Enhanced mitochondrial DNA repair and cellular survival after oxidative stress by targeting the human 8-oxoguanine glycosylase repair enzyme to mitochondria

Oxidative damage to mitochondrial DNA (mtDNA) has been implicated as a causative factor in many disease processes and in aging. We have recently discovered that different cell types vary in their capacity to repair this damage, and this variability correlates with their ability to withstand oxidative stress. To explore strategies to enhance repair of oxidative lesions in mtDNA, we have constructed a vector containing a mitochondrial transport sequence upstream of the sequence for human 8-oxoguanine DNA glycosylase. This enzyme is the glycosylase/AP lyase that participates in repair of purine lesions, such as 8-oxoguanine. Western blot analysis confirmed that this recombinant protein was targeted to mitochondria. Enzyme activity assays showed that mitochondrial extracts from cells transfected with the construct had increased enzyme activity compared with cells transfected with vector only, whereas nuclear enzyme activity was not changed. Repair assays showed that there was enhanced repair of oxidative lesions in mtDNA. Additional studies revealed that this augmented repair led to enhanced cellular viability as determined by reduction of the tetrazolium compound to formazan, trypan blue dye exclusion, and clonogenic assays. Therefore, targeting of DNA repair enzymes to mitochondria may be a viable approach for the protection of cells against some of the deleterious effects of oxidative stress.     

Repair of Oxidative DNA Damage in Saccharomyces cerevisiae

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae.     

Oxidative damage-induced PCNA complex formation is efficient in xeroderma pigmentosum group A but reduced in Cockayne syndrome group B cells.

Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases delta and epsilon, is essential for both DNA replication and repair. PCNA is required in the resynthesis step of nucleotide excision repair (NER). After UV irradiation, PCNA translocates into an insoluble protein complex, most likely associated with the nuclear matrix. It has not previously been investigated in vivo whether PCNA complex formation also takes place after oxidative stress. In this study, we have examined the involvement of PCNA in the repair of oxidative DNA damage. PCNA complex formation was studied in normal human cells after treatment with hydrogen peroxide, which generates a variety of oxidative DNA lesions. PCNA was detected by two assays, immunofluorescence and western blot analyses. We observed that PCNA redistributes from a soluble to a DNA-bound form during the repair of oxidative DNA damage. PCNA complex formation was analyzed in two human natural mutant cell lines defective in DNA repair: xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). XP-A cells are defective in overall genome NER while CS-B cells are defective only in the preferential repair of active genes. Immunofluorescent detection of PCNA complex formation was similar in normal and XP-A cells, but was reduced in CS-B cells. Consistent with this observation, western blot analysis in CS-B cells showed a reduction in the ratio of PCNA relocated as compared to normal and XP-A cells. The efficient PCNA complex formation observed in XP-A cells following oxidative damage suggests that formation of PCNA-dependent repair foci may not require the XPA gene product. The reduced PCNA complex formation observed in CS-B cells suggests that these cells are defective in the processing of oxidative DNA damage.     

Protein oxidation,UVA and human DNA repair

Solar UVB is carcinogenic. Nucleotide excision repair (NER) counteracts the carcinogenicity of UVB by excising potentially mutagenic UVB-induced DNA lesions. Despite this capacity for DNA repair, non-melanoma skin cancers and apparently normal sun-exposed skin contain huge numbers of mutations that are mostly attributable to unrepaired UVB-induced DNA lesions. UVA is about 20-times more abundant than UVB in incident sunlight. It does cause some DNA damage but this does not fully account for its biological impact. The effects of solar UVA are mediated by its interactions with cellular photosensitizers that generate reactive oxygen species (ROS) and induce oxidative stress. The proteome is a significant target for damage by UVA-induced ROS. In cultured human cells, UVA-induced oxidation of DNA repair proteins inhibits DNA repair. This article addresses the possible role of oxidative stress and protein oxidation in determining DNA repair efficiency – with particular reference to NER and skin cancer risk.     

Progress in the analysis of urinary oxidative DNA damage

Oxidative DNA damage has been implicated to be important in the pathogenesis of many diseases, including cancer and heart disease. The assessment of damage in various biological matrices, such as DNA, serum, and urine, is vital to understanding this role and subsequently devising intervention strategies. Despite the numerous techniques to measure oxidative DNA damage products in urine, it remains unclear what these measurements truly represent. Sources of urinary lesions may include the diet, cell death, and, of most interest, DNA repair. Were it possible to exclude the two former contributions, a noninvasive assay for DNA repair would be invaluable in the study of DNA damage and disease. This review highlights that, although progress has been made, significant work remains. Diet, cell death, and repair need continued examination to further elucidate the kinetics of lesion formation and clearance in vivo. Studies from our laboratory and others are making appreciable progress towards the interpretation of urinary lesion measurements along with the development of urinary assays to evaluate DNA repair. Upon establishment of these details, urinary oxidative DNA damage measurements may become more than a reflection of generalized oxidative stress.     

Efficient in vitro repair of 7-hydro-8-oxodeoxyguanosine by human cell extracts: involvement of multiple pathways.

To investigate the repair of oxidative damage in DNA, we have established an in vitro assay utilizing human lymphoblastoid whole cell extracts and plasmid DNA damaged by exposure to methylene blue and visible light. This treatment has been shown to produce predominantly 7-hydro-8-oxodeoxyguanosine (8-oxodG) in double-stranded DNA at low levels of modification. DNA containing 1. 6 lesions per plasmid is substrate for efficient repair synthesis by cell extracts. The incorporation of dGMP is 2.7 +/- 0.5 times greater than the incorporation of dCMP, indicating an average repair patch of 3-4 nucleotides. Damage-specific nicking occurs within 15 min, while resynthesis is slower. The incorporation of dGMP increases linearly, while the incorporation of dCMP exhibits a distinct lag. Extracts from xeroderma pigmentosum (XP) complementation groups A and B exhibit 25 and 40%, respectively, of the incorporation of dCMP compared with normal extracts, but extracts from an XP-D cell line exhibit twice the activity. These data suggest that the efficient repair of 8-oxodG lesions observed in human cell extracts involves more than one pathway of base excision repair.     

Repair of oxidative DNA base lesions induced by fluorescent light is defective in xeroderma pigmentosum group A cells.

Fluorescent light (FL) has been shown to generate free radicals within cells, however, the specific chemical nature of DNA damage induced by FL has not previously been determined. Using gas chromatography/isotope dilution mass spectrometry, we have detected induction of the oxidative DNA lesions 5-hydroxycytosine (5-OH-Cyt), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde) in cultured cells irradiated with FL. We followed the repair of these lesions in normal and xeroderma pigmentosum group A (XP-A) cells. 5-OH-Cyt and FapyGua were repaired efficiently in normal cells within 6 h following FL exposure. XP-A cells were unable to repair these oxidative DNA base lesions. Additionally, to compare the repair of oxidative lesions induced by various sources, in vitro repair studies were performed using plasmid DNA damaged by FL, gamma-irradiation or OsO(4)treatment. Whole cell extracts from normal cells repaired damaged substrates efficiently, whereas there was little repair in XP-A extracts. Our data demon-strate defective repair of oxidative DNA base lesions in XP-A cells in vivo and in vitro.     

Complex interplay of lesion-specific DNA repair enzyme on bistranded clustered DNA damage harboring Tg:G mismatch in nucleosome core particles

5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) and 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) are major DNA damage lesions produced by endogenous oxidative stress, as well as inflicted by carcinogens and ionizing radiation. The processing of Tg:G mismatch and 8-oxodG in close proximity of each other in a bistranded clustered environment in DNA oligomer duplexes as well as in a nucleosome core particle (NCP) model are reported here. The processing of the lesions was evaluated by purified enzyme cocktails of hNTH1 and hOGG1 as well as with a HeLa cell extract. Interestingly, the yield of double-strand breaks (DSBs) resulting from the processing of the bistranded lesions are appreciably lower when the DNA is treated with the HeLa cell extract compared with the relevant purified enzyme cocktail in both models. Clustered bistranded lesions become more repair refractive when reconstituted as an NCP. This indicates a complex interplay between the repair enzymes that influence the processing of the bistranded cluster damage positively to avoid the formation of DSBs under cellular conditions. In addition to position and orientation of the lesions, the type of the lesions in the cluster environment in DNA along with the relative abundance of the lesion-specific enzymes in the cells strongly prevents the processing of the oxidized nucleobases.     

Genistein ameliorates cardiac inflammation and oxidative stress in streptozotocin-induced diabetic cardiomyopathy in rats

DNA is continuously exposed to damaging agents that can lead to changes in the genetic information with adverse consequences. Nonetheless, eukaryotic cells have mechanisms such as the DNA damage response (DDR) to prevent genomic instability. The DNA of eukaryotic cells is packaged into nucleosomes, which fold the genome into highly condensed chromatin, but relatively little is known about the role of chromatin accessibility in DNA repair. p19INK4d, a cyclin-dependent kinase inhibitor, plays an important role in cell cycle regulation and cellular DDR. Extensive data indicate that p19INK4d is a critical factor in the maintenance of genomic integrity and cell survival. p19INK4d is upregulated by various genotoxics, improving the repair efficiency for a variety of DNA lesions. The evidence of p19INK4d translocation into the nucleus and its low sequence specificity in its interaction with DNA prompted us to hypothesize that p19INK4d plays a role at an early stage of cellular DDR. In the present study, we demonstrate that upon oxidative DNA damage, p19INK4d strongly binds to and relaxes chromatin. Furthermore, in vitro accessibility assays show that DNA is more accessible to a restriction enzyme when a chromatinized plasmid is incubated in the presence of a protein extract with high levels of p19INK4d. Nuclear protein extracts from cells overexpressing p19INK4d are better able to repair a chromatinized and damaged plasmid. These observations support the notion that p19INK4d would act as a chromatin accessibility factor that allows the access of the repair machinery to the DNA damage site.     

Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions

Oxidative DNA damage has been implicated in mutagenesis, carcinogenesis and aging. Endogenous cellular processes such as aerobic metabolism generate reactive oxygen species (ROS) that interact with DNA to form dozens of DNA lesions. If unrepaired, these lesions can exert a number of deleterious effects including the induction of mutations. In an effort to understand the genetic consequences of cellular oxidative damage, many laboratories have determined the patterns of mutations generated by the interaction of ROS with DNA. Compilation of these mutational spectra has revealed that GC → AT transitions and GC → TA transversions are the most commonly observed mutations resulting from oxidative damage to DNA. Since mutational spectra convey only the end result of a complex cascade of events, which includes formation of multiple adducts, repair processing, and polymerase errors, it is difficult if not impossible to asses the mutational specificity of individual DNA lesions directly from these spectra. This problem is especially complicated in the case of oxidative DNA damage owing to the multiplicity of lesions formed by a single damaging agent. The task of assigning specific features of mutational spectra to individual DNA lesions has been made possible with the advent of a technology to analyze the mutational properties of single defined adducts, in vitro and in vivo. At the same time, parallel progress in the discovery and cloning of repair enzymes has advanced understanding of the biochemical mechanisms by which cells excise DNA damage. This combination of tools has brought our understanding of DNA lesions to a new level of sophistication. In this review, we summarize the known properties of individual oxidative lesions in terms of their structure, mutagenicity and repairability.     

Reprint of: gene susceptibility to oxidative damage: from single nucleotide polymorphisms to function

Oxidative damage to DNA can cause mutations, and mutations can lead to cancer. DNA repair of oxidative damage should therefore play a pivotal role in defending humans against cancer. This is exemplified by the increased risk of colorectal cancer of patients with germ-line mutations of the oxidative damage DNA glycosylase MUTYH. In contrast to germ-line mutations in DNA repair genes, which cause a strong deficiency in DNA repair activity in all cell types, the role of single nucleotide polymorphisms (SNPs) in sporadic cancer is unclear also because deficiencies in DNA repair, if any, are expected to be much milder. Further slowing down progress are the paucity of accurate and reproducible functional assays and poor epidemiological design of many studies. This review will focus on the most common and widely studied SNPs of oxidative DNA damage repair proteins trying to bridge the information available on biochemical and structural features of the repair proteins with the functional effects of these variants and their potential impact on the pathogenesis of disease.     

Lymphoblasts of women with BRCA1 mutations are deficient in cellular repair of 8,5'-Cyclopurine-2'-deoxynucleosides and 8-hydroxy-2'-deoxyguanosine

Mutations in breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 predispose women to a high risk of these cancers. Here, we show that lymphoblasts of women with BRCA1 mutations who had been diagnosed with breast cancer are deficient in the repair of some products of oxidative DNA damage, namely, 8-hydroxy-2'-deoxyguanosine and 8,5'-cyclopurine-2'-deoxynucleosides. Cultured lymphoblasts from 10 individuals with BRCA1 mutations and those from 5 control individuals were exposed to 5 Gy of ionizing radiation to induce oxidative DNA damage and then allowed to repair this damage. DNA samples isolated from these cells were analyzed by liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure 8-hydroxy-2'-deoxyguanosine, (5'-S)-8,5'-cyclo-2'-deoxyadenosine, (5'-R)-8,5'-cyclo-2'-deoxyguanosine, and (5'-S)-8,5'-cyclo-2'-deoxyguanosine. After irradiation and a subsequent period of repair, no significant accumulation of these lesions was observed in the DNA from control cells. In contrast, cells with BRCA1 mutations accumulated statistically significant levels of these lesions in their DNA, providing evidence of a deficiency in DNA repair. In addition, a commonly used breast tumor cell line exhibited the same effect when compared to a relevant control cell line. The data suggest that BRCA1 plays a role in cellular repair of oxidatively induced DNA lesions. The failure of cells with BRCA1 mutations to repair 8,5'-cyclopurine-2'-deoxynucleosides indicates the involvement of BRCA1 in nucleotide-excision repair of oxidative DNA damage. This work suggest that accumulation of these lesions may lead to a high rate of mutations and to deleterious changes in gene expression, increasing breast cancer risk and contributing to breast carcinogenesis.     

Gene susceptibility to oxidative damage: from single nucleotide polymorphisms to function

Oxidative damage to DNA can cause mutations, and mutations can lead to cancer. DNA repair of oxidative damage should therefore play a pivotal role in defending humans against cancer. This is exemplified by the increased risk of colorectal cancer of patients with germ-line mutations of the oxidative damage DNA glycosylase MUTYH. In contrast to germ-line mutations in DNA repair genes, which cause a strong deficiency in DNA repair activity in all cell types, the role of single nucleotide polymorphisms (SNPs) in sporadic cancer is unclear also because deficiencies in DNA repair, if any, are expected to be much milder. Further slowing down progress are the paucity of accurate and reproducible functional assays and poor epidemiological design of many studies. This review will focus on the most common and widely studied SNPs of oxidative DNA damage repair proteins trying to bridge the information available on biochemical and structural features of the repair proteins with the functional effects of these variants and their potential impact on the pathogenesis of disease.     

Mode of inhibition of short-patch base excision repair by thymine glycol within clustered DNA lesions

Clustered DNA damage, where two or more lesions are located proximally to each other, is frequently induced by ionizing radiation. Individual base lesions within a cluster are repaired by base excision repair. In this study we addressed the question of how thymine glycol (Tg) within a cluster would affect the repair of opposing lesions by human cell extracts. We have found that Tg located opposite to an abasic site does not affect cleavage of this site by apurinic/apyrimidinic (AP) endonuclease. However, Tg significantly compromised the next step of the repair. Although purified DNA polymerase beta was able to incorporate the correct nucleotide (dAMP) opposite to Tg, the rate of incorporation was reduced by 3-fold. Tg does not affect 5'-sugar phosphate removal by the 2-deoxyribose-5-phosphate (dRP) lyase activity of DNA polymerase beta, but further processing of the strand break by purified DNA ligase III was slightly diminished. In agreement with these findings, although an AP site located opposite to Tg was efficiently incised in human cell extract, only a limited amount of fully repaired product was observed, suggesting that such clustered DNA lesions may have a significantly increased lifetime in human cells compared with similar single-standing lesions.     

DNA damage levels and biochemical repair capacities associated with XRCC1 deficiency

Base excision repair (BER) is the major corrective pathway for most spontaneous, oxidative, and alkylation DNA base and sugar damage. X-ray cross-complementing 1 (XRCC1) has been suggested to function at nearly every step of this repair process, primarily through direct protein-protein interactions. Using whole cell extract (WCE) repair assays and DNA damage measurement techniques, we examined systematically the quantitative contribution of XRCC1 to specific biochemical steps of BER and single-strand break repair (SSBR). Our studies reveal that XRCC1-deficient Chinese hamster ovary WCEs exhibit normal base excision activity for 8-oxoguanine (8-OH-dG), 5-hydroxycytosine, ethenoadenine, and uracil lesions. Moreover, XRCC1 mutant EM9 cells possess steady-state levels of endogenous 8-OH-dG base damage similar to those of their wild-type counterparts. Abasic site incision activity was found to be normal in XRCC1-deficient cell extracts, as were the levels of abasic sites in isolated chromosomal DNA from mutant cells. While one- and five-nucleotide gap filling was not affected by XRCC1 status, a significant approximately 2-4-fold reduction in nick ligation activity was observed in EM9 WCEs. Our results herein suggest that the primary biochemical defect associated with XRCC1 deficiency is in the ligation step of BER/SSBR, and that XRCC1 plays no significant role in endogenous base damage and abasic site repair, or in promoting the polymerase gap-filling step.     

Detection of complex DNA damage in gamma-irradiated acute lymphoblastic leukemia Pre-b NALM-6 cells

Bistranded complex DNA damage, i.e., double-strand breaks (DSBs) and non-DSB oxidative clustered DNA lesions, is hypothesized to challenge the repair mechanisms of the cell and consequently the genomic integrity. The oxidative clustered DNA lesions may be persistent and may accumulate in human cancer cells for long times after irradiation. To evaluate the detection and possible accumulation of oxidative clustered DNA lesions in leukemia cells exposed to doses equivalent to those used in radiotherapy, we measured the induction of DSBs and three different types of oxidative clustered DNA lesions in NALM-6 cells, a human acute lymphoblastic leukemia (ALL) pre-B cell line, after exposure to (137)Cs gamma rays. For the detection and measurement of DSBs and oxidative clustered DNA lesions, we used an adaptation of the neutral comet assay (single-cell gel electrophoresis) using E. coli repair enzymes (Endo IV, Fpg and Endo III) as enzymatic probes. We found a linear dose response for the induction of DSBs and oxidative clustered DNA lesions. Clustered DNA lesions were more prevalent than prompt DSBs. For each DSB induced by radiation, approximately 2.5 oxidative clustered DNA lesions were detected. To our knowledge, this is the first study to demonstrate the detection and linear induction of oxidative clustered DNA lesions with radiation dose in an ALL cell line. These results point to the biological significance of clustered DNA lesions.     

Detection of clustered DNA lesions: Biological and clinical applications

Humans are daily exposed to background radiation and various sources of oxidative stress. My research has focused in the last 12 years on the effects of ionizing radiation on DNA, which is considered as the key target of radiation in the cell. Ionizing radiation and endogenous cellular oxidative stress can also induce closely spaced oxidatively induced DNA lesions called "clusters" of DNA damage or locally multiply damage sites, as first introduced by John Ward. I am now interested in the repair mechanisms of clustered DNA damage, which is considered as the most difficult for the cell to repair. A main part of my research is devoted to evaluating the role of clustered DNA damage in the promotion of carcinogenesis in vitro and in vivo . Currently in my laboratory, there are two main ongoing projects. (1) Study of the role of BRCA1 and DNA-dependent protein kinase catalytic subunit repair proteins in the processing of clustered DNA damage in human cancer cells. For this project, we use several tumor cell lines, such as breast cancer cell lines MCF-7 and HCC1937 (BRCA1 deficient) and human glioblastoma cells MO59J/K; and (2) Possible use of DNA damage clusters as novel cancer biomarkers for prognostic and therapeutic applications related to modulation of oxidative stress. In this project human tumor and mice tissues are being used.     

Escherichia coli Fpg glycosylase is nonrendundant and required for the rapid global repair of oxidized purine and pyrimidine damage in vivo

Endonuclease (Endo) III and formamidopyrimidine-N-glycosylase (Fpg) are two of the predominant DNA glycosylases in Escherichia coli that remove oxidative base damage. In cell extracts and purified form, Endo III is generally more active toward oxidized pyrimidines, while Fpg is more active towards oxidized purines. However, the substrate specificities of these enzymes partially overlap in vitro. Less is known about the relative contribution of these enzymes in restoring the genomic template following oxidative damage. In this study, we examined how efficiently Endo III and Fpg repair their oxidative substrates in vivo following treatment with hydrogen peroxide. We found that Fpg was nonredundant and required to rapidly remove its substrate lesions on the chromosome. In addition, Fpg also repaired a significant portion of the lesions recognized by Endo III, suggesting that it plays a prominent role in the global repair of both purine damage and pyrimidine damage in vivo. By comparison, Endo III did not affect the repair rate of Fpg substrates and was only responsible for repairing a subset of its own substrate lesions in vivo. The absence of Endo VIII or nucleotide excision repair did not significantly affect the global repair of either Fpg or Endo III substrates in vivo. Surprisingly, replication recovered after oxidative DNA damage in all mutants examined, even when lesions persisted in the DNA, suggesting the presence of an efficient mechanism to process or overcome oxidative damage encountered during replication.     

Widespread distribution of DNA glycosylases removing oxidative DNA lesions in human and rodent brains

High metabolic activity and low levels of antioxidant enzymes make neurons particularly prone to damage by reactive oxygen species. Thus, repair of oxidative DNA damage is essential for normal brain function. Base excision repair is the major pathway for repair of oxidative DNA damage, and is initiated by DNA glycosylases recognizing and removing the damaged base. In mammalian cells at least five different DNA glycosylases with overlapping substrate specificity, NEIL1, NEIL2, NEIL3, OGG1 and NTH1, remove oxidative DNA base lesions. Here we report mRNA expression and distribution of these five DNA glycosylases in human and rodent brains using in situ hybridization and Northern blotting supported by glycosylase activity assays. NEIL1, NEIL2, OGG1 and NTH1 showed widespread expression at all ages. In situ hybridization studies in mouse brain showed that expression of mNeil1 increased with age. In newborn mouse brain, mNeil3 revealed a discrete expression pattern in brain regions known to harbour stem cell populations, i.e., the subventricular zone, the rostral migratory stream, and the hilar region of the hippocampal formation. Expression of mNeil3 decreased with age, and in old mice brains could be detected only in layer V of neocortex. MNth1 was constitutively expressed during lifespan. In Northern blots, mOgg1 expression showed a transient decrease followed by an increase after 8 weeks of age. Assays for faPy DNA glycosylase activity revealed increased activity level with age in all brain regions analyzed. The widespread but differential expression of the DNA glycosylases recognizing oxidative base lesions suggests distinct and age dependent roles of these enzymes in genome maintenance in brain. The distribution of mNeil3 is particularly intriguing and points to a specific role of this enzyme in stem cell differentiation.