For many years, it was assumed that damage to mtDNA was not repaired but that damaged molecules were simply targeted for disposal. However, we know very little regarding the processes for selectively degradation of damaged mtDNA or regulated turnover of whole mitochondria. In the early 1990’s the Wilson and Bohr laboratories showed using Southern blotting methods that some types of damage to mtDNA can be repaired. The Bogenhagen laboratory was the first to show that a set of proteins purified from mitochondria are capable of conducting complete base excision repair of the sort of DNA lesions generated by oxidative damage (Bogenhagen, 1999). We are continuing studies on the mechanisms of DNA repair that operate in mitochondria. Our proteomics research developed from the observations in our lab and others that a number of mitochondrial DNA repair proteins are products of nuclear genes that provide products to both the nucleus and mitochondria. We have suggested that this situation may lead to a deficiency in mtDNA repair proteins in post-replicative tissues such as nerves and muscles, exactly the cell types that experience the highest rates of somatic mtDNA mutations leading to mitochondrial diseases.

My laboratory has been applying protein sequencing technologies to permit us to compare the mitochondrial proteomes in pairwise sets of cells or tissues. We intend to compare the spectrum of mitochondrial proteins expressed in rapidly dividing embryonal carcinoma (EC) cells to that of terminally differentiated cell types derived from the EC cells by standard hormonal treatment. Similarly, we are interested in comparing the mitochondrial proteomes of cells with and without oxidative stress. A final example of this approach is to compare breast cancer cell lines that do or do not express high levels of PPAR γ and PGC1. These comparisons will use a combination of 2D-gel methods and multidimensional chromatography with iTRAQ technology and LC-MS/MS.

A second proteomic project is to determine the protein components of the mtDNA nucleoid. We consider that it is important to understand the composition of this nucleoprotein structure, since it is considered the fundamental unit of inheritance of mtDNA in cells. We have characterized a number of proteins associated with mtDNA that appear to mediate interaction with the inner mitochondrial membrane.

An important part of our effort is to develop a comprehensive SQL database of mitochondrial proteins. This database will be unique in that it will provide a concordance to relate human mitochondrial proteins to their homologs in a number of model organisms including mouse, rat, Xenopus, C. elegans, Drosophila and yeast. Protein families will be organized using GO terminology and evidence codes tracked to the Pubmed literature database. A unique feature of this database is that it will attempt to track major publications of mitochondrial protein sequencing efforts in the selected model organisms to keep track of which proteins have been documented in which proteomic studies.

Our recent description of the structure of the accessory subunit of mtDNA polymerase γ (Carrodeguas et al., 2001) is the first step in building a database of structural information on key players in mtDNA replication and transcription. Efforts to extend this to a structure for the DNA pol γ holoenzyme are in progress. This requires expression of the catalytic subunit in insect cells infected with a recombinant baculovirus and expression of the accessory subunit in bacteria. The two proteins are reassembled into a holoenzyme of nearly 250 kDa and purified for crystallography trials. Knowledge of the structure of DNA pol γ will be important for efforts to reconstitute replication of mtDNA. Since DNA pol γ is a major target of the toxicity of nucleoside inhibitors of HIV reverse transcriptase, knowledge of this structure will be valuable to guide rational design of better drugs for AIDS therapy.