Baculovirus expression reconstitutes Drosophila mitochondrial DNA polymerase.
Drosophila mitochondrial DNA polymerase has been reconstituted and purified from baculovirus-infected insect cells. Baculoviruses encoding full-length and mature forms of the catalytic and accessory subunits were generated and used in single and co-infection studies. Recombinant heterodimeric holoenzyme was reconstituted in both the mitochondria and cytoplasm of Sf9 cells and required the mitochondrial presequences in both subunits. The recombinant holoenzyme contains DNA polymerase and 3'-5' exonuclease that are stimulated substantially by both salt and mitochondrial single-stranded DNA-binding protein. Thus, the recombinant enzyme exhibits biochemical properties indistinguishable from those of the native enzyme from Drosophila embryos. Production of the catalytic subunit alone yielded soluble protein with the chromatographic properties of the heterodimeric holoenzyme. However, the purified catalytic core has a 50-fold lower specific activity. This provides evidence of a critical role for the accessory subunit in the catalytic efficiency of Drosophila mitochondrial DNA polymerase.
Wang
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Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824-1319, USA.
Kaguni
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LS
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D000074002:DNA Polymerase gamma; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D019281:Dimerization; D004330:Drosophila / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V