An Oxidative Damage-specific Endonuclease from Rat Liver Mitochondria

Abstract

Reactive oxygen species have been shown to generate mutagenic lesions in DNA. One of the most abundant lesions in both nuclear and mitochondrial DNA is 7,8-dihydro-8-oxoguanine (8-oxoG). We report here the partial purification and characterization of a mitochondrial oxidative damage endonuclease (mtODE) from rat liver that recognizes and incises at 8-oxoG and abasic sites in duplex DNA. Rat liver mitochondria were purified by differential and Percoll gradient centrifugation, and mtODE was extracted from Triton X-100-solubilized mitochondria. Incision activity was measured using a radiolabeled double-stranded DNA oligonucleotide containing a unique 8-oxoG, and reaction products were separated by polyacrylamide gel electrophoresis. Gel filtration chromatography predicts mtODE's molecular mass to be between 25 and 30 kDa. mtODE has a monovalent cation optimum between 50 and 100 mM KCl and a pH optimum between 7.5 and 8. mtODE does not require any co-factors and is active in the presence of 5 mM EDTA. It is specific for 8-oxoG and preferentially incises at 8-oxoG:C base pairs. mtODE is a putative 8-oxoG glycosylase/lyase enzyme, because it can be covalently linked to the 8-oxoG oligonucleotide by sodium borohydride reduction. Comparison of mtODE's activity with other known 8-oxoG glycosylases/lyases and mitochondrial enzymes reveals that this may be a novel protein.Reactive oxygen species are generated in cells as a by-product of cellular respiration. Reactive oxygen species react with proteins, lipids and DNA causing cellular damage. When DNA is the target, a variety of DNA adducts are formed. Among these, 8-oxoG is one of the most abundant lesions generated (1, 2). 8-oxoG 1 is considered to be a premutagenic lesion because it can mispair with adenine during DNA replication (3-5). In the absence of DNA repair, this mispairing results in G to T transversion mutations. Since many reactive oxygen species are generated by oxidative processes that occur in mitochondria, it is of great interest to understand the oxidative DNA damage processing in these organelles.Mitochondrial DNA (mtDNA) is composed of a 16.5-kilobase pair circular genome, encoding 13 structural genes, 22 tRNAs, and two rRNAs. The DNA lies in close proximity to the free radical-producing electron transport chain, and it has been reported that mtDNA contains a higher level of oxidative DNA damage than nuclear DNA (6). Since mtDNA is subjected to relatively high levels of oxidative damage, mitochondria need DNA repair mechanisms to maintain their DNA genomes.Mitochondrial DNA repair mechanisms differ from those in the nucleus. Evidence to suggest that mitochondria lacked DNA repair mechanisms came from the observation that UV damage is not repaired in mitochondria (7, 8), while it is efficiently processed in the nucleus by nucleotide excision repair. In addition, damage caused by cisplatin and nitrogen mustard, agents that are known to induce DNA adducts that are substrates for the nuclear nucleotide excision repair pathway, is inefficiently repaired in m...

Purification and characterization of a mammalian homolog of Escherichia coli MutY mismatch repair protein from calf liver mitochondria

“…Monoamine oxidase activity was located in the outer membrane fraction whereas the majority of the malate dehydrogenase activity was detected in the matrix fraction (Table 1). A similar overflow of marker enzyme activities were found present in the sub-mitochondrial preparations of others (38) and may be due in part to the presence of other enzymes utilizing the substrates. Nevertheless, the location of the marker enzymes showed that the fractionation was effective.…”

“…It has been shown that DNA lesions caused by oxidative damage, in particular 8-oxoG, induced in Chinese hamster ovary cells are rapidly removed from the mitochondrial genome suggesting the presence of a 8-oxoG glycosylase/AP lyase (OGG1) (37). Croteau et al partially purified a 25-30 kDa base excision endonuclease that preferentially cleaved C/8-oxoG mismatches but not A/G or A/8-oxoG (38). These OGG1 or MutM-like activities are consistent with several processed forms of OGG1 enzyme being localized to the mitochondrion from a single gene (39).…”

Mitochondrial DNA repair in aging and disease

“…Mitochondrial BER has been demonstrated by numerous groups and shown to target oxidatively modified DNA bases, such as 8-oxoG and thymine glycol (29)(30)(31)(32)(33)(34)36,37). DNA repair enzymes isolated from mitochondria include several types of damage-specific DNA glycosylases (36)(37)(38)(39), AP endonuclease (40), and DNA ligase (41,42).…”

“…DNA repair enzymes isolated from mitochondria include several types of damage-specific DNA glycosylases (36)(37)(38)(39), AP endonuclease (40), and DNA ligase (41,42). DNA glycosylases that are expressed as nuclear and mitochondrial isoforms encoded by the same gene, include uracil DNA-glycosylase (UDG) (43), and 8-oxoG DNA glycosylase/AP lyase (OGG) (44).…”

Mitochondria, oxidative DNA damage, and aging

“…Several other repair enzymes have been detected in mammalian mitochondda, including a methyltransferase (134), an AP endonuclease (159), and an 8-oxo-dG glycosylase termed mtODE, for mitochondrial oxidative damage endonuclease (160). The latter enzyme excises 8-oxo-dG when it is paired with dC, but not when it is paired with dA.…”