Subunit Structure of Mitochondrial DNA Polymerase from Drosophila Embryos: PHYSICAL AND IMMUNOLOGICAL STUDIES

Matthew W Olson,Yuxun Wang,Rhoderick H Elder,Laurie S Kaguni

doi:10.1074/jbc.270.48.28932

Matthew W Olson, Yuxun Wang + Show 2 more

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https://doi.org/10.1074/jbc.270.48.28932

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Abstract

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.

Highlights

The subunit structure of mitochondrial DNA polymerase from Drosophila embryos has been examined by a combination of physical and immunological methods
Photochemical cross-linking of the complexes by UV light indicated that the ␣ but not the ␤ subunit of mitochondrial DNA polymerase makes close contact with DNA, and limited digestion of the native enzyme with trypsin showed that an ϳ65-kDa proteolytic fragment of the ␣ subunit retains the DNA binding function
The data indicate that neither the ␣ nor the ␤ subunits of Drosophila pol ␥ have been proteolyzed during the course of purification from the Fraction III to Fraction IV stage, and that the antiserum is highly specific and recognizes the native enzyme

Summary

Introduction

The subunit structure of mitochondrial DNA polymerase from Drosophila embryos has been examined by a combination of physical and immunological methods. Notwithstanding the enzyme’s low relative abundance, in surveying Drosophila at six developmental stages, we showed that the level of pol ␥ activity varies 180-fold during development and is greatest in early embryos [5]. This allowed its purification to near-homogeneity [5], and charac-. A subunit assignment for the 3Ј 3 5Ј exonuclease has not been made in any of the animal mitochondrial DNA polymerases, it is most likely that the 3Ј 3 5Ј exonuclease function resides in the polymerase catalytic subunit. While the 3Ј 3 5Ј exonuclease resides in the polymerase catalytic subunit in Bacillus subtilis DNA polymerase III, it exists as a separate subunit in E. coli DNA polymerase III [22]

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