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 immunologic al methods. A highly specific rabbit antiserum directed against the na tive 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 o f the 3' --> 5' exonuclease with the two subunit enzyme. An immunoprec ipitation analysis of crude enzyme fractions showed that the two subun its of Drosophila mitochondrial DNA polymerase are intact, and an in s itu gel proteolysis analysis showed that they are structurally distinc t. Template-primer DNA binding studies demonstrated formation of a sta ble and discrete enzyme DNA complex in the absence of accessory protei ns. Photochemical cross-linking of the complexes by UV light indicated that the alpha but not the beta subunit of mitochondrial DNA polymera se makes close contact with DNA, and limited digestion of the native e nzyme with trypsin showed that an similar to 65-kDa proteolytic fragme nt of the alpha subunit retains the DNA binding function.