Mitochondrial DNA polymerase (PolG)
Mitochondria contain their own DNA (mtDNA) which encodes a small number of essential proteins of the respiratory complexes. Each cell contains a large number of identical mtDNA molecules, which are replicated by a specific DNA polymerase, Polg, encoded in the nucleus and imported into the mitochondria. The amino acid sequence of Polg is conserved from yeast to human. In 1989, we have cloned and sequenced the yeast Polg gene (MIP1) and shown that Mip1 is an accurate enzyme playing a crucial role in the fidelity and stability of the mtDNA, in particular through its 3'-5' exonuclease proofreading activity. The loss of the proofreading activity increases the mtDNA mutation rate more than 1000 fold.
Since 2001, more than 80 Polg alleles have been reported as pathogenic in humans, with a broad spectrum of clinical presentations. We are using Saccharomyces cerevisiae as a model organism to generate and study mip1 mutations whose equivalents in humans are potentially pathogenic. Genetical and biochemical approaches are conducted. We hope to establish a correlation between mutations and clinical presentations based on the in vivo and in vitro phenotypes observed in yeast.
Accumulation of mutations in mtDNA is believed as being a cause of ageing. Using in vivo and in vitro random mutageneses in yeast, we are searching for antimutators of mtDNA in the MIP1 gene or another gene. We are expecting that this work will constitute a molecular basis for transgenic mouse studies on the role of these alleles in ageing.
Frataxin and iron-sulfur metabolism
Friedreich ataxia, the most common ataxia, is a severe neurodegenerative disease often asociated with cardiomyopathy with a prevalence of one individual out of 50, 000. It is caused by decreased levels of frataxin, a small protein conserved during evolution and localizing to mitochondria in eukaryotes. The function of frataxin, which plays an important role in the biosynthesis of iron-sulfur clusters, is a matter of intense debate.The three dimentional structure of yeast, bacterial and human frataxins is well conserved and is unique.
Frataxin is characterized by a large beta-sheet platform supported by two alpha helices, and an acidic ridge at the surface of the protein.
Frataxin is a highly acidic protein binding iron in vitro through acidic residues on helix 1 and strand 1
A drastic change in the electrostatic properties of yeast frataxin Yfh1 surface by replacing four acidic residues by alanines or two acidic residues by lysines impair the function of frataxin. Wild-type (left), D86A/E89A/D101A/E103A (middle), D86K/E103K (right) mutants. Acidic, neutral and basic residues are in red, white and blue, respectively.
We use yeast frataxin mutants to understand the roles of the acidic ridge and the beta-sheet platform in the binding of iron and interaction with the protein complex that consists of the scaffold Isu protein, that makes the assembly of iron and sulfur, and the cysteine desulfurase that provides the sulfur atom.