Mitochondrial Physiology Network 10.9: 125-126 (2005) - download pdf

Mitochondrial DNA replication and mitochondrial activity in mammals.

Mirella Trinei^1,2, M Giorgio^1,2, C Lucca¹, M Foiani¹, PG Pelicci^1,2

¹IFOM, via Adamello 16 Milano Italy; ²IEO via Ripamonti 435 Milano, Italy. - mirella.trinei@ifom-ieo-campus.it

    Mitochondria are the main intracellular source and immediate target of reactive oxygen species (ROS) which are continually generated as byproducts of aerobic metabolism in mammalian cells. Cumulative damage to  mtDNA is implicated in the aging process and in the progression of such common diseases as diabetes, cancer and heart failure.

    It has been observed that increase in the mtDNA copy number is associated with elevated oxidative stress in the aging tissues like brain and skeletal muscle. MtDNA  copy number is increased at the late passage of diploid human fibroblasts. The increase in mtDNA with aging is proposed as a feedback response that could compensate the accumulation of defective mitochondria and is also associated to increased intracellular levels of ROS [1]. We studied the effect of ROS on mtDNA replication using two-dimensional agarose gel electrophoresis of replication intermediates (RIs) [2].

    We analyzed the pattern of RIs with DNA digestions which allow  the detection of intact bubble arcs in mouse mtDNA in isolated mitochondria from mouse liver and in primary mouse fibroblasts treated with different mitochondrial drugs. We found that  increased ROS production by rotenone and antimycin A treatment directly affects mitochondrial replication activity, decreasing the percentage of intact bubbles while DNP increases mtDNA replication. These data suggest that interfering with mitochondrial electron transfer, altering mitochondrial membrane potential and modifying ROS production affect directly mtDNA replication. Since our data suggest an inverse relationship between mitochondrial activity and mtDNA replication we investigated if there is any effect at structural levels. We used confocal microscopy to detect mtDNA with anti-mtSSB (mitochondrial single strand binding proteins) and mitochondrial electron transfer complexes with anti-COX IV. With this approach we found that mitochondria show a different localization for DNA and the respiratory chain complexes suggesting a specific sub-mitochondrial compartmentalization whose organization may be crucial for mitochondrial physiology.

     This work was supported by F.I.R.C.

1.  Lee HC, Wei YH (2005) Mitochondrial biogenesis and mitochondrial DNA maintenance of mammalian cells under oxidative stress. Int. Biochem. Cell. Biol. 37: 822-834.

2.  Bowmaker M, Yang MY, Yasukawa T,  Reyes A, Jacobs HT, Huberman JA, Holt IJ (2003) Mammalian mitochondrial DNA replicates bidirectionally from an initiation zone. J. Biol. Chem. 19: 50961-50969.