Mitochondrial DNA polymerase ␥ (pol ␥) is responsible for replication and repair of mtDNA and is mutated in individuals with genetic disorders such as chronic external ophthalmoplegia and Alpers syndrome. pol ␥ is also an adventitious target for toxic side effects of several antiviral compounds, and mutation of its proofreading exonuclease leads to accelerated aging in mouse models. We have used a variety of physical and functional approaches to study the interaction of the human pol ␥ catalytic subunit with both the wild-type accessory factor, pol ␥B, and a deletion derivative that is unable to dimerize and consequently is impaired in its ability to stimulate processive DNA synthesis. Our studies clearly showed that the functional human holoenzyme contains two subunits of the processivity factor and one catalytic subunit, thereby forming a heterotrimer. The structure of pol ␥ seems to be variable, ranging from a single catalytic subunit in yeast to a heterodimer in Drosophila and a heterotrimer in mammals.Mitochondria contain a single DNA polymerase, pol 4 ␥, responsible for replication and repair of mtDNA (reviewed in Ref. 1). Human pol ␥ is isolated from mitochondria as a complex containing two subunits, a catalytic subunit, pol ␥A, of 139 kDa and an accessory subunit, pol ␥B, of 53 kDa (2-5). The catalytic subunit is a family A DNA polymerase with separate polymerase and 3Ј-5Ј exonuclease domains. Two recent developments have stressed the importance of pol ␥. Mutations in the catalytic subunit of human pol ␥ cause mitochondrial disorders (6, 7), and a transgenic mouse engineered to express an error-prone form of DNA pol ␥ lacking the 3Ј-5Ј proofreading ability accumulates errors in mtDNA and undergoes accelerated aging (8, 9).The processivity and substrate binding properties of pol ␥A are enhanced by complex formation with the accessory subunit (2, 4, 5). Most interestingly, the presence of the accessory subunit has been shown to decrease the fidelity of DNA synthesis by the catalytic subunit because it increases the ability of the enzyme to extend a mismatched primer (10). Initial characterizations of pol ␥ suggested that the enzyme forms a heterodimer containing one copy of each subunit. However, when we solved the crystal structure of mouse pol ␥B, it became apparent that this accessory factor is itself a homodimer with remarkable structural similarity to prokaryotic tRNA synthetases (Protein Data Bank code 1G5H (11)). We considered it to be very unlikely that this dimerization is a crystal packing artifact, especially because we were able to show that wild-type pol ␥B sedimented more rapidly than a deletion derivative lacking a major portion of the dimerization interface (11).The incorporation of pol ␥B in the pol ␥ holoenzyme appears to be a relatively recent event in evolutionary terms. The protein has not been reported in yeast, where pol ␥ was first cloned as a product of the mip1 gene (12), and efforts to find evidence for it have been unsuccessful (13). Drosophila pol ␥ differs from the enzyme in v...

Polymerase gamma, which replicates and repairs mitochondrial DNA, requires the Pol gamma B subunit for processivity. We determined the crystal structure of mouse Pol gamma B, a core component of the mitochondrial replication machinery. Pol gamma B shows high similarity to glycyl-tRNA synthetase and dimerizes through an unusual intermolecular four-helix bundle. A human Pol gamma B mutant lacking the four-helix bundle failed to dimerize in solution or to stimulate the catalytic subunit Pol gamma A, but retained the ability to bind with Pol gamma A to a primer-template construct, indicating that the functional holoenzyme contains two Pol gamma B molecules. Other mutants retained stimulatory activity but lost the ability to bind folded ssDNA. These results suggest that the Pol gamma B dimer contains distinct sites for Pol gamma A binding, dimerization, and DNA binding.

Peptide sequences obtained from the accessory subunit of Xenopus laevis mitochondrial DNA (mtDNA) polymerase ␥ (pol ␥) were used to clone the cDNA encoding this protein. Amino-terminal sequencing of the mitochondrial protein indicated the presence of a 44-amino-acid mitochondrial targeting sequence, leaving a predicted mature protein with 419 amino acids and a molecular mass of 47.3 kDa. This protein is associated with the larger, catalytic subunit in preparations of active mtDNA polymerase. The small subunit exhibits homology to its human, mouse, and Drosophila counterparts. Interestingly, significant homology to glycyl-tRNA synthetases from prokaryotic organisms reveals a likely evolutionary relationship. Since attempts to produce an enzymatically active recombinant catalytic subunit of Xenopus DNA pol ␥ have not been successful, we tested the effects of adding the small subunit of the Xenopus enzyme to the catalytic subunit of human DNA pol ␥ purified from baculovirus-infected insect cells. These experiments provide the first functional evidence that the small subunit of DNA pol ␥ stimulates processive DNA synthesis by the human catalytic subunit under physiological salt conditions. Mitochondrial DNA (mtDNA) is replicated by a DNA polymerase, DNA polymerase ␥ (pol ␥), that is distinct from nuclear DNA polymerases ␣, ␤, ␦, ε, and . Since DNA pol ␥ represents only a small fraction of total cellular DNA polymerase, purification and characterization of the subunit composition of this enzyme have been difficult. Molecular cloning has contributed greatly to understanding the structure of DNA pol ␥ in different organisms. The catalytic subunits of DNA pol ␥ have been cloned for several organisms (6,16,17,27,34) and have been found to resemble family A of DNA polymerases, related to Escherichia coli DNA pol I. In Saccharomyces cerevisiae DNA pol ␥ is composed of a single polypeptide, while in Drosophila melanogaster DNA pol ␥ is comprised of two different polypeptides, a catalytic subunit of 125 kDa and an accessory subunit of 41 kDa (24, 33). The Drosophila subunits copurify and have been shown to interact, but the recombinant proteins have not yet been shown to be functional. The function of the small subunit, which we refer to as pol ␥B, is unknown. It has been proposed to influence the processivity of the catalytic subunit. Putative mammalian homologs of the Drosophila accessory subunit have been identified in sequence databases. One published purification scheme for human DNA pol ␥ suggested the existence of a small subunit (11), but the potential relationship between this polypeptide and Drosophila pol ␥B has not been established. Recently, the catalytic subunit of human DNA pol ␥ was expressed in an active form (10,19). Surprisingly, the recombinant catalytic subunit alone displayed most of the characteristics of the enzyme purified from human cells, which did not appear to contain a stoichiometric amount of a small subunit. Thus, it is not clear what role, if any, is played by putative human DNA pol ␥B.We hav...