an electrochemical detector to measure the concentrations of the reduced and oxidized forms of coenzyme Q10 (CoQ10) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the cerebrospinal fluid (CSF) of 17 patients with sALS and 17 age-matched controls with no neurological diseases. The percentage of oxidized CoQ10 in the CSF of sALS patients was greater than that in the CSF of controls (p<0.002) and was negatively correlated with the duration of illness (rho=-0.61, p<0.01). The concentration of 8-OHdG in the CSF of sALS patients was greater than that in the CSF of controls (p<0.005) and was positively correlated with the duration of illness (rho=0.53, p<0.005). The percentage of oxidized CoQ10 was correlated with the concentrations of 8-OHdG in the CSF of sALS patients (rho=-0.53, p<0.05). These results suggest that both mitochondrial oxidative damage and oxidative DNA damage play important roles in the pathogenesis of sporadic amyotrophic lateral sclerosis.     

A putative Leishmania DNA polymerase theta protects the parasite against oxidative damage

Leishmania infantum is a protozoan parasite that is phagocytized by human macrophages. The host macrophages kill the parasite by generating oxidative compounds that induce DNA damage. We have identified, purified and biochemically characterized a DNA polymerase θ from L. infantum (LiPolθ), demonstrating that it is a DNA-dependent DNA polymerase involved in translesion synthesis of 8oxoG, abasic sites and thymine glycol lesions. Stably transfected L. infantum parasites expressing LiPolθ were significantly more resistant to oxidative and interstrand cross-linking agents, e.g. hydrogen peroxide, cisplatin and mitomycin C. Moreover, LiPolθ-overexpressing parasites showed an increased infectivity toward its natural macrophage host. Therefore, we propose that LiPolθ is a translesion synthesis polymerase involved in parasite DNA damage tolerance, to confer resistance against macrophage aggression.     

The mitochondrial protease OMA1 acts as a metabolic safeguard upon nuclear DNA damage

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RecQL4 cytoplasmic localization: Implications in mitochondrial DNA oxidative damage repair

RecQL4, one of the five human RecQ helicases, is crucial for genomic stability and RecQL4 when mutated leads to premature aging phenotypes in humans. Unlike other human RecQ helicases, RecQL4 is found both in the nucleus and the cytoplasm. While the nuclear localization signal (NLS) and the retention domain at the N-terminus are responsible for the nuclear localization of RecQL4, the signal for its cytoplasmic localization is essentially unknown. In this study, two functional nuclear exporting signals (NESs; pNES2 and pNES3) were identified at the C-terminus of RecQL4. Deletion of pNES2 drastically diminished the cytoplasmic localization of RecQL4. Strikingly, addition of ubiquitination tail at the C-terminus of RecQL4 substantially enriched the cytoplasmic fraction of RecQL4 only in the presence of functional pNES2. Immunofluorescence studies revealed that the cytoplasmic RecQL4 was localized in mitochondria. Consistent with its mitochondrial localization, a regulatory role for RecQL4 in the maintenance of mitochondrial DNA (mtDNA) copy number was demonstrated. Elevation of ectopic expression of RecQL4 increased the mtDNA copy number in HEK293 cells while RecQL4 knock down markedly decreased the mtDNA copy number in U2OS cells. Additionally, a substantially increased level of mitochondrial superoxide production, and a markedly decreased repair capacity for oxidative DNA damage were observed in the mitochondria of both RecQL4 deficient human fibroblasts and RecQL4-suppressed cancer cells. These data strongly suggest a regulatory role for RecQL4 in mitochondrial stability and function. Collectively, our study demonstrates that NES-mediated RecQL4 export to the cytoplasm is essential for the maintenance of mitochondrial genome stability.     

Increased oxidative damage in nuclear and mitochondrial DNA in mild cognitive impairment

Increasing evidence suggests that oxidative damage is associated with normal aging and several neurodegenerative diseases. Mild cognitive impairment (MCI), the phase between normal aging and early dementia, is a common problem in the elderly with many subjects going on to develop Alzheimer's disease (AD). Although increased DNA oxidation is observed in the AD brain, it is unclear when the oxidative damage begins. To determine if DNA oxidation occurs in the brain of subjects with MCI, we quantified multiple oxidized bases in nuclear and mitochondrial DNA isolated from frontal, parietal and temporal lobes and cerebellum of short post-mortem interval autopsies of eight amnestic patients with MCI and six age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring. We found statistically significant elevations (p < 0.05) of 8-hydroxyguanine, a widely studied biomarker of DNA damage, in MCI nuclear DNA from frontal and temporal lobe and in mitochondrial DNA from the temporal lobe compared with age-matched control subjects. Levels of 8-hydroxyadenine and 4,6-diamino-5-formamidopyrimidine were significantly elevated in nuclear DNA from all three neocortical regions in MCI. Statistically significant elevations of 4,6-diamino-5-formamidopyrimidine were also observed in mitochondrial DNA of MCI temporal, frontal and parietal lobes. These results suggest that oxidative damage to nuclear and mitochondrial DNA occurs in the earliest detectable phase of AD and may play a meaningful role in the pathogenesis of this disease.     

Filamin-A as a marker and target for DNA damage based cancer therapy

Filamin-A, also called actin binding protein 280 (ABP-280), cross-links the actin filaments into dynamic orthogonal network to serve as scaffolds in multiple signaling pathways. It has been reported that filamin-A interacts with DNA damage response proteins BRCA1 and BRCA2. Defects of filamin-A impair the repair of DNA double strand breaks (DSBs), resulting in sensitization of cells to ionizing radiation. In this study, we sought to test the hypothesis that filamin-A can be used as a target for cancer chemotherapy and as a biomarker to predict cancer response to therapeutic DNA damage. We found that reduction of filamin-A sensitizes cancer cells to chemotherapy reagents bleomycin and cisplatin, delays the repair of not only DSBs but also single strand breaks (SSBs) and interstrand crosslinks (ICLs), and increases chromosome breaks after the drug treatment. By treating a panel of human melanoma cell lines with variable filamin-A expression, we observed a correlation between expression level of filamin-A protein and drug IC(50). We further inhibited the expression of filamin-A in melanoma cells, and found that this confers an increased sensitivity to bleomycin and cisplatin treatment in a mouse xenograft tumor model. These results suggest that filamin-A plays a role in repair of a variety of DNA damage, that lack of filamin-A is a prognostic marker for a better outcome after DNA damage based treatment, and filamin-A can be inhibited to sensitize filamin-A positive cancer cells to therapeutic DNA damage. Thus filamin-A can be used as a biomarker and a target for DNA damage based cancer therapy.     

Origins of human mitochondrial point mutations as DNA polymerase gamma-mediated errors

Mitochondrial mutational spectra in human cells, tissues and derived tumors for bp 10,030-10,130 are essentially identical, suggesting a predominant mutagenic role for endogenous processes. We hypothesized that errors mediated by mitochondrial DNA polymerase gamma were the primary sources of mutations. Point mutations created in this sequence by human DNA pol gamma in vitro were thus compared to the eighteen mutational hotspots, all single base substitutions, previously found in human tissues. The set of concordant hotspots accounted for 83% of these in vivo mutational events. About half of these mutations are insensitive to prolonged heating of DNA during PCR and half increase proportionally with heating time at 98 degrees C. Primary misincorporation errors and miscopying errors past thermal denaturing products such as deaminated cytosines (uracils) thus appear to be of approximately equal importance. For the sequence studied, these data support the conclusion that, endogenous error mediated by DNA pol gamma constitutes the primary source of mitochondrial point mutations in human tissues.     

Repair of oxidative DNA damage

Cellular genomes suffer extensive damage from exogenous agents and reactive oxygen species formed during normal metabolism. The MutT homologs (MutT/MTH) remove oxidized nucleotide precursors so that they cannot be incorporated into DNA during replication. Among many repair pathways, the base excision repair (BER) pathway is the most important cellular protection mechanism responding to oxidative DNA damage. The 8-oxoG glycosylases (Fpg or MutM/OGG) and the MutY homologs (MutY/MYH) glycosylases along with MutT/MTH protect cells from the mutagenic effects of 8-oxoG, the most stable and deleterious product known caused by oxidative damage to DNA. The key enzymes in the BER process are DNA glycosylases, which remove different damaged bases by cleavage of the N-glycosylic bonds between the bases and the deoxyribose moieties of the nucleotide residues. Biochemical and structural studies have demonstrated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of strated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of several glycosylases show that the substrate base flips out of the sharply bent DNA helix and the minor groove is widened to be accessed by the glycosylases. To complete the repair after glycosylase action, the apurinic/apyrimidinic (AP) site is further processed by an incision step, DNA synthesis, an excision step, and DNA ligation through two alternative pathways. The short-patch BER (1-nucleotide patch size) and long-patch BER (2–6-nucleotide patch size) pathways need AP endonuclease to generate a 3′ hydroxyl group but require different sets of enzymes for DNA synthesis and ligation. Protein-protein interactions have been reported among the enzymes involved in BER. It is possible that the successive players in the repair pathway are assembled in a complex to perform concerted actions. The BER pathways are proposed to protect cells and organisms from mutagenesis and carcinogenesis.     

Carbohydrate restriction does not change mitochondrial free radical generation and oxidative DNA damage

Many previous investigations have consistently reported that caloric restriction (40%), which increases maximum longevity, decreases mitochondrial reactive species (ROS) generation and oxidative damage to mitochondrial DNA (mtDNA) in laboratory rodents. These decreases take place in rat liver after only seven weeks of caloric restriction. Moreover, it has been found that seven weeks of 40% protein restriction, independently of caloric restriction, also decrease these two parameters, whereas they are not changed after seven weeks of 40% lipid restriction. This is interesting since it is known that protein restriction can extend longevity in rodents, whereas lipid restriction does not have such effect. However, before concluding that the ameliorating effects of caloric restriction on mitochondrial oxidative stress are due to restriction in protein intake, studies on the third energetic component of the diet, carbohydrates, are needed. In the present study, using semipurified diets, the carbohydrate ingestion of male Wistar rats was decreased by 40% below controls without changing the level of intake of the other dietary components. After seven weeks of treatment the liver mitochondria of the carbohydrate restricted animals did not show changes in the rate of mitochondrial ROS production, mitochondrial oxygen consumption or percent free radical leak with any substrate (complex I- or complex II-linked) studied. In agreement with this, the levels of oxidative damage in hepatic mtDNA and nuclear DNA were not modified in carbohydrate restricted animals. Oxidative damage in mtDNA was one order of magnitude higher than that in nuclear DNA in both dietary groups. These results, together with previous ones, discard lipids and carbohydrates, and indicate that the lowered ingestion of dietary proteins is responsible for the decrease in mitochondrial ROS production and oxidative damage in mtDNA that occurs during caloric restriction.