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

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