Subunit structure of mitochondrial DNA polymerase from Drosophila embryos. Physical and immunological studies.
The subunit structure of mitochondrial DNA polymerase from Drosophila embryos has been examined by a combination of physical and immunological methods. A highly specific rabbit antiserum directed against the native enzyme was developed and found to recognize specifically its two subunits in immunoblot and immunoprecipitation analyses. That and the potent inhibition by the rabbit antiserum of the DNA polymerase and 3'-->5' exonuclease activities of the nearly homogeneous mitochondrial DNA polymerase provide strong evidence for the physical association of the 3'-->5' exonuclease with the two subunit enzyme. An immunoprecipitation analysis of crude enzyme fractions showed that the two subunits of Drosophila mitochondrial DNA polymerase are intact, and an in situ gel proteolysis analysis showed that they are structurally distinct. Template-primer DNA binding studies demonstrated formation of a stable and discrete enzyme-DNA complex in the absence of accessory proteins. Photochemical cross-linking of the complexes by UV light indicated that the alpha but not the beta subunit of mitochondrial DNA polymerase makes close contact with DNA, and limited digestion of the native enzyme with trypsin showed that an approximately 65-kDa proteolytic fragment of the alpha subunit retains the DNA binding function.
D000818:Animals; D001483:Base Sequence; D004258:DNA Polymerase III / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D004330:Drosophila / Q000196:embryology / Q000201:enzymology*; D007106:Immune Sera / Q000276:immunology; D007814:Larva / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011817:Rabbits; D013698:Templates, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D007106:Immune Sera; D009838:Oligodeoxyribonucleotides; D004258:DNA Polymerase III