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John Kuriyan - One of the best experts on this subject based on the ideXlab platform.
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Clamp loaders and sliding clamps.
Current opinion in structural biology, 2002Co-Authors: David Jeruzalmi, Mike O'donnell, John KuriyanAbstract:A coherent view of the structure and function of DNA polymerase Processivity factors (sliding clamps and clamp loaders) is emerging from recent structural studies. Crystal structures of sliding clamps from the T4 and RB69 bacteriophages, and from an archaebacterium expand the gallery of ring-shaped Processivity factors and clarify how the clamp interacts with the DNA polymerase. Crystallographic and electron microscopic views of clamp loaders from bacteria, archaebacteria and eukaryotes emphasize their common architecture and have produced models of how ATPbinding might be coupled to clamp opening/loading.
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crystal structure of the eukaryotic dna polymerase Processivity factor pcna
Cell, 1995Co-Authors: Talluru S R Krishna, John Kuriyan, Xiangpeng Kong, Sonja L Gary, Peter M J BurgersAbstract:Abstract The crystal structure of the Processivity factor required by eukaryotic DNA polymerase δ, proliferating cell nuclear antigen (PCNA) from S. cerevisiae, has been determined at 2.3 A resolution. Three PCNA molecules, each containing two topologically identical domains, are tightly associated to form a closed ring. The dimensions and electrostatic properties of the ring suggest that PCNA encircles duplex DNA, providing a DNA-bound platform for the attachment of the polymerase. The trimeric PCNA ring is strikingly similar to the dimeric ring formed by the β subunit (Processivity factor) of E. coli DNA polymerase III holoenzyme, with which it shares no significant sequence identity. This structural correspondence further substantiates the mechanistic connection between eukaryotic and prokaryotic DNA replication that has been suggested on biochemical grounds.
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crystal structure of the eukaryotic dna polymerase Processivity factor pcna
Cell, 1995Co-Authors: Talluru S R Krishna, John Kuriyan, Xiangpeng Kong, Sonja L Gary, Peter M J BurgersAbstract:The crystal structure of the Processivity factor required by eukaryotic DNA polymerase delta, proliferating cell nuclear antigen (PCNA) from S. cerevisiae, has been determined at 2.3 A resolution. Three PCNA molecules, each containing two topologically identical domains, are tightly associated to form a closed ring. The dimensions and electrostatic properties of the ring suggest that PCNA encircles duplex DNA, providing a DNA-bound platform for the attachment of the polymerase. The trimeric PCNA ring is strikingly similar to the dimeric ring formed by the beta subunit (Processivity factor) of E. coli DNA polymerase III holoenzyme, with which it shares no significant sequence identity. This structural correspondence further substantiates the mechanistic connection between eukaryotic and prokaryotic DNA replication that has been suggested on biochemical grounds.
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three dimensional structure of the β subunit of e coli dna polymerase iii holoenzyme a sliding dna clamp
Cell, 1992Co-Authors: Xiangpeng Kong, John Kuriyan, Mike Odonnell, Rene OnrustAbstract:Abstract The crystal structure of the β subunit (Processivity factor) of DNA polymerase III holoenzyme has been determined at 2.5 A resolution. A dimer of the β subunit (M r = 2 × 40.6 kd, 2 × 366 amino acid residues) forms a ring-shaped structure lined by 12 α helices that can encircle duplex DNA. The structure is highly symmetrical, with each monomer containing three domains of identical topology. The charge distribution and orientation of the helices indicate that the molecule functions by forming a tight clamp that can slide on DNA, as shown biochemically. A potential structural relationship is suggested between the β subunit and proliferating cell nuclear antigen (PCNA, the eukaryotic polymerase δ [and e] Processivity factor), and the gene 45 protein of the bacteriophage T4 DNA polymerase.
Charles C. Richardson - One of the best experts on this subject based on the ideXlab platform.
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Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA
Proceedings of the National Academy of Sciences, 2010Co-Authors: Candice M. Etson, Charles C. Richardson, Samir M. Hamdan, Antoine M. Van OijenAbstract: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.
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Rescue of Bacteriophage T7 DNA Polymerase of Low Processivity by Suppressor Mutations Affecting Gene 3 Endonuclease
Journal of Virology, 2009Co-Authors: Seung-joo Lee, Stanley Tabor, Kajal Chowdhury, Charles C. RichardsonAbstract: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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The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers Processivity on Escherichia coli DNA polymerase I.
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Ella Bedford, Stanley Tabor, Charles C. RichardsonAbstract:Bacteriophage T7 DNA polymerase shares extensive sequence homology with Escherichia coli DNA polymerase I. However, in vivo, E. coli DNA polymerase I is involved primarily in the repair of DNA whereas T7 DNA polymerase is responsible for the replication of the viral genome. In accord with these roles, T7 DNA polymerase is highly processive while E. coli DNA polymerase I has low Processivity. The high Processivity of T7 DNA polymerase is achieved through tight binding to its Processivity factor, E. coli thioredoxin. We have identified a unique 76-residue domain in T7 DNA polymerase responsible for this interaction. Insertion of this domain into the homologous site in E. coli DNA polymerase I results in a dramatic increase in the Processivity of the chimeric DNA polymerase, a phenomenon that is dependent upon its binding to thioredoxin.
Frédéric Iseni - One of the best experts on this subject based on the ideXlab platform.
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The vaccinia virus DNA polymerase structure provides insights into the mode of Processivity factor binding
Nature Communications, 2017Co-Authors: Nicolas Tarbouriech, Corinne Ducournau, Stephanie Hutin, Eric Forest, Darren J. Hart, Christophe N. Peyrefitte, Wim P. Burmeister, Frédéric IseniAbstract:The catalytic subunit E9 of the vaccinia virus DNA polymerase forms a functional polymerase holoenzyme by interacting with the heterodimeric Processivity factor A20/D4. Here the authors present the structure of full-length E9 and show that an insertion within its palm domain binds A20, in a mode different from other family B polymerases. Vaccinia virus (VACV), the prototype member of the Poxviridae , replicates in the cytoplasm of an infected cell. The catalytic subunit of the DNA polymerase E9 binds the heterodimeric Processivity factor A20/D4 to form the functional polymerase holoenzyme. Here we present the crystal structure of full-length E9 at 2.7 Å resolution that permits identification of important poxvirus-specific structural insertions. One insertion in the palm domain interacts with C-terminal residues of A20 and thus serves as the Processivity factor-binding site. This is in strong contrast to all other family B polymerases that bind their co-factors at the C terminus of the thumb domain. The VACV E9 structure also permits rationalization of polymerase inhibitor resistance mutations when compared with the closely related eukaryotic polymerase delta–DNA complex.
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The vaccinia virus DNA polymerase structure provides insights into the mode of Processivity factor binding
Nature Communications, 2017Co-Authors: Nicolas Tarbouriech, Corinne Ducournau, Stephanie Hutin, Eric Forest, Darren J. Hart, Christophe N. Peyrefitte, Wim P. Burmeister, Philippe J. Mas, Petr Man, Frédéric IseniAbstract:Vaccinia virus (VACV), the prototype member of the Poxviridae, replicates in the cytoplasm of an infected cell. The catalytic subunit of the DNA polymerase E9 binds the heterodimeric Processivity factor A20/D4 to form the functional polymerase holoenzyme. Here we present the crystal structure of full-length E9 at 2.7 Å resolution that permits identification of important poxvirus-specific structural insertions. One insertion in the palm domain interacts with C-terminal residues of A20 and thus serves as the Processivity factor-binding site. This is in strong contrast to all other family B polymerases that bind their co-factors at the C terminus of the thumb domain. The VACV E9 structure also permits rationalization of polymerase inhibitor resistance mutations when compared with the closely related eukaryotic polymerase delta-DNA complex.
C Richardson - One of the best experts on this subject based on the ideXlab platform.
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conformational dynamics of bacteriophage t7 dna polymerase and its Processivity factor escherichia coli thioredoxin
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Barak Akabayov, Stanley Tabor, Seung-joo Lee, Sabine R Akabayov, Arkadiusz W Kulczyk, C RichardsonAbstract:Gene 5 of bacteriophage T7 encodes a DNA polymerase (gp5) responsible for the replication of the phage DNA. Gp5 polymerizes nucleotides with low Processivity, dissociating after the incorporation of 1 to 50 nucleotides. Thioredoxin (trx) of Escherichia coli binds tightly (Kd = 5 nM) to a unique segment in the thumb subdomain of gp5 and increases Processivity. We have probed the molecular basis for the increase in Processivity. A single-molecule experiment reveals differences in rates of enzymatic activity and Processivity between gp5 and gp5/trx. Small angle X-ray scattering studies combined with nuclease footprinting reveal two conformations of gp5, one in the free state and one upon binding to trx. Comparative analysis of the DNA binding clefts of DNA polymerases and DNA binding proteins show that the binding surface contains more hydrophobic residues than other DNA binding proteins. The balanced composition between hydrophobic and charged residues of the binding site allows for efficient sliding of gp5/trx on the DNA. We propose a model for trx-induced conformational changes in gp5 that enhance the Processivity by increasing the interaction of gp5 with DNA.
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two modes of interaction of the single stranded dna binding protein of bacteriophage t7 with the dna polymerase thioredoxin complex
Journal of Biological Chemistry, 2010Co-Authors: Sharmistha Ghosh, Samir M. Hamdan, C RichardsonAbstract: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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a covalent linkage between the gene 5 dna polymerase of bacteriophage t7 and escherichia coli thioredoxin the Processivity factor fate of thioredoxin during dna synthesis
Journal of Biological Chemistry, 2003Co-Authors: Donald E Johnson, C RichardsonAbstract:Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase, which acquires high Processivity by binding to Escherichia coli thioredoxin. The gene 5 protein-thioredoxin complex (gp5/trx) polymerizes thousands of nucleotides before dissociating from a primer-template. We have engineered a disulfide linkage between the gene 5 protein and thioredoxin within the binding surface of the two proteins. The polymerase activity of the covalently linked complex (gp5-S-S-trx) is similar to that of gp5/trx on poly(dA)/oligo(dT). However, gp5-S-S-trx has only one third the polymerase activity of gp5/trx on single-stranded M13 DNA. gp5-S-S-trx has difficulty polymerizing nucleotides through sites of secondary structure on M13 DNA and stalls at these sites, resulting in lower Processivity. However, gp5-S-S-trx has an identical Processivity and rate of elongation when E. coli single-stranded DNA-binding protein (SSB protein) is used to remove secondary structure from M13 DNA. Upon completing synthesis on a DNA template lacking secondary structure, both complexes recycle intact, without dissociation of the Processivity factor, to initiate synthesis on a new DNA template. However, a complex stalled at secondary structure becomes unstable, and both subunits dissociate from each other as the polymerase prematurely releases from M13 DNA.
Mike O'donnell - One of the best experts on this subject based on the ideXlab platform.
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Processivity Clamps in DNA Replication
Encyclopedia of Biological Chemistry, 2013Co-Authors: N.y. Yao, Mike O'donnellAbstract:Cells from all domains of life contain ring-shaped sliding clamp proteins that encircle DNA and slide along it. Sliding clamps were initially discovered by their ability to hold the chromosomal replicase to DNA for high Processivity during replication of long chromosomes. Sliding clamps are opened and closed around DNA by a clamp-loading machine driven by ATP hydrolysis. Sliding clamps also bind many other proteins including ligase, mismatch repair factors, certain nucleases, and numerous DNA polymerases involved in DNA repair. We briefly review the structure and function of sliding clamps and clamp-loading machines. We then describe how clamps are utilized by the replicating apparatus to circumvent various obstacles encountered during replication.
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Clamp loaders and sliding clamps.
Current opinion in structural biology, 2002Co-Authors: David Jeruzalmi, Mike O'donnell, John KuriyanAbstract:A coherent view of the structure and function of DNA polymerase Processivity factors (sliding clamps and clamp loaders) is emerging from recent structural studies. Crystal structures of sliding clamps from the T4 and RB69 bacteriophages, and from an archaebacterium expand the gallery of ring-shaped Processivity factors and clarify how the clamp interacts with the DNA polymerase. Crystallographic and electron microscopic views of clamp loaders from bacteria, archaebacteria and eukaryotes emphasize their common architecture and have produced models of how ATPbinding might be coupled to clamp opening/loading.
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Processivity of DNA polymerases: two mechanisms, one goal
Structure, 1998Co-Authors: Zvi Kelman, Jerard Hurwitz, Mike O'donnellAbstract:Replicative DNA polymerases are highly processive enzymes that polymerize thousands of nucleotides without dissociating from the DNA template. The recently determined structure of the Escherichia coli bacteriophage T7 DNA polymerase suggests a unique mechanism that underlies Processivity, and this mechanism may generalize to other replicative polymerases.
