mip1 containing mutations associated with mitochondrial disease causes mutagenesis and depletion of mtDNA in Saccharomyces cerevisiae.
DNA polymerase gamma (pol gamma) is responsible for replication and repair of mitochondrial DNA (mtDNA). Over 150 mutations in POLG (which encodes pol gamma) have been discovered in patients with mitochondrial disorders including Alpers, progressive external ophthalmoplegia and ataxia-neuropathy syndrome. However, the severity and dominance of many POLG disease-associated mutations are unclear, because they have been reported in sporadic cases. To understand the consequences of pol gamma disease-associated mutations in vivo, we identified dominant and recessive changes in mtDNA mutagenesis, depletion and mitochondrial dysfunction caused by 31 mutations in the conserved regions of the gene, MIP1, which encodes the Saccharomyces cerevisiae ortholog of human pol gamma. Twenty mip1 mutant enzymes were shown to disrupt mtDNA replication and may be sufficient to cause disease. Previously uncharacterized sporadic mutations, Q308H, R807C, G1076V, R1096H and S1104C, caused decreased polymerase activity leading to mtDNA depletion and mitochondrial dysfunction. We present evidence showing a limited role of point mutagenesis by these POLG mutations in mitochondrial dysfunction and disease progression. Instead, most mitochondrial defective mip1 mutants displayed reduced or depleted mtDNA. We also determined that the severity of the phenotype of the mip1 mutant strain correlates with the age of onset of disease associated with the human ortholog. Finally, we demonstrated that increasing nucleotide pools by overexpression of ribonucleotide reductase (RNR1) suppressed mtDNA replication defects caused by several dominant mip1 mutations, and the orthologous human mutations revealed severe nucleotide binding defects.
Stumpf
Jeffrey D
JD
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes ofHealth, Research, Triangle Park, NC 27709, USA.
Bailey
Christopher M
CM
Spell
Diana
D
Stillwagon
Matthew
M
Anderson
Karen S
KS
Copeland
William C
WC
D000367:Age Factors; D004256:DNA Polymerase I / Q000235:genetics*; D000074002:DNA Polymerase gamma; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D028361:Mitochondrial Diseases / Q000235:genetics*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*
D017931:DNA Primers; D004272:DNA, Mitochondrial; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D004256:DNA Polymerase I; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C068352:MIP1 protein, S cerevisiae