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C Richardson - One of the best experts on this subject based on the ideXlab platform.
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identification of dna Primase inhibitors via a combined fragment based and virtual screening
Scientific Reports, 2016Co-Authors: Stefan Ilic, C Richardson, Gerhard Wagner, Sabine R Akabayov, Haribabu Arthanari, Barak AkabayovAbstract:The structural differences between bacterial and human Primases render the former an excellent target for drug design. Here we describe a technique for selecting small molecule inhibitors of the activity of T7 DNA Primase, an ideal model for bacterial Primases due to their common structural and functional features. Using NMR screening, fragment molecules that bind T7 Primase were identified and then exploited in virtual filtration to select larger molecules from the ZINC database. The molecules were docked to the Primase active site using the available Primase crystal structure and ranked based on their predicted binding energies to identify the best candidates for functional and structural investigations. Biochemical assays revealed that some of the molecules inhibit T7 Primase-dependent DNA replication. The binding mechanism was delineated via NMR spectroscopy. Our approach, which combines fragment based and virtual screening, is rapid and cost effective and can be applied to other targets.
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zinc binding domain of the bacteriophage t7 dna Primase modulates binding to the dna template
Journal of Biological Chemistry, 2012Co-Authors: Barak Akabayov, C RichardsonAbstract:The zinc-binding domain (ZBD) of prokaryotic DNA Primases has been postulated to be crucial for recognition of specific sequences in the single-stranded DNA template. To determine the molecular basis for this role in recognition, we carried out homolog-scanning mutagenesis of the zinc-binding domain of DNA Primase of bacteriophage T7 using a bacterial homolog from Geobacillus stearothermophilus. The ability of T7 DNA Primase to catalyze template-directed oligoribonucleotide synthesis is eliminated by substitution of any five-amino acid residue-long segment within the ZBD. The most significant defect occurs upon substitution of a region (Pro-16 to Cys-20) spanning two cysteines that coordinate the zinc ion. The role of this region in Primase function was further investigated by generating a protein library composed of multiple amino acid substitutions for Pro-16, Asp-18, and Asn-19 followed by genetic screening for functional proteins. Examination of proteins selected from the screening reveals no change in sequence-specific recognition. However, the more positively charged residues in the region facilitate DNA binding, leading to more efficient oligoribonucleotide synthesis on short templates. The results suggest that the zinc-binding mode alone is not responsible for sequence recognition, but rather its interaction with the RNA polymerase domain is critical for DNA binding and for sequence recognition. Consequently, any alteration in the ZBD that disturbs its conformation leads to loss of DNA-dependent oligoribonucleotide synthesis.
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the roles of tryptophans in primer synthesis by the dna Primase of bacteriophage t7
Journal of Biological Chemistry, 2012Co-Authors: Huidong Zhang, C RichardsonAbstract:DNA Primases catalyze the synthesis of oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Prokaryotic Primases consist of a zinc-binding domain (ZBD) necessary for recognition of a specific template sequence and a catalytic RNA polymerase domain. Interactions of both domains with the DNA template and ribonucleotides are required for primer synthesis. Five tryptophan residues are dispersed in the Primase of bacteriophage T7: Trp-42 in the ZBD and Trp-69, -97, -147, and -255 in the RNA polymerase domain. Previous studies showed that replacement of Trp-42 with alanine in the ZBD decreases primer synthesis, whereas substitution of non-aromatic residues for Trp-69 impairs both primer synthesis and delivery. However, the roles of tryptophan at position 97, 147, or 255 remain elusive. To investigate the essential roles of these residues, we replaced each tryptophan with the structurally similar tyrosine and examined the effect of this subtle alteration on primer synthesis. The substitution at position 42, 97, or 147 reduced primer synthesis, whereas substitution at position 69 or 255 did not. The functions of the tryptophans were further examined at each step of primer synthesis. Alteration of residue 42 disturbed the conformation of the ZBD and resulted in partial loss of the zinc ion, impairing binding to the ssDNA template. Replacement of Trp-97 with tyrosine reduced the binding affinity to NTP and the catalysis step. The replacement of Trp-147 with tyrosine also impaired the catalytic step. Therefore, Trp-42 is important in maintaining the conformation of the ZBD for template binding; Trp-97 contributes to NTP binding and the catalysis step; and Trp-147 maintains the catalysis step.
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gene 5 5 protein of bacteriophage t7 in complex with escherichia coli nucleoid protein h ns and transfer rna masks transfer rna priming in t7 dna replication
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Enduo Wang, C RichardsonAbstract:DNA Primases provide oligoribonucleotides for DNA polymerase to initiate lagging strand synthesis. A deficiency in the Primase of bacteriophage T7 to synthesize primers can be overcome by genetic alterations that decrease the expression of T7 gene 5.5, suggesting an alternative mechanism to prime DNA synthesis. The product of gene 5.5 (gp5.5) forms a stable complex with the Escherichia coli histone-like protein H-NS and transfer RNAs (tRNAs). The 3′-terminal sequence (5′-ACCA-3′) of tRNAs is identical to that of a functional primer synthesized by T7 Primase. Mutations in T7 that suppress the inability of Primase reduce the amount of gp5.5 and thus increase the pool of tRNA to serve as primers. Alterations in T7 gene 3 facilitate tRNA priming by reducing its endonuclease activity that cleaves at the tRNA–DNA junction. The tRNA bound to gp5.5 recruits H-NS. H-NS alone inhibits reactions involved in DNA replication, but the binding to gp5.5–tRNA complex abolishes this inhibition.
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mechanism of sequence specific template binding by the dna Primase of bacteriophage t7
Nucleic Acids Research, 2010Co-Authors: Samir M Hamdan, C RichardsonAbstract:ABSTRACTDNA Primases catalyze the synthesis of theoligoribonucleotides required for the initiation oflagging strand DNA synthesis. Biochemical studieshave elucidated the mechanism for the sequence-specific synthesis of primers. However, thephysical interactions of the Primase with the DNAtemplate to explain the basis of specificity havenot been demonstrated. Using a combination ofsurface plasmon resonance and biochemicalassays, we show that T7 DNA Primase has only aslightly higher affinity for DNA containing thePrimase recognition sequence (5 0 -TGGTC-3 ) thanfor DNA lacking the recognition site. However, thisbinding is drastically enhanced by the presenceof the cognate Nucleoside triphosphates (NTPs),Adenosine triphosphate (ATP) and Cytosinetriphosphate (CTP) that are incorporated into theprimer, pppACCA. Formation of the dimer, pppAC,the initial step of sequence-specific primer synthe-sis, is not sufficient for the stable binding.Preformed primers exhibit significantly less select-ive binding than that observed with ATP and CTP.Alterations in subdomains of the Primase result inloss of selective DNA binding. We present a model inwhich conformational changes induced duringprimer synthesis facilitate contact between thezinc-binding domain and the polymerase domain.INTRODUCTIONThe replication of a duplex DNA molecule is a complexprocess requiring an assembly of numerous proteins, thereplisome (1,2). At the replication fork, DNA helicaseunwinds dsDNA to provide a single-stranded DNA(ssDNA) template on which DNA polymerase polymer-izes nucleotides for leading strand DNA synthesis. Thesimultaneous synthesis of both DNA strands is intricatesince the two strands have opposite polarities, yet DNApolymerases function in only one direction by adding nu-cleotides to the 3
Barak Akabayov - One of the best experts on this subject based on the ideXlab platform.
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DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling.
Journal of Visualized Experiments, 2019Co-Authors: Stefan Ilic, Shira Cohen, Ariel Afek, David B. Lukatsky, Raluca Gordan, Barak AkabayovAbstract:DNA Primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like Primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA Primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA Primase to its template DNA, and 2) stronger binding of Primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and Primase activity assay and is designated as high-throughput Primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA Primase in unprecedented time and scalability.
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DnaG Primase—A Target for the Development of Novel Antibacterial Agents
Antibiotics, 2018Co-Authors: Stefan Ilic, Shira Cohen, Meenakshi Singh, Adi Dayan, Barak AkabayovAbstract:The bacterial Primase—an essential component in the replisome—is a promising but underexploited target for novel antibiotic drugs. Bacterial Primases have a markedly different structure than the human Primase. Inhibition of Primase activity is expected to selectively halt bacterial DNA replication. Evidence is growing that halting DNA replication has a bacteriocidal effect. Therefore, inhibitors of DNA Primase could provide antibiotic agents. Compounds that inhibit bacterial DnaG Primase have been developed using different approaches. In this paper, we provide an overview of the current literature on DNA Primases as novel drug targets and the methods used to find their inhibitors. Although few inhibitors have been identified, there are still challenges to develop inhibitors that can efficiently halt DNA replication and may be applied in a clinical setting.
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DNA Sequence Context Controls the Binding and Processivity of the T7 DNA Primase.
iScience, 2018Co-Authors: Ariel Afek, Stefan Ilic, John Horton, David B. Lukatsky, Raluca Gordan, Barak AkabayovAbstract:Summary Primases are key enzymes involved in DNA replication. They act on single-stranded DNA and catalyze the synthesis of short RNA primers used by DNA polymerases. Here, we investigate the DNA binding and activity of the bacteriophage T7 Primase using a new workflow called high-throughput Primase profiling (HTPP). Using a unique combination of high-throughput binding assays and biochemical analyses, HTPP reveals a complex landscape of binding specificity and functional activity for the T7 Primase, determined by sequences flanking the Primase recognition site. We identified specific features, such as G/T-rich flanks, which increase Primase-DNA binding up to 10-fold and, surprisingly, also increase the length of newly formed RNA (up to 3-fold). To our knowledge, variability in primer length has not been reported for this Primase. We expect that applying HTPP to additional enzymes will reveal new insights into the effects of DNA sequence composition on the DNA recognition and functional activity of Primases.
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DNA sequence context controls the binding and processivity of T7 DNA Primase
bioRxiv, 2018Co-Authors: Ariel Afek, Stefan Ilic, John Horton, David B. Lukatsky, Raluca Gordan, Barak AkabayovAbstract:Primases are key enzymes involved in DNA replication. They act on single-stranded DNA, and catalyze the synthesis of short RNA primers used by DNA polymerases. Here, we investigate the DNA-binding and activity of the bacteriophage T7 Primase using a new workflow called High-Throughput Primase Profiling (HTPP). Using a unique combination of high-throughput binding assays and biochemical analyses, HTPP reveals a complex landscape of binding specificity and functional activity for the T7 Primase, determined by sequences flanking the Primase recognition site. We identified specific features, such as G/T-rich flanks, which increase Primase-DNA binding up to 10-fold and, surprisingly, also increase the length of newly formed RNA (up to 3-fold). To our knowledge, variability in primer length has not been reported for this Primase. We expect that applying HTPP to additional enzymes will reveal new insights into the effects of DNA sequence composition on the DNA recognition and functional activity of Primases.
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modulation of rna primer formation by mn ii substituted t7 dna Primase
Scientific Reports, 2017Co-Authors: Stefan Ilic, Sabine R Akabayov, Haribabu Arthanari, Roy Froimovici, Ron Meiry, Dan Vilenchik, Alfredo J Hernandez, Barak AkabayovAbstract:Lagging strand DNA synthesis by DNA polymerase requires RNA primers produced by DNA Primase. The N-terminal Primase domain of the gene 4 protein of phage T7 comprises a zinc-binding domain that recognizes a specific DNA sequence and an RNA polymerase domain that catalyzes RNA polymerization. Based on its crystal structure, the RNA polymerase domain contains two Mg(II) ions. Mn(II) substitution leads to elevated RNA primer synthesis by T7 DNA Primase. NMR analysis revealed that upon binding Mn(II), T7 DNA Primase undergoes conformational changes near the metal cofactor binding site that are not observed when the enzyme binds Mg(II). A machine-learning algorithm called linear discriminant analysis (LDA) was trained by using the large collection of Mn(II) and Mg(II) binding sites available in the protein data bank (PDB). Application of the model to DNA Primase revealed a preference in the enzyme’s second metal binding site for Mn(II) over Mg(II), suggesting that T7 DNA Primase activity modulation when bound to Mn(II) is based on structural changes in the enzyme.
Luca Pellegrini - One of the best experts on this subject based on the ideXlab platform.
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an archaeal Primase functions as a nanoscale caliper to define primer length
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Sandro Holzer, Luca Pellegrini, Stephen D. BellAbstract:The cellular replicative DNA polymerases cannot initiate DNA synthesis without a priming 3′ OH. During DNA replication, this is supplied in the context of a short RNA primer molecule synthesized by DNA Primase. The Primase of archaea and eukaryotes, despite having varying subunit compositions, share sequence and structural homology. Intriguingly, archaeal Primase has been demonstrated to possess the ability to synthesize DNA de novo, a property shared with the eukaryotic PrimPol enzymes. The dual RNA and DNA synthetic capabilities of the archaeal DNA Primase have led to the proposal that there may be a sequential hand-off between these synthetic modes of Primase. In the current work, we dissect the functional interplay between DNA and RNA synthetic modes of Primase. In addition, we determine the key determinants that govern primer length definition by the archaeal Primase. Our results indicate a primer measuring system that functions akin to a caliper.
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primer synthesis by a eukaryotic like archaeal Primase is independent of its fe s cluster
Nature Communications, 2017Co-Authors: Sandro Holzer, M L Kilkenny, Stephen D. Bell, Luca PellegriniAbstract:DNA replication depends on Primase, the specialised polymerase responsible for synthesis of the RNA primers that are elongated by the replicative DNA polymerases. In eukaryotic and archaeal replication, Primase is a heterodimer of two subunits, PriS and PriL. Recently, a third Primase subunit named PriX was identified in the archaeon Sulfolobus solfataricus. PriX is essential for primer synthesis and is structurally related to the Fe–S cluster domain of eukaryotic PriL. Here we show that PriX contains a nucleotide-binding site required for primer synthesis, and demonstrate equivalence of nucleotide-binding residues in PriX with eukaryotic PriL residues that are known to be important for primer synthesis. A Primase chimera, where PriX is fused to a truncated version of PriL lacking the Fe–S cluster domain retains wild-type levels of primer synthesis. Our evidence shows that PriX has replaced PriL as the subunit that endows Primase with the unique ability to initiate nucleic acid synthesis. Importantly, our findings reveal that the Fe–S cluster is not required for primer synthesis. Primase is the specialised DNA-dependent RNA polymerase responsible for the initiation of DNA synthesis during DNA replication. Here the authors use a structural biology approach to identify the initiation site in the S. solfataricus PriSLX Primase and to demonstrate that its Fe-S cluster is dispensable for primer synthesis.
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structures of human Primase reveal design of nucleotide elongation site and mode of pol α tethering
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: M L Kilkenny, Rajika L Perera, Michael Longo, Luca PellegriniAbstract:Initiation of DNA synthesis in genomic duplication depends on Primase, the DNA-dependent RNA polymerase that synthesizes de novo the oligonucleotides that prime DNA replication. Due to the discontinuous nature of DNA replication, Primase activity on the lagging strand is required throughout the replication process. In eukaryotic cells, the presence of Primase at the replication fork is secured by its physical association with DNA polymerase α (Pol α), which extends the RNA primer with deoxynucleotides. Our knowledge of the mechanism that primes DNA synthesis is very limited, as structural information for the eukaryotic enzyme has proved difficult to obtain. Here, we describe the crystal structure of human Primase in heterodimeric form consisting of full-length catalytic subunit and a C-terminally truncated large subunit. We exploit the crystallographic model to define the architecture of its nucleotide elongation site and to show that the small subunit integrates primer initiation and elongation within the same set of functional residues. Furthermore, we define in atomic detail the mode of association of Primase to Pol α, the critical interaction that keeps Primase tethered to the eukaryotic replisome.
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a conserved motif in the c terminal tail of dna polymerase α tethers Primase to the eukaryotic replisome
Journal of Biological Chemistry, 2012Co-Authors: M L Kilkenny, Giacomo De Piccoli, Rajika L Perera, Karim Labib, Luca PellegriniAbstract:Abstract The DNA polymerase α/Primase complex forms an essential part of the eukaryotic replisome. The catalytic subunits of Primase and Pol α synthesise composite RNA-DNA primers that initiate the leading and lagging DNA strands at replication forks. The physical basis and physiological significance of tethering Primase to the eukaryotic replisome via Pol α remain poorly characterised. We have identified a short conserved motif at the extreme C-terminus of Pol α that is critical for interaction of the yeast orthologue Pol1 with Primase. We show that truncation of the C-terminal residues 1452-1468 of Pol1 abrogates the interaction with the Primase, as does mutation to alanine of the invariant amino acid F1463. Conversely, a Pol1 peptide spanning the last 16 residues binds Primase with high affinity, and the equivalent peptide from human Pol α binds Primase in an analogous fashion. These in vitro data are mirrored by experiments in yeast cells, as Primase does not interact in cell extracts with Pol1 that either terminates at residue 1452 or has the F1463A mutation. The ability to disrupt the association between Primase and Pol α allowed us to assess the physiological significance of Primase being tethered to the eukaryotic replisome in this way. We find that the F1463A mutation in Pol1 renders yeast cells dependent on the S-phase checkpoint, whereas truncation of Pol1 at amino acid 1452 blocks yeast cell proliferation. These findings indicate that tethering of Primase to the replisome by Pol α is critical for the normal action of DNA replication forks in eukaryotic cells.
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the pol α Primase complex
Sub-cellular biochemistry, 2012Co-Authors: Luca PellegriniAbstract:Initiation of DNA synthesis in eukaryotic replication depends on the Pol α-Primase complex, a multi-protein complex endowed with polymerase and Primase activity. The Pol α-Primase complex assembles the RNA-DNA primers required by the processive Pol δ and Pol e for bulk DNA synthesis on the lagging and leading strand, respectively. During primer synthesis, the Primase subunits synthesise de novo an oligomer of 7–12 ribonucleotides in length, which undergoes limited extension with deoxyribonucleotides by Pol α. Despite its central importance to DNA replication, little is known about the mechanism of primer synthesis by the Pol α-Primase complex, which comprises the steps of initiation, ‘counting’ and hand-off of the RNA primer by the Primase to Pol α, followed by primer extension with dNTPs and completion of the RNA-DNA hybrid primer. Recent biochemical and structural work has started to provide some insight into the molecular basis of initiation of DNA synthesis. Important advances include the structural characterisation of the evolutionarily related archaeal Primase, the elucidation of the mechanism of interaction between Pol α and its B subunit and the observation that the regulatory subunit of the Primase contains an iron-sulfur cluster domain that is essential for primer synthesis.
Tom Ellenberger - One of the best experts on this subject based on the ideXlab platform.
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a molecular handoff between bacteriophage t7 dna Primase and t7 dna polymerase initiates dna synthesis
Journal of Biological Chemistry, 2004Co-Authors: Masato Kato, Gerhard Wagner, Tom EllenbergerAbstract: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.
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the crystal structure of the bifunctional Primase helicase of bacteriophage t7
Molecular Cell, 2003Co-Authors: Eric A Toth, Ying Li, Michael R Sawaya, Yifan Cheng, Tom EllenbergerAbstract:Within minutes after infecting Escherichia coli, bacteriophage T7 synthesizes many copies of its genomic DNA. The lynchpin of the T7 replication system is a bifunctional Primase-helicase that unwinds duplex DNA at the replication fork while initiating the synthesis of Okazaki fragments on the lagging strand. We have determined a 3.45 A crystal structure of the T7 Primase-helicase that shows an articulated arrangement of the Primase and helicase sites. The crystallized Primase-helicase is a heptamer with a crown-like shape, reflecting an intimate packing of helicase domains into a ring that is topped with loosely arrayed Primase domains. This heptameric isoform can accommodate double-stranded DNA in its central channel, which nicely explains its recently described DNA remodeling activity. The double-jointed structure of the Primase-helicase permits a free range of motion for the Primase and helicase domains that suggests how the continuous unwinding of DNA at the replication fork can be periodically coupled to Okazaki fragment synthesis.
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modular architecture of the bacteriophage t7 Primase couples rna primer synthesis to dna synthesis
Molecular Cell, 2003Co-Authors: Masato Kato, C Richardson, Gerhard Wagner, Tom EllenbergerAbstract:Abstract DNA Primases are template-dependent RNA polymerases that synthesize oligoribonucleotide primers that can be extended by DNA polymerase. The bacterial Primases consist of zinc binding and RNA polymerase domains that polymerize ribonucleotides at templating sequences of single-stranded DNA. We report a crystal structure of bacteriophage T7 Primase that reveals its two domains and the presence of two Mg 2+ ions bound to the active site. NMR and biochemical data show that the two domains remain separated until the Primase binds to DNA and nucleotide. The zinc binding domain alone can stimulate primer extension by T7 DNA polymerase. These findings suggest that the zinc binding domain couples primer synthesis with primer utilization by securing the DNA template in the Primase active site and then delivering the primed DNA template to DNA polymerase. The modular architecture of the Primase and a similar mechanism of priming DNA synthesis are likely to apply broadly to prokaryotic Primases.
James M Berger - One of the best experts on this subject based on the ideXlab platform.
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identification of a dna Primase template tracking site redefines the geometry of primer synthesis
Nature Structural & Molecular Biology, 2008Co-Authors: Jacob E Corn, Jeffrey G Pelton, James M BergerAbstract:Primases are essential RNA polymerases required for the initiation of DNA replication, lagging strand synthesis and replication restart. Many aspects of Primase function remain unclear, including how the enzyme associates with a moving nucleic acid strand emanating from a helicase and orients primers for handoff to replisomal components. Using a new screening method to trap transient macromolecular interactions, we determined the structure of the Escherichia coli DnaG Primase catalytic domain bound to single-stranded DNA. The structure reveals an unanticipated binding site that engages nucleic acid in two distinct configurations, indicating that it serves as a nonspecific capture and tracking locus for template DNA. Bioinformatic and biochemical analyses show that this evolutionarily constrained region enforces template polarity near the active site and is required for Primase function. Together, our findings reverse previous proposals for primer–template orientation and reconcile disparate studies to re-evaluate replication fork organization.
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regulation of bacterial priming and daughter strand synthesis through helicase Primase interactions
Nucleic Acids Research, 2006Co-Authors: Jacob E Corn, James M BergerAbstract:The replisome is a multi-component molecular machine responsible for rapidly and accurately copying the genome of an organism. A central member of the bacterial replisome is DnaB, the replicative helicase, which separates the parental duplex to provide templates for newly synthesized daughter strands. A unique RNA polymerase, the DnaG Primase, associates with DnaB to repeatedly initiate thousands of Okazaki fragments per replication cycle on the lagging strand. A number of studies have shown that the stability and frequency of the interaction between DnaG and DnaB determines Okazaki fragment length. More recent work indicates that each DnaB hexamer associates with multiple DnaG molecules and that these Primases can coordinate with one another to regulate their activities at a replication fork. Together, disparate lines of evidence are beginning to suggest that Okazaki fragment initiation may be controlled in part by crosstalk between multiple Primases bound to the helicase.
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crosstalk between Primase subunits can act to regulate primer synthesis in trans
Molecular Cell, 2005Co-Authors: Jacob E Corn, Paul J Pease, Greg L Hura, James M BergerAbstract:Summary The coordination of Primase function within the replisome is an essential but poorly understood feature of lagging strand synthesis. By using crystallography and small-angle X-ray scattering (SAXS), we show that functional elements of bacterial Primase transition between two dominant conformations: an extended form that uncouples a regulatory domain from its associated RNA polymerase core and a compact state that sequesters the regulatory region from the site of primer synthesis. FRET studies and priming assays reveal that the regulatory domain of one Primase subunit productively associates with nucleic acid that is bound to the polymerase domain of a second protomer in trans . This intersubunit interaction allows Primase to select initiation sites on template DNA and implicates the regulatory domain as a "molecular brake" that restricts primer length. Our data suggest that the replisome may cooperatively use multiple Primases and this conformational switch to control initiation frequency, processivity, and ultimately, Okazaki fragment synthesis.
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structure of the rna polymerase domain of e coli Primase
Science, 2000Co-Authors: James L Keck, Daniel D Roche, Simon A Lynch, James M BergerAbstract:All cellular organisms use specialized RNA polymerases called “Primases” to synthesize RNA primers for the initiation of DNA replication. The high-resolution crystal structure of a Primase, comprising the catalytic core of the Escherichia coli DnaG protein, was determined. The core structure contains an active-site architecture that is unrelated to other DNA or RNA polymerase palm folds, but is instead related to the “toprim” fold. On the basis of the structure, it is likely that DnaG binds nucleic acid in a groove clustered with invariant residues and that DnaG is positioned within the replisome to accept single-stranded DNA directly from the replicative helicase.