C-Terminal Extension of the Yeast Mitochondrial DNA Polymerase Determines the Balance between Synthesis and Degradation

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

Polymerase/exonuclease balance of Mip1 and C-terminal deletion mutants.

<p>The balance between polymerase and exonuclease activities was assayed on a 45 nt template primed with a radiolabeled 25 nt primer. 4 nM DNA polymerase and 10 nM substrate were incubated for 5 min at 30°C in the presence of indicated concentrations of dNTP. Reactions were stopped with an equal volume of 80% formamide, 25 mM EDTA. A. Reaction products were resolved on 8% urea polyacrylamide gel. Positions of the 25 nt primer and 45 nt reaction product are indicated. B. The percentage of the polymerization products out of total reaction products were plotted against dNTP concentration. FL-Mip1 – empty circle, Mip1Δ175 –filled square, Mip1Δ216 – filled triangle.</p

Processivity of Mip1 and C-terminal deletion mutants.

<p>Processivity was measured under single-hit conditions with 4 nM of substrate M13 ssDNA singly primed with radiolabeled USP and 1 mg/ml calf thymus activated DNA. The reaction was performed at 30°C with 4 nM DNA polymerase in the presence of 100 µM dNTP. The reaction was stopped with 0.5 mg/ml Proteinase K, 1% SDS, 20 mM EDTA after indicated time points. A. Reaction products were separated on 0.8% alkaline agarose gel. Arrows indicate positions of M13mp18 unit length (7250 nt) and 17 nt USP. B. Processivity of FL-Mip1, Mip1Δ175 and Mip1Δ216 was calculated as the average length of the product (nt) synthesized by the polymerase per one binding event. Weighted mean method based on the product intensity and length was used for analysis. Data from three independent experiments was used to calculate the average processivity and standard deviation values.</p

DNA polymerase activity of Mip1 and C-terminal deletion mutants.

<p>DNA polymerase activity was measured using 40 nM DNA polymerase, 4 nM M13 circular ssDNA singly primed with radiolabeled USP primer and 100 µM dNTP. The reaction was carried out at 30°C and stopped after 0 s, 10 s, 20 s, 30 s, 45 s, 1 min, 2 min and 5 min with equal volume of 80% deionized formamide, 25 mM EDTA. A. Reaction products were resolved on 8% denaturating polyacrylamide gel. The position of 17 nt USP is marked with an arrow. B. Maximum product length was determined using the DNA marker as a standard and plotted against time. FL-Mip1 – empty circle, Mip1Δ175 –filled square, Mip1Δ216 – filled triangle.</p

Exonuclease activity of Mip1 and C-terminal deletion mutants.

<p>Exonuclease activity was assayed on a 45 nt template primed with a radiolabeled 25 nt primer. 4 nM DNA polymerase was incubated with 2 nM 45/25 substrate in the total absence of dNTP or in the presence of 5 nM dATP. Reactions were carried out at 30°C and stopped at indicated time points with equal volume of 80% formamide, 25 mM EDTA. A. Reaction products without dNTP were resolved on 8% urea polyacrylamide gel. B. Reaction products with 5 nM dATP were resolved on 8% urea polyacrylamide gel. Position of the 25 nt primer is indicated. C. Exonuclease activity was calculated as the amount of released dNMP from the reaction products. The proportional amount of the products was calculated from the intensity of the signal and the amount of the dNMP released during the reaction, taking into account the size of each of the exonuclease products. The amount of released dNMP was plotted against time. Filled square – reaction in the absence of the dNTP, empty circle – reaction in the presence of 5 nM dATP.</p

DNA binding affinity of Mip1 and C-terminal deletion mutants.

<p>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 K_D (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 K_D and standard deviation values.</p

Length and amino acid sequence alignment of C-terminal extension of mitochondrial DNA polymerases from <i>Saccharomycetes</i>.

<p>A. Length of the CTE of <i>Saccharomycetes</i> species. The C-terminal extension was defined as the protein sequence starting from the 16^th amino acid past the γ6 motif. B. Amino acid sequence alignment of <i>Saccharomycetes</i> CTEs was performed with the PRALINE software available at <a href="http://www.ibi.vu.nl/programs" target="_blank">www.ibi.vu.nl/programs</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033482#pone.0033482-Simossis1" target="_blank">[42]</a> using the PAM250 weights matrix. Alignment of the first 150 residues is shown and the positions of <i>S. cerevisiae</i> Mip1Δ175, Mip1Δ216 and Mip1Δ279 deletion mutants are indicated.</p