Accessory Subunit of Mitochondrial DNA Polymerase fromDrosophila Embryos: CLONING, MOLECULAR ANALYSIS, AND ASSOCIATION IN THE NATIVE ENZYME

Yuxun Wang,Carol L Farr,Laurie S Kaguni

doi:10.1074/jbc.272.21.13640

Abstract

A full-length cDNA of the accessory (beta) 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 361 residues and a predicted molecular mass of 41 kDa. The gene encoding the beta subunit lies within 4 kilobase pairs of that for the catalytic subunit in the Drosophila genome, on the left arm of chromosome 2. The two genes have similar structural features and share several common DNA sequence elements in their upstream regions, suggesting the possibility of coordinate regulation. A human cDNA homolog of the accessory subunit was identified, and its nucleotide sequence was determined. The human sequence encodes a polypeptide with a predicted molecular mass of 43 kDa that shows a high degree of amino acid sequence similarity to the Drosophila beta subunit. Subunit-specific rabbit antisera, directed against the recombinant catalytic and accessory subunit polypeptides overexpressed and purified from Escherichia coli, recognize specifically and immunoprecipitate the native enzyme from Drosophila embryos. Demonstration of the physical association of the two subunits in the Drosophila enzyme and identification of a human accessory subunit homolog provide evidence for a common heterodimeric structure for animal mitochondrial DNA polymerases.

Highlights

A full-length cDNA of the accessory (␤) subunit of mitochondrial DNA polymerase from Drosophila embryos has been obtained, and its nucleotide sequence was determined
Cloning of the ␤ Subunit of D. melanogaster DNA Polymerase ␥—D. melanogaster mitochondrial DNA polymerase holoenzyme was purified to near homogeneity from embryonic mitochondria as described by Wernette and Kaguni [5]
The enzyme derived from 1120 g of embryos was subjected to SDS-polyacrylamide gel electrophoresis, and the ␤ subunit polypeptide was transferred to polyvinylidene difluoride membrane

Summary

Introduction

A full-length cDNA of the accessory (␤) 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 361 residues and a predicted molecular mass of 41 kDa. The gene encoding the ␤ subunit lies within 4 kilobase pairs of that for the catalytic subunit in the Drosophila genome, on the left arm of chromosome 2. Demonstration of the physical association of the two subunits in the Drosophila enzyme and identification of a human accessory subunit homolog provide evidence for a common heterodimeric structure for animal mitochondrial DNA polymerases. Nuclear encoded DNA polymerase (pol ␥) is involved in the replication of animal mtDNA [2]. The subunit structure of native pol ␥ from these sources remains an unresolved issue

Methods

Results

Conclusion