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. 2013 Jun;194(2):519-22.
doi: 10.1534/genetics.113.150920. Epub 2013 Apr 15.

The exonuclease activity of the yeast mitochondrial DNA polymerase γ suppresses mitochondrial DNA deletions between short direct repeats in Saccharomyces cerevisiae

Jeffrey D Stumpf  1 , William C Copeland
Affiliations

The exonuclease activity of the yeast mitochondrial DNA polymerase γ suppresses mitochondrial DNA deletions between short direct repeats in Saccharomyces cerevisiae

Jeffrey D Stumpf et al. Genetics. 2013 Jun.

Abstract

The importance of mitochondrial DNA (mtDNA) deletions in the progeroid phenotype of exonuclease-deficient DNA polymerase γ mice has been intensely debated. We show that disruption of Mip1 exonuclease activity increases mtDNA deletions 160-fold, whereas disease-associated polymerase variants were mostly unaffected, suggesting that exonuclease activity is vital to avoid deletions during mtDNA replication.

Keywords: Mip1; Pol Gamma; direct repeats; exonuclease; mtDNA deletions.

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Figures

Figure 1
Figure 1
MtDNA deletions between 21-mer direct repeats in strains with Mip1-proficient or -deficient exonuclease. Heteroallelic mip1 strains were created by transforming TRP1-containing centromeric plasmids PFL39, containing wild-type MIP1 (Foury and Vanderstraeten 1992) (MIP1 [Het]; JSY113), or mip1 encoding an exonuclease-deficient mutant variant (Strand et al. 2003) (mip1-exo [Het]; JSY114) into trp1::G418 NPY75 (Phadnis et al. 2005) (JSY77). JSY113 and JSY114 were made monoallelic by transforming a MIP1::HYG cassette, selecting for colonies that grew on minimal plates lacking arginine and tryptophan and were resistant to hygromycin and G418, creating JSY131 (MIP1 [Mono]) and JSY133 (mip1-exo [Mono]), respectively. Monoallelic strains were confirmed by their inability to maintain mtDNA after plasmid loss. All strains were grown independently from at least 20 colonies at 30° in YP (yeast extract 1%, peptone 2%) with 2% glucose and 0.01% adenine sulfate for 2 days. Appropriate dilutions from saturated cultures were plated on synthetic complete media lacking arginine to determine total number of cells with mtDNA. Approximately 108 cells were plated onto YP with 2% glycerol and deletion mutants were counted after 4 days. Deletions were confirmed phenotypically in several mutants from each strain; all rho+ colonies tested were Arg− (data not shown). The mutant frequency was determined as the median number of mutant colonies per 108 Arg+ cells. The 95% confidence levels are represented as error bars and were determined using the method of the median.
Figure 2
Figure 2
Deletion mutagenesis of mip1 mutants with disease-associated POLG mutations. All strains are NPY75-derived heteroallelic strains with wild-type chromosomal MIP1 and a centromeric plasmid containing either wild-type MIP1 (Foury and Vanderstraeten 1992), the exonuclease-deficient mip1 allele (mip-exo), or a disease-associated variant denoted by the amino acid change in Mip1 (Stumpf et al. 2010). All strains were grown independently from at least 20 colonies at 30° in YP (yeast extract 1%, peptone 2%) with 2% glucose and 0.01% adenine sulfate for 2 days. Appropriate dilutions of samples from the saturated cultures were plated on synthetic complete media lacking arginine to determine total number of cells with mtDNA. The cultures were plated onto YP with 2% glycerol and deletion mutants were counted after 4 days. Deletions were confirmed phenotypically in several mutants from each strain; all rho+ colonies tested were Arg− (data not shown). The mutant frequency was determined as the median number of mutant colonies per 108 Arg+ cells. Error bars represent 95% confidence levels and were determined using the method of the median. P-values relative to wild type were 6 × 10−5 (Exo−), 0.9 (L211P), 0.7 (Q264H), 0.3 (R265L), 0.6 (R607C), 0.5 (R853C), 0.6 (V863I), and 0.5 (S861C).
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