Mitochondrial dysfunction and mutations in human mitochondrial mtDNA can be linked to several diseases and disorders. Some examples are cancer or neurological diseases such as Parkinson’s disease, and Alzheimer’s dementia. The function of several organs (e.g. heart, kidney, liver, eyes, ears, hair, and skin) may be compromised by mitochondrial dysfunction. Even the natural aging process in mammals is characterized by mutations in mtDNA.

The main function of mitochondria is to provide eukaryotic cells with cellular energy. Despite the importance of the mtDNA genome, it is rather small. The genome only encodes thirteen essential proteins of the oxidative phosphorylation complexes, 22 transfer RNAs, and 2 ribosomal RNAs, all of which are required to translate these thirteen mRNAs in the mitochondrial matrix. The function of mtDNA can be affected by base modifications, point mutations, deletions and insertions, and, under certain conditions, mtDNA may also be completely lost. The resulting phenotypes are caused mostly by environmental carcinogens, reactive oxygen species, and mutated or missing genetic factors.

Previously, during my post-doctoral studies in the laboratory of Dr. Virginia Zakian at Princeton University, Princeton, NJ, I identified a DNA helicase, Rrm3, that facilitates replication fork movement past non-histone, multi-protein complexes that are bound to specific sites on yeast chromosomes. Such sites are present within ribosomal DNA (see figure below), telomeres, centromeres, transfer RNA genes, and inactive replication origins.

Replication of DNA is visualized using a two-dimensional neutral/neutral agarose gel electrophoresis technique. This technique separates branched DNA molecules, such as replication intermediates, from linear, non-replicating DNA molecules. In the absence of the DNA helicase Rrm3, replication forks slow down at specific locations within yeast ribosomal DNA (rDNA) as indicated in the cartoon in panel B. Examples of such locations are at the 5S or 35S rRNA genes, and at inactive replication origins. This effect can be seen in the above figures. For this purpose, yeast genomic DNA was cut with restriction enzymes and separated by agarose gel-electrophoresis according to mass (labeled as 1 in panel A). Individual agarose gel slices containing the cut genomic DNA were transferred to a separate electrophoresis gel-tray, and followed by separation of the DNA in a second dimension agarose gel under conditions that separate DNA according to mass and shape (labeled as 2 in panel A). The resulting agarose gel is processed using the Southern transfer technique. The individual replication intermediates are depicted as different triangular shaped structures in panel A. The arrows above the structures indicate the direction of fork movement. As shown in panel B, replication forks may slow down at specific locations on the chromosomes, which are indicated by the darker hybridization areas.
Abbreviations: specific non-replicated DNA fragment (1N); almost fully replicated DNA fragment (2N); replication fork barrier (RFB); autonomous replicative sequence (ARS); 5S rRNA gene (5S); 35S rRNA gene (35S).