Purification and characterization of porcine liver DNA polymerase γ: utilitzation of dUTP and dTTP during<i>in vitro</i>DNA synthesis

Mosbaugh, Dale W.

doi:10.1093/nar/16.12.5645

Cited by 47 publications

(48 citation statements)

References 35 publications

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“…Such a scenario could also help to explain the apparently poor recovery of second subunit with respect to the catalytic subunit (8). Additionally, because only the catalytic subunit is sensitive to inhibition by NEM, we believe that previous reports of NEM sensitivity for DNA polymerase ␥ are the natural conclusions for preparations containing excess catalytic subunit (6,8,24).…”

Section: Discussionmentioning

confidence: 84%

The Mitochondrial p55 Accessory Subunit of Human DNA Polymerase γ Enhances DNA Binding, Promotes Processive DNA Synthesis, and Confers N-Ethylmaleimide Resistance

Lim¹,

Longley²,

Copeland

1999

Journal of Biological Chemistry

200

252

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Human DNA polymerase ␥ is composed of a 140-kDa catalytic subunit and a smaller accessory protein variously reported to be 43-54 kDa. Immunoblot analysis of the purified, heterodimeric native human polymerase ␥ complex identified the accessory subunit as 55 kDa. We isolated the full-length cDNA encoding a 55-kDa polypeptide, expressed the cDNA in Escherichia coli and purified the 55-kDa protein to homogeneity. Recombinant Hp55 forms a high affinity, salt-stable complex with Hp140 during protein affinity chromatography. Immunoprecipitation, gel filtration, and sedimentation analyses revealed a 190-kDa complex indicative of a native heterodimer. Reconstitution of Hp140⅐Hp55 raises the salt optimum of Hp140, stimulates the polymerase and exonuclease activities, and increases the processivity of the enzyme by several 100-fold. Similar to Hp140, isolated Hp55 binds DNA with moderate strength and was a specificity for double-stranded primer-template DNA. However, Hp140⅐Hp55 has a surprisingly high affinity for DNA, and kinetic analyses indicate Hp55 enhances the affinity of Hp140 for primer termini by 2 orders of magnitude. Thus the enhanced DNA binding caused by Hp55 is the basis for the salt tolerance and high processivity characteristic of DNA polymerase ␥. Observation of native DNA polymerase ␥ both as an Hp140 monomer and as a heterodimer with Hp55 supports the notion that the two forms act in mitochondrial DNA repair and replication. Additionally, association of Hp55 with Hp140 protects the polymerase from inhibition by N-ethylmaleimide.Human mitochondrial DNA is a 16,569-base pair closed circular molecule encoding 13 polypeptides required for oxidative phosphorylation and 24 specialized tRNA and rRNAs needed for translation within the organelle (for review see Refs. 1 and 2). Point mutations or deletions in mitochondrial DNA cause of a wide range of neurological and cardiopathological diseases (3). The human mitochondrial genome is replicated by the nuclear encoded DNA polymerase ␥ (4). DNA polymerase ␥ has been characterized as a processive, salt-tolerant, dideoxynucleotide-sensitive, aphidicolin-resistant, Family A type DNA polymerase that can utilize a wide variety of DNA substrates including poly(rA)⅐oligo(dT) (5). Animal cell DNA polymerase ␥ from Drosophila melanogaster was first shown unequivocally by Wernette and Kaguni (6) to consist of two subunits of 125 and 35 kDa in the highly purified fraction. Highly purified DNA polymerase ␥ from Xenopus laevis contains two subunit of 140 and 50 kDa (7). An initial report on human HeLa cell DNA polymerase ␥ identified a 140-kDa polypeptide and a 54-kDa polypeptide in the most purified fraction (8).We have previously cloned the cDNAs for the human, chicken, Drosophila, and Schizosaccharomyces pombe DNA polymerase ␥ catalytic subunit (9, 10). The overexpressed human 140-kDa catalytic subunit contains DNA polymerase, 3Ј 3 5Ј exonuclease, and 5ЈdRP lyase activities in the absence of the accessory subunit (11, 12). Native heterodimeric Drosophila polymerase ␥ is high...

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Section: Discussionmentioning

confidence: 84%

The Mitochondrial p55 Accessory Subunit of Human DNA Polymerase γ Enhances DNA Binding, Promotes Processive DNA Synthesis, and Confers N-Ethylmaleimide Resistance

Lim¹,

Longley²,

Copeland

1999

Journal of Biological Chemistry

200

252

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“…In mammalian cells, DNA polymerases ␣ and ␤, which function predominantly in chromosomal replication and repair, respectively, have been reported to exhibit similar kinetic constants for dTTP and dUTP (22). In contrast, DNA polymerase ␥, the principal replication enzyme in mitochondria, showed a significantly higher affinity for dTTP than for dUTP (17). Moreover, several thermostable DNA polymerases use dUTP highly inefficiently during PCR amplification (23).…”

Section: Discussionmentioning

confidence: 99%

“…In bacteria, a considerable amount of dUTP is produced because it is an obligatory intermediate in the de novo synthesis of dTMP (16). This fact, together with the ability of numerous prokaryotic DNA polymerases to use dUTP in place of dTTP during DNA synthesis (17,18), make incorporation of uracil into the bacterial chromosome unavoidable.…”

Section: Detection Of Uracil Residues In 29 Dna Molecules Synthesizedmentioning

confidence: 99%

Phage φ29 protein p56 prevents viral DNA replication impairment caused by uracil excision activity of uracil-DNA glycosylase

Serrano‐Heras

Bravo

Salas

2008

Proc. Natl. Acad. Sci. U.S.A.

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Protein p56 encoded by the Bacillus subtilis phage 29 inhibits host uracil-DNA glycosylase (UDG) activity. In previous studies, we suggested that this inhibition is likely a defense mechanism developed by phage 29 to prevent the action of UDG if uracilation occurs in DNA either from deamination of cytosine or the incorporation of dUMP during viral DNA replication. In this work, we analyzed the ability of 29 DNA polymerase to insert dUMP into DNA. Primer extension analysis showed that viral DNA polymerase incorporates dU opposite dA with a catalytic efficiency only 2-fold lower than that for dT. Using the 29 DNA amplification system, we found that 29 DNA polymerase is also able to carry out the extension of the dA:dUMP pair and replicate past uracil. Additionally, UDG and apurinic-apyrimidinic endonuclease treatment of viral DNA isolated from 29-infected cells revealed that uracil residues arise in 29 DNA during replication, probably as a result of misincorporation of dUMP by the 29 DNA polymerase. On the other hand, the action of UDG on uracil-containing 29 DNA impaired in vitro viral DNA replication, which was prevented by the presence of protein p56. Furthermore, transfection activity of uracil-containing 29 DNA was significantly higher in cells that constitutively synthesized p56 than in cells lacking this protein. Thus, our data support a model in which protein p56 ensures an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracil residues present in the 29 genome.29 DNA polymerase ͉ DNA repair ͉ protein-primed replication ͉ dUMP incorporation

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“…In bacteria such as Escherichia coli and Salmonella typhimurium, dUTP biosynthesis is an obligatory intermediate in the de novo synthesis of dTTP [3][4][5]. Since DNA polymerases can utilize dUTP in place of dTTP [6,7], some incorporation of dUMP into the bacterial chromosome is unavoidable. Studies performed in vivo or with cell-free systems have shown that the frequency of dUMP incorporation depends largely on the relative sizes of the intracellular dUTP and dTTP pools [8,9].…”

Section: Introductionmentioning

confidence: 99%

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

et al. 2006

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The steady-state levels of uracil residues in DNA extracted from strains of Escherichia coli were measured and the influence of defects in the genes for uracil-DNA glycosylase (ung), doublestrand uracil-DNA glycosylase (dug), and dUTP pyrophosphatase (dut) on uracil accumulation was determined. A sensitive method, called the Ung-ARP assay, was developed that utilized E. coli Ung, T4pdg, and the Aldehyde Reactive Probe reagent to label a basic sites resulting from uracil excision with biotin. The limit of detection was one uracil residue per million DNA nucleotides (U/10 6 nt). Uracil levels in the genomic DNA of E. coli JM105 (ung + dug + ) were at the limit of detection, as were those of an isogenic dug mutant, regardless of growth phase. Inactivation of ung in JM105 resulted in 31 ± 2.6 U/10 6 nt during early log growth and 19 ± 1.7 U/ 10 6 nt in saturated phase. An ung dug double mutant (CY11) accumulated 33 ± 2.9 U/10 6 nt and 23 ± 1.8 U/10 6 nt during early log and saturated phase growth, respectively. When cultures of CY11 were supplemented with 20 ng/ml of 5-fluoro-2′-deoxyuridine, uracil levels in early log phase growth DNA rose to 125 ± 1.7 U/10 6 nt. Deoxyuridine supplementation reduced the amount of uracil in CY11 DNA, but uridine did not. Levels of uracil in DNA extracted from CJ236 (dut-1 ung-1) were determined to be 3000-8000 U/10 6 nt as measured by the Ung-ARP assay, twodimensional thin-layer chromatography of metabolically-labeled 32 P DNA, and LC/MS of uracil and thymine deoxynucleosides. DNA sequencing revealed that the sole molecular defect in the CJ236 dUTP pyrophosphatase gene was a C→T transition mutation that resulted in a Thr24Ile amino acid change.

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Purification and characterization of porcine liver DNA polymerase γ: utilitzation of dUTP and dTTP duringin vitroDNA synthesis

Cited by 47 publications

References 35 publications

The Mitochondrial p55 Accessory Subunit of Human DNA Polymerase γ Enhances DNA Binding, Promotes Processive DNA Synthesis, and Confers N-Ethylmaleimide Resistance

The Mitochondrial p55 Accessory Subunit of Human DNA Polymerase γ Enhances DNA Binding, Promotes Processive DNA Synthesis, and Confers N-Ethylmaleimide Resistance

Phage φ29 protein p56 prevents viral DNA replication impairment caused by uracil excision activity of uracil-DNA glycosylase

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

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