Age-dependent deficiency in import of mitochondrial DNA glycosylases required for repair of oxidatively damaged bases

Abstract

The mitochondria are the major source of chronic oxidative stress, which has been implicated in the aging process. Along with other cellular changes, aged cells accumulate mutations in both their nuclear and mitochondrial genomes, and they contain increased amounts of oxidatively damaged mutagenic bases such as 7,8-dihydro-8-oxoguanine, suggesting age-dependent inhibition of its repair. Surprisingly, the level and activity of 8-oxoguanine-DNA glycosylase (OGG1), responsible for repair of 7,8-dihydro-8-oxoguanine, was found to be higher in the liver mitochondrial extract from old rodents than in that from young ones. We addressed this paradox by analyzing OGG1 in the mitochondria of young vs. old mouse livers, as well as in replicating vs. presenescent human fibroblasts. We show here that although the total OGG1 activity is higher in old mitochondria, a large fraction of the enzyme is stuck to the membrane in the precursor form, which could not be translocated to and processed in the mitochondrial matrix. A nearly identical phenomenon was observed with the mitochondrial uracil-DNA glycosylase responsible for repair of mutagenic uracil. These results indicate an age-dependent decline in the mitochondrial import of proteins needed for DNA repair and possibly for other functions.

Fig. 1.

Age-dependent change in OGG1-α. (A) Western blot analysis of OGG1-α polypeptide levels in nuclei of 4- and 20-mo-old mouse livers. Two animals were used for each age group. The blots were reprobed with antilamin b Ab. (B) OGG1-α activity in liver nuclear extracts (15, 30, and 45 μg for each) of 4- and 20-mo-old mice. (C) The average activity for six animals of each group relative to the young animal. (D) Western analysis of OGG1-α levels in nuclear extracts (in duplicate) from proliferating (PDL 41) and presenescent (PDL 78) MRC5 cells. Other details are given in Materials and Methods.

Fig. 2.

Western blot analysis of precursor and mature OGG1-β in mitochondria and submitochondrial fractions of young and old mouse livers. (A) Identification of 38-kDa OGG1-α and 45-kDa OGG1-β in the mitochondrial extract (ME) and nuclear extract (NE), respectively, from 4-mo-old mouse livers. Lamin b and cytochrome c were analyzed in these extracts to confirm the lack of cross-contamination between nuclear and mitochondrial preparations. (B) Presence of 47-kDa OGG1-β precursor in mitochondrial extracts of 20-mo-old mouse livers. (C) Presence of OGG1-β precursor in high PDL (>50) MRC5 cells. Mitochondrial extracts from MRC5 cells collected at various PDL were analyzed for OGG1.

Fig. 3.

Sensitivity of mitochondrial OGG1-β to trypsin. (A) Presence of precursor OGG1-β in OM/IMS and its sensitivity to trypsin. The precursor and mature OGG1-β bands are indicated. (B) Trypsin sensitivity of OGG1-β in presenescent PDL 77 vs. PDL 41 MRC5 cells. The 47-kDa OGG1-β precursor was not separated from the mature 45-kDa band. Cytochrome c oxidase subunit IV (COX IV) analyzed in the same blot served as an internal control.

Fig. 4.

Effect of mitochondrial trypsin treatment on OGG1-β activity. (A) 8-oxoG excision activity in extracts (50 μg each) of mitochondria pretreated with or without trypsin. Probe indicates oligonucleotide substrate without extract. (B) The average OGG activity from six animals expressed relative to untreated samples is shown in a bar graph.

Fig. 5.

Effects of trypsin on UDG activity associated with mitochondria from young and old mice. Uracil excision activity was measured, as described in Materials and Methods, with extracts (15 and 30 μg) of mitochondria with or without trypsin pretreatment from young (A) and old (B) mouse livers. The relative activity is shown in C. (D) Cytochrome c oxidase activity in mitochondrial extracts from young and old mouse livers.