Expression, purification, and initial kinetic characterization of the large 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 mitochondrial 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 cloned a cDNA encoding the large subunit of human DNA polymerase gamma, the enzyme that replicates the mitochondrial genome. The large subunit was recombinantly expressed and purified to near homogeneity. The purified 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 rate 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 constants for the enzyme binding to DNA and nucleoside triphosphate were 39 nM and 14 microM, respectively. The 3'-5' exonuclease rate was measured at 0. 18 s-1. Though the slow rate of polymerization suggests that the large subunit of human DNA polymerase gamma may require accessory factors to increase its processivity of polymerization, the kinetic parameters indicate that the large subunit of DNA polymerase gamma could replicate the mitochondrial genome in a physiologically relevant time frame. This study provides the initial characterization of the large subunit of DNA polymerase gamma and establishes the baseline for examination of the effects of accessory proteins such as the putative small subunit.
Graves
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SW
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park 16802, USA.
Johnson
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AA
Johnson
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KA
D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D003001:Cloning, Molecular; D000074002:DNA Polymerase gamma; D018076:DNA, Complementary / Q000302:isolation & purification; D004272:DNA, Mitochondrial / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000096:biosynthesis; D007700:Kinetics; D010957:Plasmids / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility; D018243:Teratocarcinoma; D013942:Thymine Nucleotides / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004272:DNA, Mitochondrial; D007106:Immune Sera; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C024157:thymidine 5'-triphosphate