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CR counteracts the age-related increase in mitochondrial abasic sites

Posted Nov 21 2008 10:11am

In addition the 23 chromosome nuclear genome most of us are familiar with, mitochondria contain their own, distinct genome. Each mitochondrion contains several copies of this genome, and most cell types contain hundreds of mitochondria per cell. In contrast to the linear nuclear chromosomes, the mitochondrial genome is circular; contains about 16,500 bases, compared to ~3 billion bases in the nuclear genome; and encodes 13 protein-coding genes, compared to ~25,000 protein-coding genes in the nuclear genome. Other characteristics of the mitochondrial genome provide the basis for the endosymbiotic theory.

Lezza et al. measured the number of abasic sites (AP, referring to apurinic and apyrimidinic) in the mitochondrial genome of young and aged rat brain. AP sites are positions along the DNA backbone where no adenine, guanine, cytosine, or thymine is attached; they are among the most frequent damages to DNA.  The authors showed that in normally feeding animals, the number of AP sites increases with age — but calorie restricted (CR) mice did not show such an increase.

Localization of Abasic Sites and Single-Strand Breaks in Mitochondrial DNA from Brain of Aged Rat, Treated or not with Caloric Restriction Diet.

According to the “mitochondrial theory of aging” the lifelong accumulation of various kinds of damage to mitochondrial DNA (mtDNA) has been related to the age-dependent mitochondrial bioenergetic dysfunction. Caloric restriction (CR) diet is able to prevent or delay the onset of several age-related damages to mtDNA. The effects of aging and CR on the presence of abasic sites and single-strand breaks of the sugar-phosphate backbone in mtDNA have been analyzed by applying Ligation Mediated-PCR to a H strand region of brain mtDNA from young and old ad libitum-fed and old CR-treated rats. The region, encompassing the Direct Repeat 1 of the 4,834 bp-long deletion, is highly damaged in the old ad libitum-fed animals with respect to the young ones, whereas in the CR rats it shows a much lower extent of damage. The data confirm, at single nucleotide resolution, the protective effect of CR on the age-related mtDNA damage.

The “various kinds of damage” include those caused by reactive oxygen species (ROS), which are a byproduct of oxidative phosphorylation in the mitochondria. ROS have been shown to cause point mutations, deletions, single and double stranded breaks, base oxidation, and AP sites in DNA. Because mitochondria are the major source of ROS, mtDNA is especially susceptible to oxidative damage. There is evidence that the mitochondrial DNA (mtDNA) repair system is less robust than the nuclear genome repair systems. Because the genes on the mitochondrial genome encode proteins for the all important electron transport chain, mtDNA damage may play a role in aging and age-related diseases.

The researchers examined a 33 base pair region surrounding the Direct Repeat 1, which is a hotspot for mtDNA damage and deletions. They measured the number of AP sites in mtDNA using ligation-mediated PCR, which is a genome sequencing technique that can distinguish between cut and uncut DNA. The mtDNA was treated with sodium hydroxide, which cuts DNA specifically at AP sites.

Mitochondrial DNA from 6 month old rats fed ad libitum was compared to mtDNA from 26 month old rats fed either 40% CR for most of their life or ad libitum. Young rats’ mtDNA exhibited fewer AP sites per genome. In the old ad libitum -fed rats, 19 of 33 positions were abasic at a high frequency, with up to 21 AP sites found in any one old ad libitum -fed rat. Any one old CR rat had a maximum of 13 AP sites, with 12 of these AP sites shared by all CR old rats. These sites occurred at a much lower frequency than in the old rats fed ad libitum

CR decreases ROS production in aged rat brains, and this decrease might be responsible for the decrease in AP sites.

      
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