Abstract
A full-length cDNA of the catalytic subunit of mitochondrial DNA polymerase from Drosophila embryos has been obtained, and its nucleotide sequence was determined. The cDNA clone encodes a polypeptide with a deduced amino acid sequence of 1145 residues and a predicted molecular mass of 129.9 kDa. Amino-terminal sequence analysis of the mature catalytic subunit of the heterodimeric mitochondrial enzyme from Drosophila embryos identified the amino-terminal amino acid at position +10 in the deduced amino acid sequence, indicating a mitochondrial presequence peptide of only nine amino acids. Alignment of the catalytic subunit sequence with that of Escherichia coli DNA polymerase I Klenow fragment indicated a high degree of amino acid sequence conservation in each of the three DNA polymerase and three 3' --> 5' exonuclease domains identified by biochemical studies in the latter enzyme. Bacterial overexpression, purification, and biochemical analysis demonstrated both 5' --> 3' DNA polymerase and 3' --> 5' exonuclease in the recombinant polypeptide. This represents the first demonstration of 3' --> 5' exonuclease activity in the polymerase catalytic subunit of animal mitochondrial DNA polymerase.
Highlights
Mitochondria are essential in animal cells and are the only organelles outside the nucleus that contain a DNA genome
Cloning of the Catalytic Subunit of D. melanogaster DNA Polymerase ␥—The heterodimeric mitochondrial DNA polymerase from D. melanogaster was purified to near-homogeneity from embryonic mitochondria as described by Wernette and Kaguni [2]
Mitochondrial DNA polymerase is the key enzyme involved in replication of the mtDNA genome
Summary
Mitochondria are essential in animal cells and are the only organelles outside the nucleus that contain a DNA genome. We have studied the key replicative enzyme in mitochondria, mitochondrial DNA polymerase (pol ␥). In biochemical studies to date, the role of the small subunit has not been determined nor has a subunit assignment for the 3Ј 3 5Ј exonuclease been made. To address these issues and to elucidate structure-function relationships in animal mitochondrial DNA polymerase, we have pursued the molecular cloning of the two subunits of Drosophila pol ␥. We report here the cloning, bacterial overexpression, and biochemical characterization of the polymerase catalytic subunit
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