Mitochondrial and nuclear DNA base excision repair are affected differently by caloric restriction

Abstract

Aging is strongly correlated with the accumulation of oxidative damage in DNA, particularly in mitochondria. Oxidative damage to both mitochondrial and nuclear DNA is repaired by the base excision repair (BER) pathway. The "mitochondrial theory of aging" suggests that aging results from declining mitochondrial function, due to high loads of damage and mutation in mitochondrial DNA (mtDNA). Restriction of caloric intake is the only intervention so far proven to slow the aging rate. However, the molecular mechanisms underlying such effects are still unclear. We used caloric-restricted (CR) mice to investigate whether lifespan extension is associated with changes in mitochondrial BER activities. Mice were divided into two groups, receiving 100% (PF) or 60% (CR) of normal caloric intake, a regime that extends mean lifespan by approximately 40% in CR mice. Mitochondria isolated from CR mice had slightly higher uracil (UDG) and oxoguanine DNA glycosylase (OGG1) activities but marginally lower abasic endonuclease and polymerase gamma gap-filling activities, although these differences were tissue-specific. Uracil-initiated BER synthesis incorporation activities were significantly lower in brain and kidney from CR mice but marginally enhanced in liver. However, nuclear repair synthesis activities were increased by CR, indicating differential regulation of BER in the two compartments. The results indicate that a general up-regulation of mitochondrial BER does not occur in CR.

Activities of DNA base excision repair enzymes in liver and brain correlate with body mass, but not lifespan

“…Tissue was powdered using a mortar and pestle and rinsed twice in 1× PBS. Subsequent steps in nuclear isolation were essentially as described in Stuart et al (2004). Powdered tissue was further minced in 20 volumes of MSHE buffer (0.21 M mannitol, 70 mM sucrose, 10 mM HEPES pH 7.4, 1 mM EGTA, 2 mM EDTA, with 5 mM DTT and 1 μg/ml aproptinin added immediately prior to use) and homogenized with ten strokes of a PotterElvejhem homogenizer.…”

“…Gels were visualized with PhosphoImager (Fujifilm FLA-3000) and analyzed with ImageGuage TM software. APE activity was quantified as the intensity of the product bands relative to the combined intensity of the product and substrate bands, essentially as in Stuart et al (2004). Polβ-mediated nucleotide incorporation was measured by incubating 1 μg nuclear extract protein with 1 pmol of an oligonucleotide with a single nucleotide gap (GAP ; Table 2) for 10, 20, 30 min at 37°C, in a total volume of 10 μl reaction buffer (40 mM HEPES, 0.1 mM EDTA, 5 mM MgCl 2 , 0.2 mg/ml BSA, 50 mM KCl, 1 mM DTT, 40 mM phosphocreatine, 100 μg/ml phosphocreatine kinase, 3% glycerol, 2 mM ATP, 40 μM dCTP, and 4 μCi [α-32 P]dCTP).…”

“…While there is evidence that DNA BER is upregulated concomitantly with increased longevity in mice (e.g., Cabelof et al 2003b;Stuart et al 2004;Kisby et al 2010), other results question the relationship between BER and longevity. For example, ∼50% reduction in either APE or polβ activity is not associated with reduced lifespan in unstressed mice (Meira et al 2001;Cabelof et al 2006).…”

Mitochondrial DNA repair in aging and disease

“…Caloric restriction has been reported to lower ROS generation in brain mitochondria (88,89) and prevent age-dependent accumulation of 8-oxoG in rat brain (90,91). Calorie restricted mice were used to investigate whether lifespan extension is associated with changes in mitochondrial BER activities (92). The most significant effect of caloric restriction was a reduction in uracil-initiated BER activity in brain and kidney mitochondria, while this activity in liver was maintained or perhaps slightly enhanced.…”

“…The most significant effect of caloric restriction was a reduction in uracil-initiated BER activity in brain and kidney mitochondria, while this activity in liver was maintained or perhaps slightly enhanced. The authors propose that mitochondrial polymerase γ might be involved in regulation of mitochondrial BER activity in calorie restricted mice (92). Since polymerase γ is the only known polymerase in mitochondria and is responsible for both repair and replication, it is reasonable to expect that it is involved in aging associated repair of mtDNA damage.…”