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Detection of DNA base‐excision repair activity for oxidative lesions in adult rat brain mitochondria
Author(s) -
Chen Dexi,
Lan Jing,
Pei Wei,
Chen Jun
Publication year - 2000
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/1097-4547(20000715)61:2<225::aid-jnr13>3.0.co;2-0
Subject(s) - base excision repair , ap site , dna repair , dna ligase , mitochondrion , biology , dna damage , nucleotide excision repair , mitochondrial dna , biochemistry , microbiology and biotechnology , xrcc1 , dna , oxidative phosphorylation , gene , genotype , single nucleotide polymorphism
Abstract Endogenous oxidative damage to brain mitochondrial DNA and consequential disturbances of gene expression and mitochondrial dysfunction have long been implicated in aging and the pathogenesis of neurodegenerative diseases. It has yet to be determined, however, whether mitochondria in brain cells contain an active DNA repair system and, if so, how this system functions. Therefore, the capacity for the repair of defined types of oxidative DNA lesions has been investigated in adult rat brain mitochondria. Using in vitro DNA incorporation repair assay, we have detected base excision repair (BER) activity for the common oxidative DNA adduct 8‐hydroxyl‐2′‐deoxyguanine (8‐oxodG) in mitochondria protein extracts from cortical tissues and cultured primary cortical neurons and astrocytes. The levels of BER activity were both protein concentration‐dependent and repair‐incubation time‐dependent. To resolve the BER pathway, the activity of essential BER enzymes was examined in mitochondria using oligonucleotide incision assay, DNA polymerase assay, and DNA ligase assay employing specific DNA substrates. Mitochondrial extracts were able to remove specifically 8‐oxodG, uracil, and the apurinic/apyrimidinic abasic site from substrates. Moreover, a gamma‐like DNA polymerase activity and a DNA ligase activity were detected in mitochondiral extracts, based on the formation of specific repair products. These results demonstrate that adult brain mitochondria possess an active BER system for repairing oxidative DNA lesions. This repair system appears to function by sequential actions of DNA repair enzymes that are homologous to, but not identical to, that in the nucleus. Thus, BER may represent an endogenous protective mechanism against oxidative damage to mitochondrial, as well as nuclear, genomes in brain cells. J. Neurosci. Res. 61:225–236, 2000. © 2000 Wiley‐Liss, Inc.