EXPRESSION, PURIFICATION, AND INITIAL KINETIC CHARACTERIZATION OF THELARGE SUBUNIT OF THE HUMAN MITOCHONDRIAL-DNA POLYMERASE

Faulty replication of the human mitochondrial genome is thought to be the cause of many diseases, moreover, the low selectivity of the mitoc hondrial DNA polymerase has been implicated as the cause of many side effects observed in the treatment of viral infections such as HIV. To better understand how the mitochondrial genome is replicated, we clone d a cDNA encoding the large subunit of human DNA polymerase gamma, the enzyme that replicates the mitochondrial genome. The large subunit wa s recombinantly expressed and purified to near homogeneity. The purifi ed enzyme demonstrated both polymerase and 3'-5' exonuclease activity. The purified protein was examined in single nucleotide incorporation assays, demonstrating that the enzyme had a maximum polymerization rat e of 3.5 s(-1) and a dissociation rate from the DNA substrate of 0.03 s(-1), affording a calculated processivity of 116. The dissociation co nstants for the enzyme binding to DNA and nucleoside triphosphate were 39 nM and 14 mu M, respectively. The 3'-5' exonuclease rate was measu red at 0.18 s(-1). Though the slow rate of polymerization suggests tha t the large subunit of human DNA polymerase gamma may require accessor y factors to increase its processivity of polymerization, the kinetic parameters indicate that the large subunit of DNA polymerase gamma cou ld replicate the mitochondrial genome in a physiologically relevant ti me frame. This study provides the initial characterization of the larg e subunit of DNA polymerase gamma and establishes the baseline for exa mination of the effects of accessory proteins such as the putative sma ll subunit.