The Experts below are selected from a list of 291 Experts worldwide ranked by ideXlab platform

Peter H. Von Hippel - One of the best experts on this subject based on the ideXlab platform.

  • A single-molecule view of the assembly pathway, subunit stoichiometry, and unwinding activity of the bacteriophage T4 Primosome (helicase-primase) complex.
    Biochemistry, 2013
    Co-Authors: Wonbae Lee, Davis Jose, Carey Phelps, Andrew H. Marcus, Peter H. Von Hippel
    Abstract:

    Single-molecule fluorescence resonance energy transfer (smFRET) methods were used to study the assembly pathway and DNA unwinding activity of the bacteriophage T4 helicase–primase (Primosome) complex. The helicase substrates used were surface-immobilized model DNA replication forks “internally” labeled in the duplex region with opposed donor/acceptor (iCy3/iCy5) chromophore pairs in the lagging and leading strands. The time dependence of the smFRET signals was monitored during the unwinding process, and helicase rates and processivities were measured as a function of GTP concentration. This smFRET approach was also used to investigate the subunit stoichiometry of the Primosome and the assembly pathway required to form functional and fully active Primosome–DNA complexes. We confirmed that gp41 helicase monomer subunits form stable hexameric helicases in the presence of GTP and that the resulting (gp41)6 complexes bind only weakly at DNA fork junctions. The addition of a single subunit of gp61 primase stabi...

  • A Single-Molecule View of the Assembly Pathway, Subunit Stoichiometry, and Unwinding Activity of the Bacteriophage T4 Primosome (helicase–primase) Complex
    2013
    Co-Authors: Wonbae Lee, Davis Jose, Carey Phelps, Andrew H. Marcus, Peter H. Von Hippel
    Abstract:

    Single-molecule fluorescence resonance energy transfer (smFRET) methods were used to study the assembly pathway and DNA unwinding activity of the bacteriophage T4 helicase–primase (Primosome) complex. The helicase substrates used were surface-immobilized model DNA replication forks “internally” labeled in the duplex region with opposed donor/acceptor (iCy3/iCy5) chromophore pairs in the lagging and leading strands. The time dependence of the smFRET signals was monitored during the unwinding process, and helicase rates and processivities were measured as a function of GTP concentration. This smFRET approach was also used to investigate the subunit stoichiometry of the Primosome and the assembly pathway required to form functional and fully active Primosome–DNA complexes. We confirmed that gp41 helicase monomer subunits form stable hexameric helicases in the presence of GTP and that the resulting (gp41)6 complexes bind only weakly at DNA fork junctions. The addition of a single subunit of gp61 primase stabilized the resulting Primosome complex at the fork and resulted in fully active and processive Primosome helicases with gp41:gp61 subunit ratios of 6:1, while higher and lower subunit ratios substantially reduced the Primosome unwinding activity. The use of alternative assembly pathways resulted in a loss of helicase activity and the formation of metastable DNA–protein aggregates, which were easily detected in our smFRET experiments as intense light-scattering foci. These single-molecule experiments provide a detailed real-time visualization of the assembly pathway and duplex DNA unwinding activity of the T4 Primosome and are consistent with more indirect equilibrium and steady state results obtained in bulk solution studies

  • Breathing fluctuations in position-specific DNA base pairs are involved in regulating helicase movement into the replication fork
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Davis Jose, Steven E. Weitzel, Peter H. Von Hippel
    Abstract:

    We previously used changes in the near-UV circular dichroism and fluorescence spectra of DNA base analogue probes placed site specifically to show that the first three base pairs at the fork junction in model replication fork constructs are significantly opened by “breathing” fluctuations under physiological conditions. Here, we use these probes to provide mechanistic snapshots of the initial interactions of the DNA fork with a tight-binding replication helicase in solution. The Primosome helicase of bacteriophage T4 was assembled from six (gp41) helicase subunits, one (gp61) primase subunit, and nonhydrolyzable GTPγS. When bound to a DNA replication fork construct this complex advances one base pair into the duplex portion of the fork and forms a stably bound helicase “initiation complex.” Replacement of GTPγS with GTP permits the completion of the helicase-driven unwinding process. Our spectroscopic probes show that the Primosome in this stable helicase initiation complex binds the DNA of the fork primarily via backbone contacts and holds the first complementary base pair of the fork in an open conformation, whereas the second, third, and fourth base pairs of the duplex show essentially the breathing behavior that previously characterized the first three base pairs of the free fork. These spectral changes, together with dynamic fluorescence quenching results, are consistent with a Primosome-binding model in which the lagging DNA strand passes through the central hole of the hexagonal helicase, the leading strand binds to the “outside” surfaces of subunits of the helicase hexamer, and the single primase subunit interacts with both strands.

  • Assembly and subunit stoichiometry of the functional helicase-primase (Primosome) complex of bacteriophage T4
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Davis Jose, Steven E. Weitzel, Debra H. Jing, Peter H. Von Hippel
    Abstract:

    Physical biochemical techniques are used to establish the structure, subunit stoichiometry, and assembly pathway of the Primosome complex of the bacteriophage T4 DNA replication system. Analytical ultracentrifugation and fluorescence anisotropy methods show that the functional T4 Primosome consists of six gp41 helicase subunits that assemble into a hexagon, driven by the binding of six NTPs (or six nonhydrolyzable GTPγS analogues) that are located at and stabilize the intersubunit interfaces, together with a single tightly bound gp61 primase subunit. Assembling the components of the Primosome onto a model DNA replication fork is a multistep process, but equilibrium cannot be reached along all mixing pathways. Producing a functional complex requires that the helicase hexamer be assembled in the presence of the DNA replication fork construct prior to the addition of the primase to avoid the formation of metastable DNA-protein aggregates. The gp41 helicase hexamer binds weakly to fork DNA in the absence of primase, but forms a much more stable Primosome complex that expresses full and functional helicase (and primase) activities when bound to a gp61 primase subunit at a helicase:primase subunit ratio of 6∶1. The presence of additional primase subunits does not change the molecular mass or helicase activity of the Primosome, but significantly inhibits its primase activity. We develop both an assembly pathway and a minimal mechanistic model for the structure and function of the T4 Primosome that are likely to be relevant to the assembly and function of the replication Primosome subassemblies of higher organisms as well.

  • Interactions of bacteriophage T4-coded primase (gp61) with the T4 replication helicase (gp41) and DNA in Primosome formation.
    The Journal of biological chemistry, 1999
    Co-Authors: Debra H. Jing, Feng Dong, Gary J. Latham, Peter H. Von Hippel
    Abstract:

    One primase (gp61) and six helicase (gp41) subunits interact to form the bacteriophage T4-coded Primosome at the DNA replication fork. In order to map some of the detailed interactions of the primase within the Primosome, we have constructed and characterized variants of the gp61 primase that carry kinase tags at either the N or the C terminus of the polypeptide chain. These tagged gp61 constructs have been probed using several analytical methods. Proteolytic digestion and protein kinase protection experiments show that specific interactions with single-stranded DNA and the T4 helicase hexamer significantly protect both the N- and the C-terminal regions of the T4 primase polypeptide chain against modification by these procedures and that this protection becomes more pronounced when the primase is assembled within the complete ternary Primosome complex. Additional discrete sites of both protection and apparent hypersensitivity along the gp61 polypeptide chain have also been mapped by proteolytic footprinting reactions for the binary helicase-primase complex and in the three component Primosome. These studies provide a detailed map of a number of gp61 contact positions within the Primosome and reveal interactions that may be important in the structure and function of this central component of the T4 DNA replication complex.

Kenneth J Marians - One of the best experts on this subject based on the ideXlab platform.

  • PriA-directed Assembly of a Primosome on D Loop DNA
    Journal of Biological Chemistry, 1999
    Co-Authors: Kenneth J Marians
    Abstract:

    Abstract Escherichia coli strains carrying null mutations in priA are chronically induced for the SOS response and are defective in homologous recombination, repair of UV damaged DNA, double-strand break repair, and both induced and constitutive stable DNA replication. This led to the proposal that PriA directed replication fork assembly at D loops formed by the homologous recombination machinery. The demonstration that PriA specifically recognized and bound D loop DNA supported this hypothesis. Using DNA footprinting as an assay, we show here that PriA also directs the assembly of a ϕX174-type Primosome on D loop DNA. The ability to load a complete Primosome on D loop DNA is a step necessary for replication fork assembly.

  • Two modes of PriA binding to DNA.
    The Journal of biological chemistry, 1999
    Co-Authors: Pearl Nurse, Joing Liu, Kenneth J Marians
    Abstract:

    The role of PriA, required for the assembly of the phiX174-type Primosome on DNA, in cellular DNA replication has been unclear since its discovery. Recent evidence, based on the phenotypes of strains carrying priA null mutations, has led to proposals that the Primosome assembly activity of PriA was required to load replication forks at intermediates such as D loops during homologous recombination. McGlynn et al. (McGlynn, P., Al-Deib, A. A., Liu, J., Marians, K. J., and Lloyd, R. G. (1997) J. Mol. Biol. 270, 212-221) demonstrated that PriA could, in fact, bind D loops. We show here that there are two modes of stable binding of PriA to DNA. One mode, in which the enzyme binds 3'-single-stranded extensions from duplex DNAs, presumably reflects the 3' --> 5' DNA helicase activity of PriA. The D loop DNA binding activity of PriA can be accounted for by the second mode, where the enzyme binds bent DNA at three strand junctions.

  • Replication fork assembly at recombination intermediates is required for bacterial growth
    Proceedings of the National Academy of Sciences of the United States of America, 1999
    Co-Authors: Joing Liu, Steven J. Sandler, Kenneth J Marians
    Abstract:

    PriA, a 3′ → 5′ DNA helicase, directs assembly of a Primosome on some bacteriophage and plasmid DNAs. Primosomes are multienzyme replication machines that contribute both the DNA-unwinding and Okazaki fragment-priming functions at the replication fork. The role of PriA in chromosomal replication is unclear. The phenotypes of priA null mutations suggest that the protein participates in replication restart at recombination intermediates. We show here that PriA promotes replication fork assembly at a D loop, an intermediate formed during initiation of homologous recombination. We also show that DnaC810, encoded by a naturally arising intergenic suppressor allele of the priA2∷kan mutation, bypasses the need for PriA during replication fork assembly at D loops in vitro. These findings underscore the essentiality of replication fork restart at recombination intermediates under normal growth conditions in bacteria.

  • PriA: at the crossroads of DNA replication and recombination.
    Progress in nucleic acid research and molecular biology, 1999
    Co-Authors: Kenneth J Marians
    Abstract:

    PriA is a single-stranded DNA-dependent ATPase, DNA translocase, and DNA helicase that was discovered originally because of its requirement in vitro for the conversion of bacteriophage ϕX174 viral DNA to the duplex replicative form. Studies demonstrated that PriA catalyzes the assembly of a Primosome, a multi-protein complex that primes DNA synthesis, on ϕX174 DNA. The Primosome was shown to be capable of providing both the DNA unwinding function and the Okazaki fragment priming function required for replication fork progression. However, whereas seven proteins, PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG, were required for Primosome assembly on ϕX174 DNA, only DnaB, DnaC, and DnaG were required for replication from oriC , suggesting that the other proteins were not involved in chromosomal replication. Strains carrying priA null mutations, however, were constitutively induced for the SOS response, and were defective in homologous recombination, repair of UV-damaged DNA, and double-strand breaks, and both induced and constitutive stable DNA replication. The basis for this phenotype can now be explained by the ability of PriA to load replication forks at a D loop, an intermediate that forms during homologous recombination, double-strand break-repair, and stable DNA replication. Thus, a long-theorized connection between recombination and replication is demonstrated.

  • The Ordered Assembly of the φX174-type Primosome: II. PRESERVATION OF Primosome COMPOSITION FROM ASSEMBLY THROUGH REPLICATION
    The Journal of biological chemistry, 1996
    Co-Authors: Kenneth J Marians
    Abstract:

    Abstract Gel filtration chromatography was used to isolate both preprimosomal and primosomal complexes formed on single-stranded DNA-binding protein-coated ϕX174 DNA by the combination of PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG. The presence and relative amounts of primosomal proteins in these complexes were determined by Western blotting. Protein-DNA complexes isolated (i) after assembly in the presence of 10 μM ATP, (ii) after prePrimosome movement in the presence of 1 mM ATP, (iii) after priming in the presence of the four ribonucleoside triphosphates, or (iv) after complementary strand DNA replication in the presence of the DNA polymerase III holoenzyme all had the same protein composition; prePrimosomes contained PriA, PriB, PriC, DnaT, and DnaB, whereas Primosomes included DnaG. The stable association of DnaG with the protein-DNA complex could be attributed partially to its ability to remain bound to the primers synthesized. In the absence of PriC, the efficiencies of priming and replication were reduced by one-third and one-half, respectively, even though PriC was not required for the formation of stable protein-DNA complexes on a 304-nucleotide-long single strand of DNA containing a Primosome assembly site (Ng, J. Y., and Marians, K. J. (1996) J. Biol. Chem. 271, 15642-15648). We hypothesize that maintenance of the Primosome on the replicated DNA may provide a mechanism to allow Primosomes to participate in the resolution of recombination intermediates and intermediates formed during double strand break repair by permitting the re-establishment of a replication fork.

Ellen Fanning - One of the best experts on this subject based on the ideXlab platform.

  • Structural Basis for the Interaction of a Hexameric Replicative Helicase with the Regulatory Subunit of Human DNA Polymerase α-Primase
    The Journal of biological chemistry, 2012
    Co-Authors: Bo Zhou, Diana R Arnett, Ellen Fanning, Aaron S. Brewster, Gregory A. Sowd, Charlies L. Xie, Stefan Vila, Dahai Gai, Xiaojiang S. Chen
    Abstract:

    DNA polymerase α-primase (Pol-prim) plays an essential role in eukaryotic DNA replication, initiating synthesis of the leading strand and of each Okazaki fragment on the lagging strand. Pol-prim is composed of a primase heterodimer that synthesizes an RNA primer, a DNA polymerase subunit that extends the primer, and a regulatory B-subunit (p68) without apparent enzymatic activity. Pol-prim is thought to interact with eukaryotic replicative helicases, forming a dynamic multiprotein assembly that displays Primosome activity. At least three subunits of Pol-prim interact physically with the hexameric replicative helicase SV40 large T antigen, constituting a simple Primosome that is active in vitro. However, structural understanding of these interactions and their role in viral chromatin replication in vivo remains incomplete. Here, we report the detailed large T antigen-p68 interface, as revealed in a co-crystal structure and validated by site-directed mutagenesis, and we demonstrate its functional importance in activating the SV40 Primosome in cell-free reactions with purified Pol-prim, as well as in monkey cells in vivo.

  • A Specific Docking Site for DNA Polymerase α-Primase on the SV40 Helicase Is Required for Viral Primosome Activity, but Helicase Activity Is Dispensable
    The Journal of biological chemistry, 2010
    Co-Authors: Hao Huang, Diana R Arnett, Kun Zhao, Ellen Fanning
    Abstract:

    Replication of simian virus 40 (SV40) DNA, a model for eukaryotic chromosomal replication, can be reconstituted in vitro using the viral helicase (large tumor antigen, or Tag) and purified human proteins. Tag interacts physically with two cellular proteins, replication protein A and DNA polymerase α-primase (pol-prim), constituting the viral Primosome. Like the well characterized Primosomes of phages T7 and T4, this trio of proteins coordinates parental DNA unwinding with primer synthesis to initiate the leading strand at the viral origin and each Okazaki fragment on the lagging strand template. We recently determined the structure of a previously unrecognized pol-prim domain (p68N) that docks on Tag, identified the p68N surface that contacts Tag, and demonstrated its vital role in Primosome function. Here, we identify the p68N-docking site on Tag by using structure-guided mutagenesis of the Tag helicase surface. A charge reverse substitution in Tag disrupted both p68N-binding and Primosome activity but did not affect docking with other pol-prim subunits. Unexpectedly, the substitution also disrupted Tag ATPase and helicase activity, suggesting a potential link between p68N docking and ATPase activity. To assess this possibility, we examined the Primosome activity of Tag with a single residue substitution in the Walker B motif. Although this substitution abolished ATPase and helicase activity as expected, it did not reduce pol-prim docking on Tag or Primosome activity on single-stranded DNA, indicating that Tag ATPase is dispensable for Primosome activity in vitro.

  • structure of a dna polymerase α primase domain that docks on the sv40 helicase and activates the viral Primosome
    Journal of Biological Chemistry, 2010
    Co-Authors: Hao Huang, Diana R Arnett, Brian E Weiner, Brian E Fuller, Brian M Wile, Haijiang Zhang, Walter J. Chazin, Kun Zhao, Ellen Fanning
    Abstract:

    DNA polymerase α-primase (pol-prim) plays a central role in DNA replication in higher eukaryotes, initiating synthesis on both leading and lagging strand single-stranded DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68 (also termed B-subunit), that is thought to regulate the complex. Although significant knowledge about single-subunit primases of prokaryotes has accumulated, the functions and regulation of pol-prim remain poorly understood. In the SV40 replication model, the p68 subunit is required for Primosome activity and binds directly to the hexameric viral helicase T antigen, suggesting a functional link between T antigen-p68 interaction and Primosome activity. To explore this link, we first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the T antigen helicase domain. NMR spectroscopy was used to determine the solution structure of p68N and map its interface with the T antigen helicase domain. Structure-guided mutagenesis of p68 residues in the interface diminished T antigen-p68 interaction, confirming the interaction site. SV40 Primosome activity of corresponding pol-prim mutants decreased in proportion to the reduction in p68N-T antigen affinity, confirming that p68-T antigen interaction is vital for Primosome function. A model is presented for how this interaction regulates SV40 Primosome activity, and the implications of our findings are discussed in regard to the molecular mechanisms of eukaryotic DNA replication initiation.

  • Structure of a DNA polymerase alpha-primase domain that docks on the SV40 helicase and activates the viral Primosome.
    The Journal of biological chemistry, 2010
    Co-Authors: Hao Huang, Diana R Arnett, Brian E Weiner, Brian E Fuller, Brian M Wile, Haijiang Zhang, Walter J. Chazin, Kun Zhao, Yue Gao, Ellen Fanning
    Abstract:

    DNA polymerase α-primase (pol-prim) plays a central role in DNA replication in higher eukaryotes, initiating synthesis on both leading and lagging strand single-stranded DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68 (also termed B-subunit), that is thought to regulate the complex. Although significant knowledge about single-subunit primases of prokaryotes has accumulated, the functions and regulation of pol-prim remain poorly understood. In the SV40 replication model, the p68 subunit is required for Primosome activity and binds directly to the hexameric viral helicase T antigen, suggesting a functional link between T antigen-p68 interaction and Primosome activity. To explore this link, we first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the T antigen helicase domain. NMR spectroscopy was used to determine the solution structure of p68N and map its interface with the T antigen helicase domain. Structure-guided mutagenesis of p68 residues in the interface diminished T antigen-p68 interaction, confirming the interaction site. SV40 Primosome activity of corresponding pol-prim mutants decreased in proportion to the reduction in p68N-T antigen affinity, confirming that p68-T antigen interaction is vital for Primosome function. A model is presented for how this interaction regulates SV40 Primosome activity, and the implications of our findings are discussed in regard to the molecular mechanisms of eukaryotic DNA replication initiation.

  • Mutational Analysis of Simian Virus 40 T-Antigen Primosome Activities in Viral DNA Replication
    Journal of virology, 2002
    Co-Authors: Robert D. Ott, Yingda Wang, Ellen Fanning
    Abstract:

    The recruitment of DNA polymerase α-primase (pol-prim) is a crucial step in the establishment of a functional replication complex in eukaryotic cells, but the mechanism of pol-prim loading and the composition of the eukaryotic Primosome are poorly understood. In the model system for simian virus 40 (SV40) DNA replication in vitro, synthesis of RNA primers at the origin of replication requires only the viral tumor (T) antigen, replication protein A (RPA), pol-prim, and topoisomerase I. On RPA-coated single-stranded DNA (ssDNA), T antigen alone mediates priming by pol-prim, constituting a relatively simple Primosome. T-antigen activities proposed to participate in its Primosome function include DNA helicase and protein-protein interactions with RPA and pol-prim. To test the role of these activities of T antigen in mediating priming by pol-prim, three replication-defective T antigens with mutations in the ATPase or helicase domain have been characterized. All three mutant proteins interacted physically and functionally with RPA and pol-prim and bound ssDNA, and two of them displayed some helicase activity. However, only one of these, 5030, mediated primer synthesis and elongation by pol-prim on RPA-coated ssDNA. The results suggest that a novel activity, present in 5030 T antigen and absent in the other two mutants, is required for T-antigen Primosome function.

Tadashi Ueda - One of the best experts on this subject based on the ideXlab platform.

  • Basic and aromatic residues in the C-terminal domain of PriC are involved in ssDNA and SSB binding
    Journal of biochemistry, 2015
    Co-Authors: Takahiko Aramaki, Yoshito Abe, Tsutomu Katayama, Kaori Furutani, Tadashi Ueda
    Abstract:

    In bacterial organisms, the oriC-independent Primosome plays an essential role in replication restart after dissociation of the replication DNA-protein complex following DNA damage. PriC is a key protein component in the oriC-independent replication restart Primosome. Our previous study suggested that PriC was divided into an N-terminal domain and a C-terminal domain, with the latter domain being the major contributor to single-stranded DNA (ssDNA) binding capacity. In this study, we prepared several PriC mutants in which basic and aromatic amino acid residues were mutated to alanine. Five of these residues, Arg107, Lys111, Phe118, Arg121 and Lys165 in the C-terminal domain, were shown to be involved in ssDNA binding. Moreover, we evaluated the binding of the PriC mutants to the ssDNA-binding protein (SSB) complex. Five residues, Phe118, Arg121, Arg129, Tyr152 and Arg155 in the C-terminal domain of PriC, were shown to be involved in SSB binding in the presence of ssDNA. On the basis of these results, we propose a structural model of the C-terminal domain of PriC and discuss how the interactions of PriC with SSB and ssDNA may contribute to the regulation of PriC-dependent replication restart.

  • solution structure of the n terminal domain of a replication restart Primosome factor pric in escherichia coli
    Protein Science, 2013
    Co-Authors: Takahiko Aramaki, Yoshito Abe, Tsutomu Katayama, Tadashi Ueda
    Abstract:

    In eubacterial organisms, the oriC-independent Primosome plays an essential role in replication restart after the dissociation of the replication DNA-protein complex by DNA damage. PriC is a key protein component in the replication restart Primosome. Our recent study suggested that PriC is divided into two domains: an N-terminal and a C-terminal domain. In the present study, we determined the solution structure of the N-terminal domain, whose structure and function have remained unknown until now. The revealed structure was composed of three helices and one extended loop. We also observed chemical shift changes in the heteronuclear NMR spectrum and oligomerization in the presence of ssDNA. These abilities may contribute to the PriC-ssDNA complex, which is important for the replication restart Primosome.

  • Solution structure of the N‐terminal domain of a replication restart Primosome factor, PriC, in Escherichia coli
    Protein science : a publication of the Protein Society, 2013
    Co-Authors: Takahiko Aramaki, Yoshito Abe, Tsutomu Katayama, Tadashi Ueda
    Abstract:

    In eubacterial organisms, the oriC-independent Primosome plays an essential role in replication restart after the dissociation of the replication DNA-protein complex by DNA damage. PriC is a key protein component in the replication restart Primosome. Our recent study suggested that PriC is divided into two domains: an N-terminal and a C-terminal domain. In the present study, we determined the solution structure of the N-terminal domain, whose structure and function have remained unknown until now. The revealed structure was composed of three helices and one extended loop. We also observed chemical shift changes in the heteronuclear NMR spectrum and oligomerization in the presence of ssDNA. These abilities may contribute to the PriC-ssDNA complex, which is important for the replication restart Primosome.

  • Domain separation and characterization of PriC, a replication restart Primosome factor in Escherichia coli.
    Genes to cells : devoted to molecular & cellular mechanisms, 2013
    Co-Authors: Takahiko Aramaki, Yoshito Abe, Tsutomu Katayama, Takatoshi Ohkuri, Tomonori Mishima, Shoji Yamashita, Tadashi Ueda
    Abstract:

    In Escherichia coli the oriC-independent Primosome plays an essential role in replication restart after dissociation of the replication DNA–protein complex by DNA damage. Primosome is thought to form via two pathways: one PriA dependent and the other PriA independent. PriC is a key protein in the replication restart of the PriA-independent pathway. In this study, we determined that PriC was divided into two domains. Then, we obtained information that: (i) the C-terminal domain preferentially binds to single-stranded DNA (ssDNA); (ii) the binding of PriC to ssDNA depends on salt concentration; and (iii) the binding site size of PriC is approximately 7–9 nucleotides. The protease digestion of PriC suggested that a possible DNA-binding site is the N-terminus of the C-terminal domain where basic amino acid residues are concentrated. Interestingly, α-helical induction of the C-terminal domain of PriC occurred after the addition of DNAs. Also, we examined the role of heptad repeat of leucine or valine residues in the C-terminal domain and PriC oligomerization. This study describes the structure and function analysis of PriC which forms the Primosome complex in replication restart.

  • Crystal structure of a biologically functional form of PriB from Escherichia coli reveals a potential single-stranded DNA-binding site
    Biochemical and biophysical research communications, 2005
    Co-Authors: Seijiro Shioi, Yoshito Abe, Tsutomu Katayama, Toyoyuki Ose, Katsumi Maenaka, Mitsunori Shiroishi, Daisuke Kohda, Tadashi Ueda
    Abstract:

    Abstract PriB is not only an essential protein necessary for the replication restart on the collapsed and disintegrated replication fork, but also an important protein for assembling of Primosome onto ΦX174 genomic DNA during replication initiation. Here we report a 2.0-A-resolution X-ray structure of a biologically functional form of PriB from Escherichia coli. The crystal structure revealed that despite a low level of primary sequence identity, the PriB monomer, as well as the dimeric form, are structurally identical to the N-terminal DNA-binding domain of the single-stranded DNA-binding protein (SSB) from Escherichia coli, which possesses an oligonucleotides-binding-fold. The oligonucleotide–PriB complex model based on the oligonucleotides–SSB complex structure suggested that PriB had a DNA-binding pocket conserved in SSB from Escherichia coli and might bind to single-stranded DNA in the manner of SSB. Furthermore, surface plasmon resonance analysis and fluorescence measurements demonstrated that PriB binds single-stranded DNA with high affinity, by involving tryptophan residue. The significance of these results with respect to the functional role of PriB in the assembly of Primosome is discussed.

Stephen J. Benkovic - One of the best experts on this subject based on the ideXlab platform.

  • Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 Primosome
    Nature Chemical Biology, 2009
    Co-Authors: Maria Manosas, Stephen J. Benkovic, Michelle M Spiering, Zhihao Zhuang, Vincent Croquette
    Abstract:

    The unwinding and priming activities of the bacteriophage T4 Primosome, which consists of a hexameric helicase (gp41) translocating 5′ to 3′ and an oligomeric primase (gp61) synthesizing primers 5′ to 3′, have been investigated on DNA hairpins manipulated by a magnetic trap. We find that the T4 Primosome continuously unwinds the DNA duplex while allowing for primer synthesis through a Primosome disassembly mechanism or a new DNA looping mechanism. A fused gp61-gp41 Primosome unwinds and primes DNA exclusively via the DNA looping mechanism. Other proteins within the replisome control the partitioning of these two mechanisms by disfavoring Primosome disassembly, thereby increasing primase processivity. In contrast to T4, priming in bacteriophage T7 and Escherichia coli involves discrete pausing of the Primosome and dissociation of the primase from the helicase, respectively. Thus nature appears to use several strategies to couple the disparate helicase and primase activities within Primosomes.

  • Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 Primosome
    2009
    Co-Authors: Maria Manosas, Michelle M Spiering, Zhihao Zhuang, Stephen J. Benkovic
    Abstract:

    The unwinding and priming activities of the bacteriophage T4 Primosome, which consists of a hexameric helicase (gp41) translocating 5 ′ to 3 ′ and an oligomeric primase (gp61) synthesizing primers 5 ′ to 3′, has been investigated on DNA hairpins manipulated by a magnetic trap. We find that the T4 Primosome continuously unwinds the DNA duplex while allowing for primer synthesis through a Primosome disassembly mechanism or a novel DNA looping mechanism. A fused gp61-gp41 Primosome unwinds and primes DNA exclusively via the DNA looping mechanism. Other proteins within the replisome control the partitioning of these two mechanisms disfavoring Primosome disassembly thereby increasing primase processivity. In contrast priming in bacteriophage T7 involves discrete pausing of the Primosome and in Escherichia coli appears to be associated primarily with dissociation of the primase from the helicase. Thus nature appears to use several strategies to couple the disparate helicase and primase activities within Primosomes. A model system used to study DNA replication is the bacteriophage T4 replisome. Eight proteins, corresponding to seven different activities, have been identified that together are able to reconstitute in vitro leading and lagging strand DNA synthesis1. The leading an

  • Architecture of the bacteriophage T4 Primosome: Electron microscopy studies of helicase (gp41) and primase (gp61)
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Mona T. Norcum, Faoud T. Ishmael, Michelle M Spiering, Michael A. Trakselis, J. Anthony Warrington, Stephen J. Benkovic
    Abstract:

    Replication of DNA requires helicase and primase activities as part of a Primosome assembly. In bacteriophage T4, helicase and primase are separate polypeptides for which little structural information is available and whose mechanism of association within the Primosome is not yet understood. Three-dimensional structural information is provided here by means of reconstructions from electron microscopic images. Structures have been calculated for complexes of each of these proteins with ssDNA in the presence of MgATPγS. Both the helicase (gp41) and primase (gp61) complexes are asymmetric hexagonal rings. The gp41 structure suggests two distinct forms that have been termed “open” and “closed.” The gp61 structure is clearly a six-membered ring, which may be a trimer of dimers or a traditional hexamer of monomers. This structure provides conclusive evidence for an oligomeric primase-to-ssDNA stoichiometry of 6:1.

  • Assembly of the bacteriophage T4 Primosome: Single-molecule and ensemble studies
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Zhiquan Zhang, Faoud T. Ishmael, Stephen J. Benkovic, Michelle M Spiering, Michael A. Trakselis, Gordon G. Hammes
    Abstract:

    Within replisomes for DNA replication, the Primosome is responsible for unwinding double-stranded DNA and synthesizing RNA primers. Assembly of the bacteriophage T4 Primosome on individual molecules of ssDNA or forked DNA (fDNA) has been studied by using FRET microscopy. On either DNA substrate, an ordered process of assembly begins with tight 1:1 binding of ssDNA-binding protein (gp32) and helicase-loading protein (gp59) to the DNA. Magnesium adenosine 5′-O-(3-thiotriphosphate) (MgATPγS) mediates the weak binding of helicase (gp41) to DNA coated with gp32 and gp59, whereas MgATP induces gp32 and gp59 to dissociate, leaving gp41 bound to the DNA. Finally, primase (gp61) binds to the gp41·DNA complex. Ensemble studies were used to determine protein stoichiometries and binding constants. These single-molecule studies provide an unambiguous description of the pathway for assembly of the Primosome on the lagging strand of DNA at a replication fork.

  • Protein-Protein Interactions in the Bacteriophage T4 Replisome THE LEADING STRAND HOLOENZYME IS PHYSICALLY LINKED TO THE LAGGING STRAND HOLOENZYME AND THE Primosome
    The Journal of biological chemistry, 2002
    Co-Authors: Faoud T. Ishmael, Michael A. Trakselis, Stephen J. Benkovic
    Abstract:

    The bacteriophage T4 replication complex is composed of eight proteins that function together to replicate DNA. This replisome can be broken down into four basic units: a Primosome composed of gp41, gp61, and gp59; a leading strand holoenzyme composed of gp43, gp44/62, and gp45; a lagging strand holoenzyme; and a single strand binding protein polymer. These units interact further to form the complete replisome. The leading and lagging strand polymerases are physically linked in the presence of DNA or an active replisome. The region of interaction was mapped to an extension of the finger domain, such that Cys-507 of one subunit is in close proximity to Cys-507 of a second subunit. The leading strand polymerase and the Primosome also associate, such that gp59 mediates the contact between the two complexes. Binding of gp43 to the Primosome complex causes displacement of gp32 from the gp59.gp61.gp41 Primosome complex. The resultant species is a complex of proteins that may allow coordinated leading and lagging strand synthesis, helicase DNA unwinding activity, and polymerase nucleotide incorporation.