Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA (ssDNA)-binding protein (gp2.5) that is an essential component of the phage replisome. Similar to other prokaryotic ssDNA-binding proteins, gp2.5 has an acidic C terminus that is involved in protein-protein interactions at the replication fork and in modulation of the ssDNA binding properties of the molecule. We have used genetic and biochemical approaches to identify residues critical for the function of the C terminus of gp2.5. The presence of an aromatic residue in the C-terminal position is essential for gp2.5 function. Deletion of the C-terminal residue, phenylalanine, is detrimental to its function, as is the substitution of this residue with non-aromatic amino acids. Placing the C-terminal phenylalanine in the penultimate position also results in loss of function. Moderate shortening of the length of the acidic portion of the C terminus is tolerated when the aromatic nature of the C-terminal residue is preserved. Gradual removal of the acidic C terminus of gp2.5 results in a higher affinity for ssDNA and a decreased ability to interact with T7 DNA polymerase/thioredoxin. The replacement of the charged residues in the C terminus with neutral amino acids abolishes gp2.5 function. Our data show that both the C-terminal aromatic residue and the overall acidic charge of the C terminus of gp2.5 are critical for its function.
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PMID:Essential residues in the C terminus of the bacteriophage T7 gene 2.5 single-stranded DNA-binding protein. 1680 32

T7 gene 5 DNA polymerase (gp5) and its processivity factor, Escherichia coli thioredoxin, together with the T7 gene 4 DNA helicase, catalyze strand displacement synthesis on duplex DNA processively (>17,000 nucleotides per binding event). The processive DNA synthesis is resistant to the addition of a DNA trap. However, when the polymerase-thioredoxin complex actively synthesizing DNA is challenged with excess DNA polymerase-thioredoxin exchange occurs readily. The exchange can be monitored by the use of a genetically altered T7 DNA polymerase (gp5-Y526F) in which tyrosine-526 is replaced with phenylalanine. DNA synthesis catalyzed by gp5-Y526F is resistant to inhibition by chain-terminating dideoxynucleotides because gp5-Y526F is deficient in the incorporation of these analogs relative to the wild-type enzyme. The exchange also occurs during coordinated DNA synthesis in which leading- and lagging-strand synthesis occur at the same rate. On ssDNA templates with the T7 DNA polymerase alone, such exchange is not evident, suggesting that free polymerase is first recruited to the replisome by means of T7 gene 4 helicase. The ability to exchange DNA polymerases within the replisome without affecting processivity provides advantages for fidelity as well as the cycling of the polymerase from a completed Okazaki fragment to a new primer on the lagging strand.
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PMID:Exchange of DNA polymerases at the replication fork of bacteriophage T7. 1736 50

Thioredoxin (Trx) is a highly conserved redox protein involved in several essential cellular processes. In this study, our goal was to isolate peptide ligands to Escherichia coli Trx that mimic protein-protein interactions, specifically the T7 polymerase-Trx interaction. To do this, we subjected Trx to affinity selection against a panel of linear and cysteine-constrained peptides using M13 phage display. A novel cyclized conserved peptide sequence, with a motif of C(D/N/S/T/G)D(S/T)-hydrophobic-C-X-hydrophobic-P, was isolated to Trx. These peptides bound specifically to the E. coli Trx when compared to the human and spirulina homologs. An alanine substitution of the active site cysteines (CGPC) resulted in a significant loss of peptide binding affinity to the Cys-32 mutant. The peptides were also characterized in the context of Trx's role as a processivity factor of the T7 DNA polymerase (gp5). As the interaction between gp5 and Trx normally takes place under reducing conditions, which might interfere with the conformation of the disulfide-bridged peptides, we made use of a 22 residue deletion mutant of gp5 in the thioredoxin binding domain (gp5Delta22) that bypassed the requirements of reducing conditions to interact with Trx. A competition study revealed that the peptide selectively inhibits the interaction of gp5Delta22 with Trx, under oxidizing conditions, with an IC50 of approximately 10 microM.
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PMID:Peptide ligands specific to the oxidized form of Escherichia coli thioredoxin. 1867 1

Escherichia coli thioredoxin binds to a unique flexible loop of 71 amino acid residues, designated the thioredoxin binding domain (TBD), located in the thumb subdomain of bacteriophage T7 gene 5 DNA polymerase. The initial designation of thioredoxin as a processivity factor was premature. Rather it remodels the TBD for interaction with DNA and the other replication proteins. The binding of thioredoxin exposes a number of basic residues on the TBD that lie over the duplex region of the primer-template and increases the processivity of nucleotide polymerization. Two small solvent-exposed loops (loops A and B) located within TBD electrostatically interact with the acidic C-terminal tail of T7 gene 4 helicase-primase, an interaction that is enhanced by the binding of thioredoxin. Several basic residues on the surface of thioredoxin in the polymerase-thioredoxin complex lie in close proximity to the TBD. One of these residues, lysine 36, is located proximal to loop A. The substitution of glutamate for lysine has a dramatic effect on the binding of gene 4 helicase to a DNA polymerase-thioredoxin complex lacking charges on loop B; binding is decreased 15-fold relative to that observed with wild-type thioredoxin. This defective interaction impairs the ability of T7 DNA polymerase-thioredoxin together with T7 helicase to mediate strand displacement synthesis. This is the first demonstration that thioredoxin interacts with replication proteins other than T7 DNA polymerase.
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PMID:Interactions of Escherichia coli thioredoxin, the processivity factor, with bacteriophage T7 DNA polymerase and helicase. 1875 58

Gene 2.5 of bacteriophage T7 encodes a ssDNA binding protein (gp2.5) essential for DNA replication. The C-terminal phenylalanine of gp2.5 is critical for function and mutations in that position are dominant lethal. In order to identify gp2.5 interactions we designed a screen for suppressors of gp2.5 lacking the C-terminal phenylalanine. Screening for suppressors of dominant lethal mutations of essential genes is challenging as the phenotype prevents propagation. We select for phage encoding a dominant lethal version of gene 2.5, whose viability is recovered via second-site suppressor mutation(s). Functional gp2.5 is expressed in trans for propagation of the unviable phage and allows suppression to occur via natural selection. The isolated intragenic suppressors support the critical role of the C-terminal phenylalanine. Extragenic suppressor mutations occur in several genes encoding enzymes of DNA metabolism. We have focused on the suppressor mutations in gene 5 encoding the T7 DNA polymerase (gp5) as the gp5/gp2.5 interaction is well documented. The suppressor mutations in gene 5 are necessary and sufficient to suppress the lethal phenotype of gp2.5 lacking the C-terminal phenylalanine. The affected residues map in proximity to aromatic residues and to residues in contact with DNA in the crystal structure of T7 DNA polymerase-thioredoxin.
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PMID:Mutations in the gene 5 DNA polymerase of bacteriophage T7 suppress the dominant lethal phenotype of gene 2.5 ssDNA binding protein lacking the C-terminal phenylalanine. 1940 Jul 98

The DNA polymerase encoded by gene 5 (gp5) of bacteriophage T7 has low processivity, dissociating after the incorporation of a few nucleotides. Upon binding to its processivity factor, Escherichia coli thioredoxin (Trx), the processivity is increased to approximately 800 nucleotides per binding event. Several interactions between gp5/Trx and DNA are required for processive DNA synthesis. A basic region in T7 DNA polymerase (residues K587, K589, R590, and R591) is located in proximity to the 5' overhang of the template strand. Replacement of these residues with asparagines results in a threefold reduction of the polymerization activity on primed M13 single-stranded DNA. The altered gp5/Trx exhibits a 10-fold reduction in its ability to support growth of T7 phage lacking gene 5. However, T7 phages that grow at a similar rate provided with either wild-type or altered polymerase emerge. Most of the suppressor phages contain genetic changes in or around the coding region for gene 3, an endonuclease. Altered gene 3 proteins derived from suppressor strains show reduced catalytic activity and are inefficient in complementing growth of T7 phage lacking gene 3. Results from this study reveal that defects in processivity of DNA polymerase can be suppressed by reducing endonuclease activity.
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PMID:Rescue of bacteriophage T7 DNA polymerase of low processivity by suppressor mutations affecting gene 3 endonuclease. 1953 36

Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5'-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.
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PMID:C-terminal phenylalanine of bacteriophage T7 single-stranded DNA-binding protein is essential for strand displacement synthesis by T7 DNA polymerase at a nick in DNA. 1972 88

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, 11 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.
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PMID:Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA. 2008 Jun 81

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 thioredoxin-binding 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.
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PMID:Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex. 2037 19

Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase. It achieves processivity by binding to Escherichia coli thioredoxin (trx). gp5/trx complex binds tightly to a primer-DNA template enabling the polymerization of hundreds of nucleotides per binding event. gp5 contains 10 cysteines. Under non-reducing condition, exposed cysteines form intermolecular disulfide linkages resulting in the loss of polymerase activity. No disulfide linkage is detected when Cys-275 and Cys-313 are replaced with serines. Cys-275 and Cys-313 are located on loop A and loop B of the thioredoxin binding domain, respectively. Replacement of either cysteine with serine (gp5-C275S, gp5-C313S) drastically decreases polymerase activity of gp5 on dA(350)/dT(25). On this primer-template gp5/trx in which Cys-313 or Cys-275 is replaced with serine have 50 and 90%, respectively, of the polymerase activity observed with wild-type gp5/trx. With single-stranded M13 DNA as a template gp5-C275S/trx retains 60% of the polymerase activity of wild-type gp5/trx. In contrast, gp5-C313S/trx has only one-tenth of the polymerase activity of wild-type gp5/trx on M13 DNA. Both wild-type gp5/trx and gp5-C275S/trx catalyze the synthesis of the entire complementary strand of M13 DNA, whereas gp5-C313S/trx has difficulty in synthesizing DNA through sites of secondary structure. gp5-C313S fails to form a functional complex with trx as measured by the apparent binding affinity as well as by the lack of a physical interaction with thioredoxin during hydroxyapatite-phosphate chromatography. Small angle x-ray scattering reveals an elongated conformation of gp5-C313S in comparison to a compact and spherical conformation of wild-type gp5.
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PMID:Thioredoxin, the processivity factor, sequesters an exposed cysteine in the thumb domain of bacteriophage T7 DNA polymerase. 2301 74


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