The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on
            <i>Escherichia coli</i>
            DNA polymerase I

Bedford, Ella; Tabor, Stanley; Richardson, Charles C.

doi:10.1073/pnas.94.2.479

Cited by 83 publications

(49 citation statements)

References 23 publications

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“…trx was overproduced in E. coli strain A307(DE3) that does not express trxA and then purified using the procedures described previously (2,12). gp2.5 was purified from E. coli BL21(DE3)pLysS cells overexpressing gene 2.5 as previously described (21). gp4 was overproduced and purified as described (15).…”

Section: Preparation Of Plasmids and Overproduction And Purification mentioning

confidence: 99%

Two Modes of Interaction of the Single-stranded DNA-binding Protein of Bacteriophage T7 with the DNA Polymerase-Thioredoxin Complex

Ghosh

Hamdan²,

Richardson

2010

Journal of Biological Chemistry

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The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxinbinding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD. gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions.Protein-protein interactions are essential for coordination of the multiple reactions that occur at a replication fork. The economy of proteins involved in bacteriophage T7 DNA replication has made it an attractive model for the study of these interactions. T7 DNA polymerase (gp5), an 80-kDa product of gene 5 of the phage, forms a tight 1:1 complex with the host protein, thioredoxin (trx) 3 (1, 2). This interaction stimulates gp5 activity by increasing the processivity of nucleotide polymerization (3-5). gp5/trx physically interacts with the hexameric gene 4 protein (gp4) that possesses both helicase activity, required for unwinding of duplex DNA, and primase activity, required for the initiation of Okazaki fragment synthesis on the lagging strand (1, 6). The interaction of helicase and primase is essential to coordinate DNA synthesis and unwinding on the leading strand and primer handoff to the lagging strand DNA polymerase. The gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that interacts with both gp4 and gp5/trx and stimulates their DNA unwinding and polymerization activities, respectively (7,8). We have recently shown that gp2.5 plays a role in the loading of T7 gp5/trx and helicase at a nick in duplex DNA (9).The communication between gp5, trx, gp4, and gp2.5 is dynamic and complex (4, 5, 10, 11). Although a member of the DNA polymerase I family, gp5 is unique for a 76-amino acid segment located between helices H and H1 in the thumb subdomain (see Fig. 1). Thioredoxin binds with high affinity to this segmen...

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Section: Preparation Of Plasmids and Overproduction And Purification mentioning

confidence: 99%

Two Modes of Interaction of the Single-stranded DNA-binding Protein of Bacteriophage T7 with the DNA Polymerase-Thioredoxin Complex

Ghosh

Hamdan²,

Richardson

2010

Journal of Biological Chemistry

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“…This loop is absent from other members of the Pol I family of DNA polymerases, but when inserted into E. coli DNA polymerase I at the homologous site, the resulting chimera displays a thioredoxin-dependent increase in processivity (7). The TBD also functions as a scaffold for the assembly of the replisome.…”

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confidence: 99%

Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

Etson

Hamdan

Richardson

et al. 2010

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

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The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1∶1 complex with Escherichia coli thioredoxin. By a mechanism that is not fully understood, thioredoxin acts as a processivity factor and converts gp5 from a distributive polymerase into a highly processive one. We use a single-molecule imaging approach to visualize the interaction of fluorescently labeled T7 DNA polymerase with double-stranded DNA. We have observed T7 gp5, both with and without thioredoxin, binding nonspecifically to double-stranded DNA and diffusing along the duplex. The gp5/thioredoxin complex remains tightly bound to the DNA while diffusing, whereas gp5 without thioredoxin undergoes frequent dissociation from and rebinding to the DNA. These observations suggest that thioredoxin increases the processivity of T7 DNA polymerase by suppressing microscopic hopping on and off the DNA and keeping the complex tightly bound to the duplex.T he bacteriophage T7 replisome is an elegantly simple, wellstudied model system of DNA replication (1). Replicative DNA synthesis is supported by T7 DNA polymerase (gp5), in a tight, 1∶1 complex with Escherichia coli thioredoxin, which will henceforth be referred to as gp5/trx. By itself, the 80-kDa gp5 synthesizes DNA in a distributive fashion and is capable of adding only a few nucleotides to the 3 0 end of a primer before dissociating from the primer template (2). Association of gp5 with the 12-kDa E. coli host protein thioredoxin results in a stable 1∶1 complex (K D ¼ 5 nM) and drastically increases the processivity of DNA synthesis. This interaction is critical for replication of T7 DNA-the phage cannot reproduce in thioredoxin null strains of E. coli (3, 4). The affinity of gp5/trx for primer-template DNA is increased at least 20-fold compared to that of gp5 alone (5), resulting in a complex that is capable of polymerizing hundreds of nucleotides before dissociation (2).Thioredoxin binds to gp5 via a 71-amino acid loop that resides between two alpha helices in the thumb domain and is called the thioredoxin-binding domain (TBD) (6). This loop is absent from other members of the Pol I family of DNA polymerases, but when inserted into E. coli DNA polymerase I at the homologous site, the resulting chimera displays a thioredoxin-dependent increase in processivity (7). The TBD also functions as a scaffold for the assembly of the replisome. The binding of thioredoxin structures the domain and creates two small solvent exposed loops containing binding sites for the T7 single-stranded DNA-binding protein and the T7 helicase (8, 9). Thioredoxin itself does not bind to DNA, and only one residue on thioredoxin has been implicated in binding to any of the other replisome components other than gp5 (10).Crystal structures of gp5/trx bound to primer-template DNA reveal that the TBD is extended over the dup...

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“…Genetic and biochemical studies (14,15) have shown that this 76-amino acid segment is responsible for the interaction with trx; deletion of this domain abolishes the ability of the polymerase to bind trx and consequently decreases the rate of polymerization of nucleotides due to the loss of processivity. Insertion of this region into DNA polymerase I of E. coli confers a trx-dependent processivity of the polymerase, indicating that this segment itself is sufficient for binding of trx (16). The 76-amino acid region is, therefore, also known as the thioredoxin binding domain (TBD).…”

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Thioredoxin, the Processivity Factor, Sequesters an Exposed Cysteine in the Thumb Domain of Bacteriophage T7 DNA Polymerase

Tran¹,

Lee²,

Akabayov³

et al. 2012

Journal of Biological Chemistry

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Background: Formation of disulfide bonds between exposed cysteines decreases activity of T7 DNA polymerase. Results: Only Cys-275 and Cys-313 on the thioredoxin binding domain of T7 DNA polymerase are solvent exposed. Conclusion: Cys-313 is critical for the interaction of T7 DNA polymerase with its processivity factor, thioredoxin. Significance: A single cysteine in the thioredoxin binding domain of T7 DNA polymerase is important for activity.

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The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on Escherichia coli DNA polymerase I

Cited by 83 publications

References 23 publications

Two Modes of Interaction of the Single-stranded DNA-binding Protein of Bacteriophage T7 with the DNA Polymerase-Thioredoxin Complex

Two Modes of Interaction of the Single-stranded DNA-binding Protein of Bacteriophage T7 with the DNA Polymerase-Thioredoxin Complex

Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

Thioredoxin, the Processivity Factor, Sequesters an Exposed Cysteine in the Thumb Domain of Bacteriophage T7 DNA Polymerase

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