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 fu...

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Bibliographic Details
Published in:PloS one 2012-03, Vol.7 (3), p.e33482
Main Authors: Viikov, Katrin, Jasnovidova, Olga, Tamm, Tiina, Sedman, Juhan
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino acids
Baking yeast
Biocatalysis
Biochemistry
Biodegradation
Biology
Chemical synthesis
Degradation
Deletion mutant
Deoxyribonucleic acid
Disease
DNA
DNA biosynthesis
DNA polymerase
DNA Polymerase I - chemistry
DNA Polymerase I - metabolism
DNA Replication
DNA, Fungal - metabolism
DNA, Mitochondrial - metabolism
DNA-directed DNA polymerase
Drosophila
Eukaryotes
Exonuclease
Exonucleases - metabolism
Genomes
Herpes viruses
Insects
Mitochondria - enzymology
Mitochondrial DNA
Molecular Sequence Data
Mutant Proteins - chemistry
Mutant Proteins - isolation & purification
Mutant Proteins - metabolism
Mutants
Mutation
Nucleotides
Phosphorylation
Polymerization
Primers
Protein Binding
Proteins
Replication
Saccharomyces cerevisiae
Saccharomyces cerevisiae - enzymology
Saccharomyces cerevisiae Proteins - chemistry
Saccharomyces cerevisiae Proteins - metabolism
Saccharomycetes
Sequence Alignment
Sequence Deletion
Structure-Activity Relationship
Technical education
Yeast
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0033482