T7 DNA Polymerase

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C Richardson - One of the best experts on this subject based on the ideXlab platform.

  • catalytically inactive T7 DNA Polymerase imposes a lethal replication roadblock
    Journal of Biological Chemistry, 2020
    Co-Authors: Alfredo J Hernandez, Seungwoo Chang, Joseph J Loparo, C Richardson
    Abstract:

    : Bacteriophage T7 encodes its own DNA Polymerase, the product of gene 5 (gp5). In isolation, gp5 is a DNA Polymerase of low processivity.  However, gp5 becomes highly processive upon formation of a complex with Escherichia coli thioredoxin, the product of the trxA gene. Expression of a gp5 variant in which aspartate residues in the metal-binding site of the Polymerase domain were replaced by alanine is highly toxic to E. coli cells. This toxicity is dependent on the presence of a functional E. coli trxA allele and T7 RNA Polymerase-driven expression but independent of the exonuclease activity of gp5. In vitro, the purified gp5 variant is devoid of any detectable Polymerase activity and inhibited DNA synthesis by the replisomes of E. coli and T7 in the presence of thioredoxin by forming a stable complex with DNA that prevents replication. On the other hand, the highly homologous Klenow fragment of DNA Polymerase I containing an engineered gp5 thioredoxin-binding domain did exhibit any toxicity. We conclude that gp5 alleles encoding inactive Polymerases, in combination with thioredoxin, could be useful as a shutoff mechanism in the design of a bacterial cell-growth system.

  • the c terminal residues of bacteriophage T7 gene 4 helicase primase coordinate helicase and DNA Polymerase activities
    Journal of Biological Chemistry, 2006
    Co-Authors: Boriana Marintcheva, Samir M. Hamdan, C Richardson
    Abstract:

    Abstract 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.

  • a unique loop in T7 DNA Polymerase mediates the binding of helicase primase DNA binding protein and processivity factor
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Samir M. Hamdan, Stanley Tabor, Boriana Marintcheva, Timothy R Cook, C Richardson
    Abstract:

    Bacteriophage T7 DNA Polymerase (gene 5 protein, gp5) interacts with its processivity factor, Escherichia coli thioredoxin, via a unique loop at the tip of the thumb subdomain. We find that this thioredoxin-binding domain is also the site of interaction of the phage-encoded helicase/primase (gp4) and ssDNA binding protein (gp2.5). Thioredoxin itself interacts only weakly with gp4 and gp2.5 but drastically enhances their binding to gp5. The acidic C termini of gp4 and gp2.5 are critical for this interaction in the absence of DNA. However, the C-terminal tail of gp4 is not required for binding to gp5 when the latter is bound to a primer/template. We propose that the thioredoxin-binding domain is a molecular switch that regulates the interaction of T7 DNA Polymerase with other proteins of the replisome.

  • a complex of the bacteriophage T7 primase helicase and DNA Polymerase directs primer utilization
    Journal of Biological Chemistry, 2001
    Co-Authors: Masato Kato, Stanley Tabor, C Richardson, David N Frick, Tom Ellenberger
    Abstract:

    Next Section Abstract The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA Polymerase that elongates them.Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA Polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA Polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA Polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA Polymerase and thereby delivers the RNA primer to the Polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA Polymerase.

  • the acidic carboxyl terminus of the bacteriophage T7 gene 4 helicase primase interacts with T7 DNA Polymerase
    Journal of Biological Chemistry, 1997
    Co-Authors: Stephen M. Notarnicola, Henry L. Mulcahy, Joonsoo Lee, C Richardson
    Abstract:

    The gene 4 proteins of bacteriophage T7 provide both primase and helicase activities at the replication fork. Efficient DNA replication requires that the functions of the gene 4 protein be coordinated with the movement of the T7 DNA Polymerase. We show that a carboxyl-terminal domain of the gene 4 protein is required for interaction with T7 DNA Polymerase during leading strand DNA synthesis. The carboxyl terminus of the gene 4 protein is highly acidic: of the 17 carboxyl-terminal amino acids 7 are negatively charged. Deletion of the coding region for these 17 residues results in a gene 4 protein that cannot support the growth of T7 phage. The purified mutant gene 4 protein has wild-type levels of both helicase and primase activities; however, DNA synthesis catalyzed by T7 DNA Polymerase on a duplex DNA substrate is stimulated by this mutant protein to only about 5% of the level of synthesis obtained with wild-type protein. The mutant gene 4 protein can form hexamers and bind single-stranded DNA, but as determined by native PAGE analysis, the protein cannot form a stable complex with the DNA Polymerase. The mutant gene 4 protein can prime DNA synthesis normally, indicating that for lagging strand synthesis a different set of helicase/primase-DNA Polymerase interactions are involved. These findings have implications for the mechanisms coupling leading and lagging strand DNA synthesis at the T7 replication fork.

Tom Ellenberger - One of the best experts on this subject based on the ideXlab platform.

  • Functional evidence for a small and rigid active site in a high fidelity DNA Polymerase: probing T7 DNA Polymerase with variably sized base pairs.
    Journal of Biological Chemistry, 2005
    Co-Authors: Tae Woo Kim, Tom Ellenberger, Luis G. Brieba, Eric T. Kool
    Abstract:

    Hypotheses on the origins of high fidelity in replicative DNA Polymerases have recently focused on the importance of geometric or steric effects in this selectivity. Here we reported a systematic study of the effects of base pair size in T7 DNA Polymerase (pol), the replicative enzyme for bacteriophage T7. We varied base pair size in very small (0.25 A) increments by use of a series of nonpolar thymidine shape mimics having gradually increasing size. Steady-state kinetics were evaluated for the 5A7A exonuclease-deficient mutant in a 1:1 complex with thioredoxin. For T7 pol, we studied insertion of natural nucleotides opposite variably sized T analogs in the template and, conversely, for variably sized dTTP analogs opposite natural template bases. The enzyme displayed extremely high selectivity for a specific base pair size, with drops in efficiency of as much as 280-fold for increases of 0.4 A beyond an optimum size approximating the size of a natural pair. The enzyme also strongly rejected pairs that were smaller than the optimum by as little as 0.3 A. The size preferences with T7 DNA pol were generally smaller, and the steric rejection was greater than DNA pol I Klenow fragment, correlating with the higher fidelity of the former. The hypothetical effects of varied active site size and rigidity are discussed. The data lend direct support to the concept that active site tightness is a chief determinant of high fidelity of replicative Polymerases and that a less rigid (looser) and larger active site can lead to lower fidelity.

  • A Lysine Residue in the Fingers Subdomain of T7 DNA Polymerase Modulates the Miscoding Potential of 8-Oxo-7,8-Dihydroguanosine
    Structure, 2005
    Co-Authors: Luis G. Brieba, Thomas A. Kunkel, Robert J. Kokoska, Katarzyna Bebenek, Tom Ellenberger
    Abstract:

    Summary 8-Oxo-7,8-dihydroguanosine (8oG) is a highly mutagenic DNA lesion that stably pairs with adenosine, forming 8oG( syn )·dA( anti ) Hoogsteen base pairs. DNA Polymerases show different propensities to insert dCMP or dAMP opposite 8oG, but the molecular mechanisms that determine faithful or mutagenic bypass are poorly understood. Here, we report kinetic and structural data providing evidence that, in T7 DNA Polymerase, residue Lys536 is responsible for attenuating the miscoding potential of 8oG. The Lys536Ala Polymerase shows a significant increase in mutagenic 8oG bypass versus wild-type Polymerase, and a crystal structure of the Lys536Ala mutant reveals a closed complex with an 8oG( syn )·dATP mismatch in the Polymerase active site, in contrast to the unproductive, open complex previously obtained by using wild-type Polymerase. We propose that Lys536 acts as a steric and/or electrostatic filter that attenuates the miscoding potential of 8oG by normally interfering with the binding of 8oG in a syn conformation that pairs with dATP.

  • Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA Polymerase reveal mechanisms of mutagenesis
    Proceedings of the National Academy of Sciences, 2004
    Co-Authors: Shuchismita Dutta, Charles C. Richardson, L J Romano, Donald E. Johnson, Leonid Dzantiev, Tom Ellenberger
    Abstract:

    The carcinogen 2-acetylaminofluorene forms two major DNA adducts: N-(2′-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and its deacetylated derivative, N-(2′-deoxyguanosin-8-yl)-2-aminofluorene (dG-AF). Although the dG-AAF and dG-AF adducts are distinguished only by the presence or absence of an acetyl group, they have profoundly different effects on DNA replication. dG-AAF poses a strong block to DNA synthesis and primarily induces frameshift mutations in bacteria, resulting in the loss of one or two nucleotides during replication past the lesion. dG-AF is less toxic and more easily bypassed by DNA Polymerases, albeit with an increased frequency of misincorporation opposite the lesion, primarily resulting in G → T transversions. We present three crystal structures of bacteriophage T7 DNA Polymerase replication complexes, one with dG-AAF in the templating position and two others with dG-AF in the templating position. Our crystallographic data suggest why a dG-AAF adduct blocks replication more strongly than does a dG-AF adduct and provide a possible explanation for frameshift mutagenesis during replication bypass of a dG-AAF adduct. The dG-AAF nucleoside adopts a syn conformation that facilitates the intercalation of its fluorene ring into a hydrophobic pocket on the surface of the fingers subdomain and locks the fingers in an open, inactive conformation. In contrast, the dG-AF base at the templating position is not well defined by the electron density, consistent with weak binding to the Polymerase and a possible interchange of this adduct between the syn and anti conformations.

  • a molecular handoff between bacteriophage T7 DNA primase and T7 DNA Polymerase initiates DNA synthesis
    Journal of Biological Chemistry, 2004
    Co-Authors: Masato Kato, Gerhard Wagner, Tom Ellenberger
    Abstract:

    Abstract The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA Polymerase but only on the condition that the primase stabilizes the primed DNA template in the Polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA Polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 single-stranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA Polymerase and preventing a productive interaction with the primed template. We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the Polymerase on the lagging strand of the replication fork.

  • Structure of the gene 2.5 protein, a single-stranded DNA binding protein encoded by bacteriophage T7
    Proceedings of the National Academy of Sciences, 2001
    Co-Authors: Thomas Hollis, Charles C. Richardson, James M. Stattel, Dane S. Walther, Tom Ellenberger
    Abstract:

    Abstract The gene 2.5 protein (gp2.5) of bacteriophage T7 is a single-stranded DNA (ssDNA) binding protein that has essential roles in DNA replication and recombination. In addition to binding DNA, gp2.5 physically interacts with T7 DNA Polymerase and T7 primase-helicase during replication to coordinate events at the replication fork. We have determined a 1.9-A crystal structure of gp2.5 and show that it has a conserved OB-fold (oligosaccharide/oligonucleotide binding fold) that is well adapted for interactions with ssDNA. Superposition of the OB-folds of gp2.5 and other ssDNA binding proteins reveals a conserved patch of aromatic residues that stack against the bases of ssDNA in the other crystal structures, suggesting that gp2.5 binds to ssDNA in a similar manner. An acidic C-terminal extension of the gp2.5 protein, which is required for dimer formation and for interactions with the T7 DNA Polymerase and the primase-helicase, appears to be flexible and may act as a switch that modulates the DNA binding affinity of gp2.5.

Charles C. Richardson - One of the best experts on this subject based on the ideXlab platform.

  • Hydrophobic Residue in Escherichia coli Thioredoxin Critical for the Processivity of T7 DNA Polymerase.
    Biochemistry, 2018
    Co-Authors: Seung-joo Lee, Ngoc Q. Tran, Joseph A. Lee, Charles C. Richardson
    Abstract:

    Bacteriophage T7 uses the thioredoxin of its host, Escherichia coli, to enhance the processivity of its DNA Polymerase, a requirement for the growth of phage T7. The evolutionarily conserved structure and high degree of homology of amino acid sequence of the thioredoxin family imply that homologues from other organisms might also interact with T7 DNA Polymerase to support the phage growth. Despite the structural resemblance, human thioredoxin, whose X-ray crystallographic structure overlaps with that of the E. coli protein, cannot support T7 phage growth. It does not form a complex with T7 DNA Polymerase as determined by surface plasmon resonance and thus does not increase the processivity. Homologous scanning analysis using this nonfunctional homologue reveals that the 60 N-terminal and the 12 C-terminal amino acid residues of E. coli thioredoxin can be substituted for its human counterpart without significantly affecting phage growth. Comparison of chimeric thioredoxins, followed by site-directed mutage...

  • Thioredoxin suppresses microscopic hopping of T7 DNA Polymerase on duplex DNA
    Proceedings of the National Academy of Sciences, 2010
    Co-Authors: Candice M. Etson, Charles C. Richardson, Samir M. Hamdan, Antoine M. Van Oijen
    Abstract:

    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.

  • 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
    Journal of Biological Chemistry, 2009
    Co-Authors: Sharmistha Ghosh, Boriana Marintcheva, Masateru Takahashi, Charles C. Richardson
    Abstract:

    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.

  • Rescue of Bacteriophage T7 DNA Polymerase of Low Processivity by Suppressor Mutations Affecting Gene 3 Endonuclease
    Journal of Virology, 2009
    Co-Authors: Seung-joo Lee, Stanley Tabor, Kajal Chowdhury, Charles C. Richardson
    Abstract:

    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.

  • Linear Diffusion of T7 DNA Polymerase: Thioredoxin is Required to Maintain Close Contact with DNA
    Biophysical Journal, 2009
    Co-Authors: Candice M. Etson, Charles C. Richardson, Samir M. Hamdan, Antoine M. Van Oijen
    Abstract:

    The bacteriophage T7 DNA Polymerase consists of a tight, 1:1 complex of T7 gp5, encoded by the phage, and thioredoxin, produced by the E. coli host. In the absence of thioredoxin, gp5 is capable of adding only a few nucleotides to the 3′ end of a primer before dissociating from the primer-template. But when complexed with thioredoxin, gp5 becomes highly processive, capable of polymerizing thousands of nucleotides complementary to the template strand. The mechanism by which thioredoxin acts as a processivity factor to gp5 is not fully understood. To understand the role of the thioredoxin in stabilizing Polymerase-DNA interactions, we use a single-molecule imaging approach to observe individual, fluorescently labeled T7 DNA Polymerase complexes diffusing along double-stranded DNA. Our results show that the average diffusion coefficient of T7 DNA Polymerase complexes is insensitive to ionic strength and does not exceed the theoretical diffusion limit for a protein that tracks the helical pitch and rotates as it diffuses along the DNA helix. These results suggest that the T7 DNA Polymerase slides along the DNA, remaining tightly bound to the DNA and tracking the helical pitch. However, the mean diffusion coefficients for fluorescently labeled T7 gp5 in the absence of thioredoxin increase with salt concentration, and exceed the theoretical limit for a protein tracking the DNA helix. Upon addition of unlabeled thioredoxin, the mean diffusion coefficient is restored to the value observed for the labeled T7 DNA Polymerase, and becomes salt independent. These observations indicate that, in the absence of thioredoxin, T7 gp5 intermittently dissociates from the DNA as it diffuses, and that thioredoxin binding suppresses microscopic hopping on and off the DNA.

Stanley Tabor - One of the best experts on this subject based on the ideXlab platform.

  • Rescue of Bacteriophage T7 DNA Polymerase of Low Processivity by Suppressor Mutations Affecting Gene 3 Endonuclease
    Journal of Virology, 2009
    Co-Authors: Seung-joo Lee, Stanley Tabor, Kajal Chowdhury, Charles C. Richardson
    Abstract:

    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.

  • Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine.
    Proceedings of the National Academy of Sciences, 2008
    Co-Authors: Ngoc Q. Tran, Lisa F. Rezende, Charles C. Richardson, Udi Qimron, Stanley Tabor
    Abstract:

    Bacteriophage T7 DNA Polymerase efficiently incorporates dideoxynucleotides into DNA, resulting in chain termination. Dideoxythymidine (ddT) present in the medium at levels not toxic to Escherichia coli inhibits phage T7. We isolated 95 T7 phage mutants that were resistant to ddT. All contained a mutation in T7 gene 1.7, a nonessential gene of unknown function. When gene 1.7 was expressed from a plasmid, T7 phage resistant to ddT still arose; analysis of 36 of these mutants revealed that all had a single mutation in gene 5, which encodes T7 DNA Polymerase. This mutation changes tyrosine-526 to phenylalanine, which is known to increase dramatically the ability of T7 DNA Polymerase to discriminate against dideoxynucleotides. DNA synthesis in cells infected with wild-type T7 phage was inhibited by ddT, suggesting that it resulted in chain termination of DNA synthesis in the presence of gene 1.7 protein. Overexpression of gene 1.7 from a plasmid rendered E. coli cells sensitive to ddT, indicating that no other T7 proteins are required to confer sensitivity to ddT.

  • a unique loop in T7 DNA Polymerase mediates the binding of helicase primase DNA binding protein and processivity factor
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Samir M. Hamdan, Stanley Tabor, Boriana Marintcheva, Timothy R Cook, C Richardson
    Abstract:

    Bacteriophage T7 DNA Polymerase (gene 5 protein, gp5) interacts with its processivity factor, Escherichia coli thioredoxin, via a unique loop at the tip of the thumb subdomain. We find that this thioredoxin-binding domain is also the site of interaction of the phage-encoded helicase/primase (gp4) and ssDNA binding protein (gp2.5). Thioredoxin itself interacts only weakly with gp4 and gp2.5 but drastically enhances their binding to gp5. The acidic C termini of gp4 and gp2.5 are critical for this interaction in the absence of DNA. However, the C-terminal tail of gp4 is not required for binding to gp5 when the latter is bound to a primer/template. We propose that the thioredoxin-binding domain is a molecular switch that regulates the interaction of T7 DNA Polymerase with other proteins of the replisome.

  • A unique region in bacteriophage T7 DNA Polymerase important for exonucleolytic hydrolysis of DNA.
    Journal of Biological Chemistry, 2004
    Co-Authors: Jaya K. Kumar, Stanley Tabor, Erica T. Chiu, Charles C. Richardson
    Abstract:

    Abstract A crystal structure of the bacteriophage T7 gene 5 protein/Escherichia coli thioredoxin complex reveals a region in the exonuclease domain (residues 144-157) that is not present in other members of the E. coli DNA Polymerase I family. To examine the role of this region, a genetically altered enzyme that lacked residues 144-157 (T7 Polymerase (pol) Δ144-157) was purified and characterized biochemically. The Polymerase activity and processivity of T7 pol Δ144-157 on primed M13 DNA are similar to that of wild-type T7 DNA Polymerase implying that these residues are not important for DNA synthesis. The ability of T7 pol Δ144-157 to catalyze the hydrolysis of a phosphodiester bond, as judged from the rate of hydrolysis of a p-nitrophenyl ester of thymidine monophosphate, also remains unaffected. However, the 3′-5′ exonuclease activity on polynucleotide substrates is drastically reduced; exonuclease activity on single-stranded DNA is 10-fold lower and that on double-stranded DNA is 20-fold lower as compared with wild-type T7 DNA Polymerase. Taken together, our results suggest that residues 144-157 of gene 5 protein, although not crucial for Polymerase activity, are important for DNA binding during hydrolysis of polynucleotides.

  • A Mutation in the gene-encoding bacteriophage T7 DNA Polymerase that renders the phage temperature-sensitive.
    Journal of Biological Chemistry, 2001
    Co-Authors: Jaya K. Kumar, Stanley Tabor, Robin Kremsdorf, Charles C. Richardson
    Abstract:

    Abstract Gene 5 of bacteriophage T7 encodes a DNA Polymerase essential for phage replication. A single point mutation in gene 5 confers temperature sensitivity for phage growth. The mutation results in an alanine to valine substitution at residue 73 in the exonuclease domain. Upon infection of Escherichia coli by the temperature-sensitive phage at 42 °C, there is no detectable T7 DNA synthesis in vivo. DNA Polymerase activity in these phage-infected cell extracts is undetectable at assay temperatures of 30 °C or 42 °C. Upon infection at 30 °C, both DNA synthesisin vivo and DNA Polymerase activity in cell extracts assayed at 30 °C or 42 °C approach levels observed using wild-type T7 phage. The amount of soluble gene 5 protein produced at 42 °C is comparable to that produced at 30 °C, indicating that the temperature-sensitive phenotype is not due to reduced expression, stability, or solubility. Thus the Polymerase induced at elevated temperatures by the temperature-sensitive phage is functionally inactive. Consistent with this observation, biochemical properties and heat inactivation profiles of the genetically altered enzyme over-produced at 30 °C closely resemble that of wild-type T7 DNA Polymerase. It is likely that the Polymerase produced at elevated temperatures is a misfolded intermediate in its folding pathway.

Boriana Marintcheva - One of the best experts on this subject based on the ideXlab platform.

  • 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
    Journal of Biological Chemistry, 2009
    Co-Authors: Sharmistha Ghosh, Boriana Marintcheva, Masateru Takahashi, Charles C. Richardson
    Abstract:

    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.

  • the c terminal residues of bacteriophage T7 gene 4 helicase primase coordinate helicase and DNA Polymerase activities
    Journal of Biological Chemistry, 2006
    Co-Authors: Boriana Marintcheva, Samir M. Hamdan, C Richardson
    Abstract:

    Abstract 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.

  • a unique loop in T7 DNA Polymerase mediates the binding of helicase primase DNA binding protein and processivity factor
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Samir M. Hamdan, Stanley Tabor, Boriana Marintcheva, Timothy R Cook, C Richardson
    Abstract:

    Bacteriophage T7 DNA Polymerase (gene 5 protein, gp5) interacts with its processivity factor, Escherichia coli thioredoxin, via a unique loop at the tip of the thumb subdomain. We find that this thioredoxin-binding domain is also the site of interaction of the phage-encoded helicase/primase (gp4) and ssDNA binding protein (gp2.5). Thioredoxin itself interacts only weakly with gp4 and gp2.5 but drastically enhances their binding to gp5. The acidic C termini of gp4 and gp2.5 are critical for this interaction in the absence of DNA. However, the C-terminal tail of gp4 is not required for binding to gp5 when the latter is bound to a primer/template. We propose that the thioredoxin-binding domain is a molecular switch that regulates the interaction of T7 DNA Polymerase with other proteins of the replisome.