Biomedical subjects

S Vanderstraeten

Publications and source records attributed to S Vanderstraeten.

4 recordsLinked to original sources

PubMed2004

Mitochondrial DNA mutators.

In this article we review our current knowledge of the mechanisms by which point mutations arise in the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae and discuss to what extent these mechanisms operate in human mtDNA mutagenesis. The 3'-5' exonuclease proofreading activity of Pol gamma ensures accuracy of mtDNA replication in both yeast and humans, while the role of base excision repair in mtDNA error avoidance remains debated. The mitochondrial mismatch repair Msh1 protein, which removes transitions in yeast, is absent in humans, a particularity that might cause accumulation of transitions, while the most frequent substitution in yeast mtDNA is A:T to T:A transversion. Proofreading-deficient mutator human cell lines and knockin mice have been created. They will be useful for studying the mechanisms by which mtDNA mutations accumulate in diseases, ageing, malignancy and drug therapy.

Animals↗

PubMed1998

The role of 3'-5' exonucleolytic proofreading and mismatch repair in yeast mitochondrial DNA error avoidance.

In the D171G/D230A mutant generated at conserved aspartate residues in the Exo1 and Exo2 sites of the 3'-5' exonuclease domain of the yeast mitochondrial DNA (mtDNA) polymerase (pol-gamma), the mitochondrial genome is unstable and the frequency of mtDNA point mutations is 1500 times higher than in the wild-type strain and 10 times higher than in single substitution mutants. The 10(4)-fold decrease in the 3'-5' exonuclease activity of the purified mtDNA polymerase is associated with mismatch extension and high rates of base misincorporation. Processivity of the purified polymerase on primed single-stranded DNA is decreased and the Km for dNTP is increased. The sequencing of mtDNA point mutations in the wild-type strain and in proofreading and mismatch-repair deficient mutants shows that mismatch repair contributes to elimination of the transitions while exonucleolytic proofreading preferentially repairs transversions, and more specifically A to T (or T to A) transversions. However, even in the wild-type strain, A to T (or T to A) transversions are the most frequent substitutions, suggesting that they are imperfectly repaired. The combination of both mismatch repair and proofreading deficiencies elicits a mitochondrial error catastrophe. These data show that the faithful replication of yeast mtDNA requires both exonucleolytic proofreading and mismatch repair.

Base Sequence↗

PubMed1995

Isolation and characterization of ten mutator alleles of the mitochondrial DNA polymerase-encoding MIP1 gene from Saccharomyces cerevisiae.

Ten mutator alleles of MIP1, the gene encoding mitochondrial (mt) DNA polymerase, have been isolated after in vitro random mutagenesis. Five mutations causing a 100-400-fold increase in the frequency of erythromycin-resistant (ErR) mt mutants in yeast mapped to the 3'-5' exonuclease (Exo) domain, and mainly to the three conserved motifs Exo1, Exo2 and Exo3 of this domain, highlighting the importance of proofreading in accurate mt DNA replication. The essential role of the invariant glutamate at the Exo1 site was confirmed and the participation of four amino acids (aa) in the 3'-5' Exo function revealed. Another mutation that is located between the Exo1 and Exo2 sites produced an extremely strong mutator phenotype associated with impaired DNA replication, but could be assigned neither to a conserved aa nor to a conserved portion of the 3'-5' exonuclease domain. The importance of the polymerization domain in accurate mt DNA replication was pointed out by three mutator mutations. Two of these severely impaired mt DNA replication and were assigned to a subdomain of the polymerase which probably corresponds to the 'fingers' module of the Klenow (large) fragment of Escherichia coli DNA polymerase I (PolIk). The third, which did not alter the efficiency of DNA replication, was located at the active center of the polymerization reaction. Finally, the mutation, R1001I, mapped to the C-terminal part of the MIP1 protein which has no counterpart in prokaryotic DNA polymerases.

Alleles↗

PubMed1992

Yeast mitochondrial DNA mutators with deficient proofreading exonucleolytic activity.

The MIP1 gene which encodes yeast mitochondrial DNA polymerase possesses in its N-terminal region the three motifs (Exo1, Exo2 and Exo3) which characterize the 3'-5' exonucleolytic domain of many DNA polymerases. By site directed mutagenesis we have substituted alanine or glycine residues for conserved aspartate residues in each consensus sequence. Yeast mutants were therefore generated that are capable of replicating mitochondrial DNA (mtDNA) and exhibit a mutator phenotype, as estimated by the several hundred-fold increase in the frequency of spontaneous mitochondrial erythromycin resistant mutants. By overexpressing the mtDNA polymerase from the GAL1 promoter as a major 140 kDa polypeptide, we showed that the wild-type enzyme possesses a mismatch-specific 3'-5' exonuclease activity. This activity was decreased by approximately 500-fold in the mutant D347A; in contrast, the extent of DNA synthesis was only slightly decreased. The wild-type mtDNA polymerase efficiently catalyses elongation of singly-primed M13 DNA to the full-length product. However, the mutant preferentially accumulates low molecular weight products. These data were extended to the two other mutators D171G and D230A. Glycine substitution for the Cys344 residue which is present in the Exo3 site of several polymerases generates a mutant with a slightly higher mtDNA mutation rate and a slightly lower 3'-5' exonucleolytic activity. We conclude that proofreading is an important determinant of accuracy in the replication of yeast mtDNA.

Amino Acid Sequence↗