Interactions of Escherichia coli Thioredoxin, the Processivity Factor, with Bacteriophage T7 DNA Polymerase and Helicase

Ghosh, Sharmistha; Hamdan, Samir M.; Cook, Timothy E.; Richardson, Charles C.

doi:10.1074/jbc.m805062200

Cited by 33 publications

(32 citation statements)

References 27 publications

(65 reference statements)

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“…T7 DNA polymerase interacts with T7 gp4 via the basic loops of the thioredoxin binding domain (39). The processivity factor thioredoxin binds to the thioredoxin binding domain of gp5 and causes exposure of the basic loops, which stimulate interaction between the helicase and polymerase.…”

Section: Discussionmentioning

confidence: 99%

A257T Linker Region Mutant of T7 Helicase-Primase Protein Is Defective in DNA Loading and Rescued by T7 DNA Polymerase

Patel¹,

Johnson

Sun

et al. 2011

Journal of Biological Chemistry

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The helicase and primase activities of the hexameric ringshaped T7 gp4 protein reside in two separate domains connected by a linker region. This linker region is part of the subunit interface between monomers, and point mutations in this region have deleterious effects on the helicase functions. One such linker region mutant, A257T, is analogous to the A359T mutant of the homologous human mitochondrial DNA helicase Twinkle, which is linked to diseases such as progressive external opthalmoplegia. Electron microscopy studies show that A257T gp4 is normal in forming rings with dTTP, but the rings do not assemble efficiently on the DNA. Therefore, A257T, unlike the WT gp4, does not preassemble on the unwinding DNA substrate with dTTP without Mg(II), and its DNA unwinding activity in ensemble assays is slow and limited by the DNA loading rate. Single molecule assays measured a 45 times slower rate of A257T loading on DNA compared with WT gp4. Interestingly, once loaded, A257T has almost WT-like translocation and DNA unwinding activities. Strikingly, A257T preassembles stably on the DNA in the presence of T7 DNA polymerase, which restores the ensemble unwinding activity of A257T to ϳ75% of WT, and the rescue does not require DNA synthesis. The DNA loading rate of A257T, however, remains slow even in the presence of the polymerase, which explains why A257T does not support T7 phage growth. Similar types of defects in the related human mitochondrial DNA helicase may be responsible for inefficient DNA replication leading to the disease states.

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Section: Discussionmentioning

confidence: 99%

A257T Linker Region Mutant of T7 Helicase-Primase Protein Is Defective in DNA Loading and Rescued by T7 DNA Polymerase

Patel¹,

Johnson

Sun

et al. 2011

Journal of Biological Chemistry

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“…However, when the charges are eliminated in both loops A and B (gp5-loopAB), gp4 fails to bind (5). Elimination of the charges in loops A and B of the TBD does not significantly affect the affinity of trx for the TBD (20).…”

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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“…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).…”

mentioning

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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Interactions of Escherichia coli Thioredoxin, the Processivity Factor, with Bacteriophage T7 DNA Polymerase and Helicase

Cited by 33 publications

References 27 publications

A257T Linker Region Mutant of T7 Helicase-Primase Protein Is Defective in DNA Loading and Rescued by T7 DNA Polymerase

A257T Linker Region Mutant of T7 Helicase-Primase Protein Is Defective in DNA Loading and Rescued by T7 DNA Polymerase

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

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