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C-terminal extension of the yeast mitochondrial DNA polymerase determines the balance between synthesis and degradation.

Viikov K, Jasnovidova O, Tamm T, Sedman J - PLoS ONE (2012)

Bottom Line: Mip1Δ216 also displays reduced ability to synthesize DNA through double-stranded regions.Full removal of the CTE in Mip1Δ279 results in complete loss of Mip1 polymerase activity, however the mutant retains its exonuclease activity.These results allow us to propose that CTE functions as a part of Mip1 polymerase domain that stabilizes the substrate primer end at the polymerase active site, and is therefore required for efficient mitochondrial DNA replication in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.

ABSTRACT
Saccharomyces cerevisiae mitochondrial DNA polymerase (Mip1) contains a C-terminal extension (CTE) of 279 amino acid residues. The CTE is required for mitochondrial DNA maintenance in yeast but is absent in higher eukaryotes. Here we use recombinant Mip1 C-terminal deletion mutants to investigate functional importance of the CTE. We show that partial removal of the CTE in Mip1Δ216 results in strong preference for exonucleolytic degradation rather than DNA polymerization. This disbalance in exonuclease and polymerase activities is prominent at suboptimal dNTP concentrations and in the absence of correctly pairing nucleotide. Mip1Δ216 also displays reduced ability to synthesize DNA through double-stranded regions. Full removal of the CTE in Mip1Δ279 results in complete loss of Mip1 polymerase activity, however the mutant retains its exonuclease activity. These results allow us to propose that CTE functions as a part of Mip1 polymerase domain that stabilizes the substrate primer end at the polymerase active site, and is therefore required for efficient mitochondrial DNA replication in vivo.

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Related in: MedlinePlus

DNA binding affinity of Mip1 and C-terminal deletion mutants.DNA binding was measured with an electrophoretic mobility shift assay using an oligomeric substrate of 45 nt template strand and a radiolabeled 25 nt primer. 0.2–12.5 nM polymerase was incubated for 2 min at 0°C with 1 nM substrate. A. Reaction products were resolved on a native Tris-glycine 8% polyacrylamide gel. Positions of the free and bound substrate are indicated accordingly with empty and filled triangles. B. The dissociation constant KD (nM) was calculated from the logarithmic binding curve as the concentration of the polymerase when 50% of the substrate was bound. Data from three independent experiments was used to establish the average KD and standard deviation values.
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pone-0033482-g005: DNA binding affinity of Mip1 and C-terminal deletion mutants.DNA binding was measured with an electrophoretic mobility shift assay using an oligomeric substrate of 45 nt template strand and a radiolabeled 25 nt primer. 0.2–12.5 nM polymerase was incubated for 2 min at 0°C with 1 nM substrate. A. Reaction products were resolved on a native Tris-glycine 8% polyacrylamide gel. Positions of the free and bound substrate are indicated accordingly with empty and filled triangles. B. The dissociation constant KD (nM) was calculated from the logarithmic binding curve as the concentration of the polymerase when 50% of the substrate was bound. Data from three independent experiments was used to establish the average KD and standard deviation values.

Mentions: To assess DNA binding affinity of FL-Mip1 and deletion mutants, we performed an electrophoretic mobility shift assay (Figure 5). Labeled 25/45 substrate was incubated with Mip1 at different protein concentrations varying from 0.2 nM to 12.5 nM. DNA/protein complex formation was detected as a shift of the labeled substrate during native gel electrophoresis (Figure 5A). A logarithmic binding curve was drawn from the ratio of the bound DNA substrate to all substrate, and dissociation constant KD was estimated as the protein concentration when 50% of the substrate remained unbound (Figure 5B). The KD of FL-Mip1 was estimated to be 4.3±0.68 nM. Mip1Δ175 and Mip1Δ216 both showed stronger DNA binding, with the corresponding KD values estimated to 2.2±0.75 nM and 0.95±0.18 nM, respectively. Increase in DNA binding affinity correlates well with the increase of Mip1Δ175 and Mip1Δ216 processivity as compared to FL-Mip1.


C-terminal extension of the yeast mitochondrial DNA polymerase determines the balance between synthesis and degradation.

Viikov K, Jasnovidova O, Tamm T, Sedman J - PLoS ONE (2012)

DNA binding affinity of Mip1 and C-terminal deletion mutants.DNA binding was measured with an electrophoretic mobility shift assay using an oligomeric substrate of 45 nt template strand and a radiolabeled 25 nt primer. 0.2–12.5 nM polymerase was incubated for 2 min at 0°C with 1 nM substrate. A. Reaction products were resolved on a native Tris-glycine 8% polyacrylamide gel. Positions of the free and bound substrate are indicated accordingly with empty and filled triangles. B. The dissociation constant KD (nM) was calculated from the logarithmic binding curve as the concentration of the polymerase when 50% of the substrate was bound. Data from three independent experiments was used to establish the average KD and standard deviation values.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303844&req=5

pone-0033482-g005: DNA binding affinity of Mip1 and C-terminal deletion mutants.DNA binding was measured with an electrophoretic mobility shift assay using an oligomeric substrate of 45 nt template strand and a radiolabeled 25 nt primer. 0.2–12.5 nM polymerase was incubated for 2 min at 0°C with 1 nM substrate. A. Reaction products were resolved on a native Tris-glycine 8% polyacrylamide gel. Positions of the free and bound substrate are indicated accordingly with empty and filled triangles. B. The dissociation constant KD (nM) was calculated from the logarithmic binding curve as the concentration of the polymerase when 50% of the substrate was bound. Data from three independent experiments was used to establish the average KD and standard deviation values.
Mentions: To assess DNA binding affinity of FL-Mip1 and deletion mutants, we performed an electrophoretic mobility shift assay (Figure 5). Labeled 25/45 substrate was incubated with Mip1 at different protein concentrations varying from 0.2 nM to 12.5 nM. DNA/protein complex formation was detected as a shift of the labeled substrate during native gel electrophoresis (Figure 5A). A logarithmic binding curve was drawn from the ratio of the bound DNA substrate to all substrate, and dissociation constant KD was estimated as the protein concentration when 50% of the substrate remained unbound (Figure 5B). The KD of FL-Mip1 was estimated to be 4.3±0.68 nM. Mip1Δ175 and Mip1Δ216 both showed stronger DNA binding, with the corresponding KD values estimated to 2.2±0.75 nM and 0.95±0.18 nM, respectively. Increase in DNA binding affinity correlates well with the increase of Mip1Δ175 and Mip1Δ216 processivity as compared to FL-Mip1.

Bottom Line: Mip1Δ216 also displays reduced ability to synthesize DNA through double-stranded regions.Full removal of the CTE in Mip1Δ279 results in complete loss of Mip1 polymerase activity, however the mutant retains its exonuclease activity.These results allow us to propose that CTE functions as a part of Mip1 polymerase domain that stabilizes the substrate primer end at the polymerase active site, and is therefore required for efficient mitochondrial DNA replication in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.

ABSTRACT
Saccharomyces cerevisiae mitochondrial DNA polymerase (Mip1) contains a C-terminal extension (CTE) of 279 amino acid residues. The CTE is required for mitochondrial DNA maintenance in yeast but is absent in higher eukaryotes. Here we use recombinant Mip1 C-terminal deletion mutants to investigate functional importance of the CTE. We show that partial removal of the CTE in Mip1Δ216 results in strong preference for exonucleolytic degradation rather than DNA polymerization. This disbalance in exonuclease and polymerase activities is prominent at suboptimal dNTP concentrations and in the absence of correctly pairing nucleotide. Mip1Δ216 also displays reduced ability to synthesize DNA through double-stranded regions. Full removal of the CTE in Mip1Δ279 results in complete loss of Mip1 polymerase activity, however the mutant retains its exonuclease activity. These results allow us to propose that CTE functions as a part of Mip1 polymerase domain that stabilizes the substrate primer end at the polymerase active site, and is therefore required for efficient mitochondrial DNA replication in vivo.

Show MeSH
Related in: MedlinePlus