Processivity of mitochondrial DNA polymerase from Drosophila embryos. Effects of reaction conditions and enzyme purity.

Williams, Andrea; Wernette, Catherine M.; Kaguni, Laurie S.

doi:10.1016/s0021-9258(19)74543-8

Cited by 33 publications

(9 citation statements)

References 41 publications

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“…Mutant DeriVatiVes of Human Pol γ p140. Three distinguishing characteristics of the γ polymerases are highly processive DNA synthesis (31), high-fidelity DNA synthesis partially afforded by an associated proofreading 3′ f 5′ exonuclease activity (19)(20)(21)(22)(23)(24), and extreme sensitivity to dideoxyribonucleotides (5,10). We sought to identify amino acid residues critical to exonuclease function and dideoxynucleotide discrimination.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the Native and Recombinant Catalytic Subunit of Human DNA Polymerase γ: Identification of Residues Critical for Exonuclease Activity and Dideoxynucleotide Sensitivity

et al. 1998

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The human DNA polymerase gamma catalytic subunit was overexpressed in recombinant baculovirus-infected insect cells, and the 136 000 Da protein was purified to homogeneity. Application of the same purification protocol to HeLa mitochondrial lysates permitted isolation of native DNA polymerase gamma as a single subunit, allowing direct comparison of the native and recombinant enzymes without interference of other polypeptides. Both forms exhibited identical properties, and the DNA polymerase and 3' --> 5' exonuclease activities were shown unambiguously to reside in the catalytic polypeptide. The salt sensitivity and moderate processivity of the isolated catalytic subunit suggest other factors could be required to restore the salt tolerance and highly processive DNA synthesis typical of gamma polymerases. To facilitate our understanding of mitochondrial DNA replication and mutagenesis as well as cytotoxicity mediated by antiviral nucleotide analogues, we also constructed two site-directed mutant proteins of the human DNA polymerase gamma. Substituting alanine for two essential acidic residues in the exonuclease motif selectively eliminated the 3' --> 5' exonucleolytic function of the purified mutant polymerase gamma. Replacement of a tyrosine residue critical for sugar recognition with phenylalanine in polymerase motif B reduced dideoxynucleotide inhibition by a factor of 5000 with only minor effects on overall polymerase function.

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Section: Resultsmentioning

confidence: 99%

Characterization of the Native and Recombinant Catalytic Subunit of Human DNA Polymerase γ: Identification of Residues Critical for Exonuclease Activity and Dideoxynucleotide Sensitivity

et al. 1998

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“…Given the large difference in primary structure compared with DNA polymerase I, it is not feasible to identify individual DNA contact residues in DNA polymerase γ without direct structural analysis. We speculate that the large size of the domain separating the exo and pol active sites in DNA polymerase γ may allow this enzyme to enclose the DNA primer-template more tightly and may contribute to the high degree of processivity observed for the mitochondrial polymerase (30).…”

Section: Discussionmentioning

confidence: 99%

The gamma subfamily of DNA polymerases: cloning of a developmentally regulated cDNA encoding Xenopus laevis mitochondrial DNA polymerase gamma

Carrodeguas

Bogenhagen

1996

Nucleic Acids Research

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We used the known sequence of the Saccharomyces cerevisiae DNA polymerase gamma to clone the genes or cDNAs encoding this enzyme in two other yeasts, Pychia pastoris and Schizosaccharomyces pombe, and one higher eukaryote, Xenopus laevis. To confirm the identity of the final X.laevis clone, two antisera raised against peptide sequences were shown to react with DNA polymerase gamma purified from X.laevis oocyte mitochondria. A developmentally regulated 4.6 kb mRNA is recognized on Northern blots of oocyte RNA using the X.laevis cDNA. Comparison of the four DNA polymerase gamma gene sequences revealed several highly conserved sequence blocks, comprising an N-terminal 3'-->5'exonuclease domain and a C-terminal polymerase active center interspersed with gamma-specific gene sequences. The consensus sequences for the DNA polymerase gamma exonuclease and polymerase domains show extensive sequence similarity to DNA polymerase I from Escherichia coli. Sequence conservation is greatest for residues located near the active centers of the exo and pol domains of the E.coli DNA polymerase I structure. The domain separating the exonuclease and polymerase active sites is larger in DNA polymerase gamma than in other members of family A (DNA polymerase I-like) polymerases. The S.cerevisiae DNA polymerase gamma is atypical in that it includes a 240 residue C-terminal extension that is not found in the other members of the DNA polymerase gamma family, or in other family A DNA polymerases.

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“…Recent studies of the recombinant Drosophila melanogaster large subunit have shown that it is capable of both polymerase activity and 3′-5′ exonuclease activity, but further characterization of the large subunit from Drosophila has been hampered by the low solubility of the recombinant protein (11). Purified D. melanogaster DNA polymerase γ can exhibit low processivity (defined as the number of nucleotides incorporated per binding event), and homogeneous fractions of DNA polymerase γ are less active than less pure preparations of the enzyme (15), suggesting the possible loss of an accessory protein during purification.…”

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confidence: 99%

Expression, Purification, and Initial Kinetic Characterization of the Large Subunit of the Human Mitochondrial DNA Polymerase

Graves

Johnson

1998

Biochemistry

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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.

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Processivity of mitochondrial DNA polymerase from Drosophila embryos. Effects of reaction conditions and enzyme purity.

Cited by 33 publications

References 41 publications

Characterization of the Native and Recombinant Catalytic Subunit of Human DNA Polymerase γ: Identification of Residues Critical for Exonuclease Activity and Dideoxynucleotide Sensitivity

Characterization of the Native and Recombinant Catalytic Subunit of Human DNA Polymerase γ: Identification of Residues Critical for Exonuclease Activity and Dideoxynucleotide Sensitivity

The gamma subfamily of DNA polymerases: cloning of a developmentally regulated cDNA encoding Xenopus laevis mitochondrial DNA polymerase gamma

Expression, Purification, and Initial Kinetic Characterization of the Large Subunit of the Human Mitochondrial DNA Polymerase

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