The Highly Processive DNA Polymerase of Bacteriophage T5

Andraos, Nathalie; Tabor, Stanley; Richardson, Charles C.

doi:10.1074/jbc.m408428200

Cited by 26 publications

(25 citation statements)

References 36 publications

(30 reference statements)

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“…The blunt end primer-template and the primer with six mismatched bases with the same DNA template (mispair-6) are subjected to nucleolytic degradation of the DNA in the absence of PP i . The prominent nucleolytic degradation is due to the preference of the exonuclease activity for doublestranded DNA as shown previously (27). Interestingly, mispair-6 primer undergoes both enhanced nucleolytic degradation and enhanced pyrophosphorolytic activity relative to the primer with three mismatched bases and the primer with the bubble.…”

Section: Effect Of Configuration Of the Primer-template Onmentioning

confidence: 77%

Pyrovanadolysis, a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate, Mn2+, and DNA Polymerase of Bacteriophage T7

Akabayov¹,

Kulczyk²,

Akabayov³

et al. 2011

Journal of Biological Chemistry

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DNA polymerases catalyze the 3-5-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP i ). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP i , a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP i complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn 2؉ , larger than Mg 2؉ , fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.Gene 5 of bacteriophage T7 encodes a DNA polymerase essential for replication of the T7 genome (1). T7 DNA polymerase forms a complex with Escherichia coli thioredoxin (trx), 2 an interaction that increases its processivity of nucleotide polymerization several hundredfold (2). The structure of T7 DNA polymerase in complex with trx is shown in Fig. 1. The structure closely resembles that of E. coli DNA polymerase I, thus placing it in the polymerase I family of DNA polymerases (3). In this study, we designate the polymerase in complex with trx as T7 DNA polymerase. Like most other prokaryotic DNA polymerases, T7 DNA polymerase has a proofreading 3Ј-5Ј-exonuclease activity located in the amino-terminal half of the protein (3). The crystal structure shows that its active site is located between the "fingers" and the "palm" subdomains and contains two magnesium ions (Mg 2ϩ ). During polymerization of nucleotides, the Mg 2ϩ closest to the primer (catalytic Mg 2ϩ ) is responsible for deprotonation of the 3Ј-hydroxyl group of the primer prior to its nucleophilic attack on the ␣-phosphate of the incoming dNTP. The second Mg 2ϩ (structural Mg 2ϩ ) is associated with stabilization of the reactive state of the ␤-and ␥-phosphates of the incoming deoxynucleoside 5Ј-triphosphate (dNTP) (4).In the presence of PP i , DNA polymerases catalyze the 3Ј-5Ј-degradation of a DNA primer annealed to a DNA template (5). In this reaction, known as pyrophosphorolysis, the deoxynucleotides (dNMP) of the DNA are converted to dNTP. Pyrophosphorolysis is a true reversal of the polymerization reaction in that the products are dNTPs, and it has the same requirements for a template and a 3Ј-hydroxyl-terminated primer (5). The 3Ј-5Ј-exonuclease associated with DNA polymerase, however, catalyzes the 3Ј-5Ј hydrolysis of the primer to yield dNMPs without a strict requirement for proper base pairing. Pyrophosphorolysis o...

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Section: Effect Of Configuration Of the Primer-template Onmentioning

confidence: 77%

Pyrovanadolysis, a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate, Mn2+, and DNA Polymerase of Bacteriophage T7

Akabayov¹,

Kulczyk²,

Akabayov³

et al. 2011

Journal of Biological Chemistry

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“…For example, DNA polymerase from T7 bacteriophage requires stimulation by SSB to perform strand displacement DNA synthesis (He et al, 2003;Andraos et al, 2004). Human Polγ requires the helicase Twinkle as well as the accessory subunit PolγB to perform rolling circle DNA synthesis in vitro (Korhonen et al, 2004;Farge et al, 2007).…”

Section: Mip1 Is Capable Of Strand Displacement Dna Synthesismentioning

confidence: 99%

“…In this case, elongation of the primer to products larger than 81 nt would indicate strand displacement synthesis. T7 DNA polymerase was used as a control in the reaction, as it was previously shown to be incapable of strand displacement activity (Andraos et al, 2004). Complete stalling of DNA synthesis at the product size of 81 nt was observed with T7 DNA polymerase (Fig.…”

Section: Mip1 Is Capable Of Strand Displacement Dna Synthesismentioning

confidence: 99%

Yeast mitochondrial DNA polymerase is a highly processive single-subunit enzyme

2011

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“…Both Phi29 and T5 DNA polymerases contain unique domains not found in polymerase superfamilies (15,16), and they alone can generate the ssDNA template for leading strand DNA synthesis. Deletion of these non-conserved regions results in loss of the ability of the polymerases to catalyze strand displacement DNA synthesis (16,17). However, the T7 DNA pol/trx complex, like most other replicative DNA polymerases, requires a DNA helicase to catalyze the unwinding of the duplex DNA.…”

Section: Discussionmentioning

confidence: 99%

The C-terminal Residues of Bacteriophage T7 Gene 4 Helicase-Primase Coordinate Helicase and DNA Polymerase Activities

Lee

Marintcheva

Hamdan

et al. 2006

Journal of Biological Chemistry

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The gene 4 protein of bacteriophage T7 plays a central role in DNA replication by providing both helicase and primase activities. The C-terminal helicase domain is not only responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding, but it also interacts with T7 DNA polymerase to coordinate helicase and polymerase activities. The C-terminal 17 residues of gene 4 protein are critical for its interaction with the T7 DNA polymerase/thioredoxin complex. This C terminus is highly acidic; replacement of these residues with uncharged residues leads to a loss of interaction with T7 DNA polymerase/ thioredoxin and an increase in oligomerization of the gene 4 protein. Such an alteration on the C terminus results in a reduced efficiency in strand displacement DNA synthesis catalyzed by gene 4 protein and T7 DNA polymerase/thioredoxin. Replacement of the C-terminal amino acid, phenylalanine, with non-aromatic residues also leads to a loss of interaction of gene 4 protein with T7 DNA polymerase/thioredoxin. However, neither of these modifications of the C terminus affects helicase and primase activities. A chimeric gene 4 protein containing the acidic C terminus of the T7 gene 2.5 single-stranded DNA-binding protein is more active in strand displacement synthesis. Gene 4 hexamers containing even one subunit of a defective C terminus are defective in their interaction with T7 DNA polymerase.

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The Highly Processive DNA Polymerase of Bacteriophage T5

Cited by 26 publications

References 36 publications

Pyrovanadolysis, a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate, Mn2+, and DNA Polymerase of Bacteriophage T7

Pyrovanadolysis, a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate, Mn2+, and DNA Polymerase of Bacteriophage T7

Yeast mitochondrial DNA polymerase is a highly processive single-subunit enzyme

The C-terminal Residues of Bacteriophage T7 Gene 4 Helicase-Primase Coordinate Helicase and DNA Polymerase Activities

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