Catalytic subunit of mitochondrial DNA polymerase from Drosophila embryos. Cloning, bacterial overexpression, and biochemical characterization.
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.
Lewis
D L
DL
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824-1319, USA.
Farr
C L
CL
Wang
Y
Y
Lagina
A T
AT
Kaguni
L S
LS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism*; D004330:Drosophila / Q000196:embryology / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V