[CANCER RESEARCH 61, 5378 –5381, July 15, 2001] Advances in Brief Repair of 8-Oxodeoxyguanosine Lesions in Mitochondrial DNA Depends on the Oxoguanine DNA Glycosylase (OGG1) Gene and 8-Oxoguanine Accumulates in the Mitochondrial DNA of OGG1-defective Mice Nadja C. de Souza-Pinto, Lars Eide, Barbara A. Hogue, Tanja Thybo, Tinna Stevnsner, Erling Seeberg, Arne Klungland, and Vilhelm A. Bohr 1 Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, Maryland 21224 [N. C. d. S-P., B. A. H., V. A. B.]; Department of Molecular Biology, The National Hospital, University of Oslo, 0027 Oslo, Norway [L. E., E. S., A. K.]; Danish Center for Molecular Gerontology, Aarhus University, DK-8000 Aarhus, Denmark [T. S.]; BioCentrum-DTU, Technical University of Denmark, 2800 Lyngby, Denmark [T. T.] Abstract Mitochondria are not only the major site for generation of reactive oxygen species, but also one of the main targets of oxidative damage. One of the major products of DNA oxidation, 8-oxodeoxyguanosine (8-oxodG), accumulates in mitochondrial DNA (mtDNA) at levels three times higher than in nuclear DNA. The main pathway for the repair of 8-oxodG is the base excision repair pathway initiated by oxoguanine DNA glycosylase (OGG1). We previously demonstrated that mammalian mitochondria from mice efficiently remove 8-oxodG from their genomes and isolated a protein from rat liver mitochondria with 8-oxoguanine (8-oxodG) DNA glycosylase/apurinic DNA lyase activity. In the present study, we demon- strated that the mitochondrial 8-oxodG DNA glycosylase/apurinic DNA lyase activity is the mitochondrial isoform of OGG1. Using mouse liver mitochondria isolated from ogg1 / mice, we showed that the OGG1 gene encodes for the mitochondrial 8-oxodG glycosylase because these extracts have no incision activity toward an oligonucleotide containing a single 8-oxodG DNA base lesion. Consistent with an important role for the OGG1 protein in the removal of 8-oxodG from the mitochondrial genome, we found that mtDNA isolated from liver from OGG1-null mutant ani- mals contained 20-fold more 8-oxodG than mtDNA from wild-type animals. Introduction Mitochondria are the major cellular source of ROS. 2 Calcula- tions based on early observations suggest that up to 5% of the oxygen consumed by the electron transport chain may be converted to ROS by incomplete oxygen reduction (1). Hence, mitochondria are also one of the main cellular targets of ROS-induced oxidative damage, and in fact relatively high levels of oxidized proteins, lipids, and nucleic acids are detected in mammalian mitochondria under normal metabolic conditions (2). The mtDNA is highly prone to oxidative damage because it sits on the inner mitochon- drial membrane in close proximity to the electron transport chain, and indeed, the levels of oxidized bases in mtDNA are two to three times higher than in nuclear DNA (3). 8-oxodG is one of the most prevalent products of the oxidative attack of DNA. It accumulates in mtDNA with age and under certain pathological conditions, e.g., associated with some types of cancer and neurodegenerative diseases. This adduct may be of particular biological relevance because, unless repaired, it induces G:C to T:A transversions with high frequencies (4). Aerobic or- ganisms, from bacteria to humans, have developed sophisticated repair pathways to remove and prevent the formation of 8-oxodG from their genomes. In mammals, BER is the main pathway for the repair of 8-oxodG (5). BER is initiated by a DNA glycosylase, which recognizes and removes the base in a free form by cleavage of the glycosylic bond between the damaged base and the deoxy- ribose residue. The resulting abasic site is cleaved to generate a gap that, after appropriate processing of the termini, will be filled by DNA polymerase and ligated (6). The OGG1 protein is the main DNA glycosylase for the repair of 8-oxodG lesions in DNA. This enzyme is a 38 – 44 KDa protein with functional homology to bacterial formamidopyrimidine glycosylase. OGG1 is a bifunctional DNA glycosylase, with an associated AP lyase activity that cleaves DNA at abasic sites through a -elimination mechanism (7, 8). Mammalian mitochondria efficiently remove 8-oxodG from their genome. Using a Southern blot technique that allows repair rates to be measured in specific parts of the genome, Taffe et al. (9) demon- strated that mitochondria from Chinese hamster ovary cells remove formamidopyrimidine glycosylase-sensitive sites from their DNA at rates comparable to an actively transcribed gene in nuclear DNA. Recently, our laboratory reported the isolation of an oxidative damage endonuclease from rat liver mitochondria, with a molecular mass estimated by gel-filtration chromatography to be between 25 and 30 kDa. This activity was specific for 8-oxodG, with a preference for 8-oxodG:C bp. We also demonstrated that this enzyme is a putative 8-oxodG glycosylase/AP lyase because it can be covalently linked to an 8-oxodG oligonucleotide by sodium borohydride reduction (10). Here, we demonstrate that the mitochondrial 8-oxodG glycosylase/AP lyase activity is attributable to an isoform of OGG1. Using knockout mice for the OGG1 gene, we found that extracts prepared from liver mitochondria isolated from those animals have no incision activity toward an 8-oxodG-containing substrate and that DNA from the mitochondria of such mice has a 20-fold increase in the steady-state levels 8-oxodG. Materials and Methods Materials. HEPES, benzamidine HCl, DTT, BSA, and acrylamide/bisac- rylamide (19:1) were from Sigma Chemical Co. (Saint Louis, MO). Protease inhibitors and UDG were from Boehringer Mannheim (Indianapolis, IN). Isotopes were from NEN Life Science Products (Wilmington, DE). G25 spin columns were from Pharmacia (Peapack, NJ). T4 polynucleotide kinase was from Stratagene (Austin, TX). All other reagents were ACS grade from Sigma Chemical Co. Received 4/17/01; accepted 5/31/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Laboratory of Molecular Gerontology, Box 1, National Institute on Aging, GRC, NIH, 5600 Nathan Shock Drive, Baltimore MD 21224. Phone: (410) 558-8580; Fax: (410) 558-8157; E-mail: vbohr@ nih.gov. 2 The abbreviations used are: ROS, reactive oxygen species; mtDNA, mitochondrial DNA; 8-oxodG, 7,8-dihydro-8-oxodeoxyguanosine; BER, base excision repair; OGG1, oxoguanine DNA glycosylase; AP, apurinic DNA; UDG, uracil DNA glycosylase; MLM, mouse liver mitochondria; HPLC, high-performance liquid chromatography; EC, electro- chemical; wt, wild type; mtUDG, mitochondrial UDG. 5378 Research. on October 28, 2021. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from