In eukaryotic organisms, the vast majority of genetic material resides in the nucleus. However, eukaryotic cells also contain organelles with their own genetic material. One of these organelles is the mitochondrion, in which the cell synthesizes ATP by a reaction known as oxidative phosphorylation. Other DNA-containing organelles are the chloroplasts, which are involved in photosynthesis, and specialized secondary plastids such as the apicoplast of Plasmodium, which are not involved in photosynthesis but are essential to the organism. The long-term goal of our laboratory is to contribute to an understanding of DNA metabolism in organelles. This goal is based on the belief that studies of the organelles' nucleic acid transactions could help us develop rational drug design against diseases such as malaria and Chagas' disease, which are caused by parasitic protozoa with specialized DNA-containing organelles (the apicoplast in Plasmodium falciparum and the kinetoplast in Trypanosoma cruzi). Our investigations also aim to help in understanding the role of mitochondria in human diseases.
Despite the relatively small amount of genomic material in mitochondria, mitochondrial DNA encodes key components of the respiratory machinery. Mutations in the human mitochondrial genome are linked to various inherited diseases, such as Keans-Sayre and Leigh syndromes, progressive external ophthalmoplegia, and Leber hereditary optic neuropathy. These diseases are characterized by rearrangements of or point mutations in mitochondrial DNA. Furthermore, mitochondrial dysfunction may be associated with multifactor diseases, such as diabetes, or complex phenomena, such as aging. Despite the importance of mitochondria in human health, little is known about the mitochondrion's nucleic acid metabolism, in particular replication and transcription. Mitochondrial RNA and DNA polymerases are encoded in the nucleus and imported into the mitochondria, and are both related to single-subunit bacteriophage nucleic acid polymerases.
Mitochondrial DNA polymerase, known as DNA polymerase γ, belongs to the family of DNA A polymerases and contains two domains: a polymerization domain involved in nucleotide addition and an exonuclease domain involved in proofreading. Mitochondrial RNA polymerases are modular proteins containing three motifs involved in catalysis that are shared with family DNA A polymerases.
We believe that mitochondrial DNA transactions occur by mechanisms that are similar to those of the classic bacteriophage T7 system. Bacteriophage T7 contains the simplest replisome fully characterized to date; in this system, three proteins (T7 DNA polymerase, T7 helicase primase, and the T7 single-stranded DNA binding protein) are sufficient to carry out coordinated leading- and lagging-strand DNA synthesis, and a single-subunit RNA polymerase is sufficient to perform regulated transcription. DNA polymerase γ, which is remarkably similar to T7 DNA polymerase, carries out mitochondrial DNA replication; an RNA polymerase that resembles T7 RNA polymerase carries out mitochondrial transcription.
To understand how DNA replication and transcription occur in the mitochondrion, we plan to use the budding yeast Saccharomyces cerevisiae as a model because its mitochondrial DNA replication is similar to that of human transcription systems and because it is amenable to genetic tools. We use a multidisciplinary approach that combines molecular biology, yeast genetics, biochemistry, and X-ray crystallography to address fundamental questions of mitochondrial DNA metabolism.
The mitochondrial theory of aging postulates that accumulated mutations in mitochondrial DNA generate a vicious cycle in which the damaged DNA increases the production of reactive oxidative species, leading to complex phenomena like apoptosis and aging. We are particularly interested in how DNA polymerase γ negotiates DNA lesions such as bulky adducts or those caused by oxidative metabolism and, in our model system, are investigating whether there is a clear correlation between DNA fidelity of mitochondrial genome replication and aging.
We are also interested in understanding the basic mechanisms of mitochondrial transcription. As it is a multistep reaction involving promoter recognition, opening, unwinding, and so forth, we are establishing the kinetics of each transcriptional step and the putative conformational changes that yeast mitochondrial RNA polymerase undergoes during the overall transcription reaction. This is particularly challenging because yeast mitochondrial RNA polymerase has two more elements than T7 RNA polymerase: a transcription factor possibly involved in proper promoter recognition and an N-terminal region known as the Amino Terminal Domain, which is involved in coupling transcription and translation.
Given that the yeast mitochondrial replisome is simpler than the bacteriophage T7 replisome, we argue that mitochondrial RNA polymerase may have alternative roles in replisome assembly. It is believed that T7 RNA polymerase synthesizes initial RNA primers at origins of replication, which are extended by T7 DNA polymerase during leading-strand DNA synthesis. At the lagging strand, T7 primase synthesizes primers that are extended by T7 DNA polymerase in a transient complex called the primosome. We believe that mitochondrial RNA polymerase may synthesize the RNA primers at origins of replication. As a DNA primase has not been identified in S. cerevisiae mitochondria, yeast mitochondrial RNA polymerase may synthesize an RNA that is extended by DNA polymerase γ. We plan to characterize this putative interaction by using biochemical and structural approaches.
In the T7 bacteriophage, the T7 helicase is involved in unwinding the double-stranded DNA and coordinating leading- and lagging-strand DNA synthesis. As a DNA helicase has not been found in S. cerevisiae's mitochondria, we wonder if DNA polymerase γ, in combination with the mitochondrial single-stranded binding protein, is able to displace duplex DNA and thus obviate the need for a DNA helicase in S. cerevisiae'smitochondria.
We expect that understanding the basic mechanisms of mitochondrial DNA metabolism will help us to elucidate mitochondrial DNA maintenance and, in the future, facilitate genetic interventions against mitochondrial diseases.
Last updated September 2010
