Primer synthesis kinetics by Escherichia coli primase on single-stranded DNA templates

Swart, John R.; Griep, Mark A.

doi:10.1021/bi00049a025

Cited by 59 publications

(115 citation statements)

References 38 publications

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“…The same di-, tri-, and tetranucleotide products are produced in reactions containing 5Ј-GGGTCA-3Ј and 5Ј-GGGTCAA-3Ј as are produced with the sequence 5Ј-GGGTC-3Ј (Fig. 1, lanes [1][2][3][4][5]. This result demonstrates that, like the cryptic C, these nucleotides are not copied into the oligoribonucleotide primer.…”

Section: Resultsmentioning

confidence: 83%

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Interaction of Bacteriophage T7 Gene 4 Primase with Its Template Recognition Site

Frick

Richardson

1999

Journal of Biological Chemistry

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The primase fragment of the bacteriophage T7 63-kDa gene 4 helicase/primase protein contains the 271 N-terminal amino acid residues and lacks helicase activity. The primase fragment catalyzes the synthesis of oligoribonucleotides at rates similar to those catalyzed by the full-length protein in the presence of a 5-nucleotide DNA template containing a primase recognition site (5-GGGTC-3, 5-TGGTC-3, 5-GTGTC-3, or 5-TTGTC-3). Although it is not copied into the oligoribonucleotides, the cytosine at the 3-position is essential for synthesis and template binding. Two nucleotides flanking the 3-end of the recognition site are required for tight DNA binding and rapid oligoribonucleotide synthesis. Nucleotides added to the 5-end have no effect on the rate of oligoribonucleotide synthesis or the affinity of the primase for DNA. The binding of either ATP or CTP significantly increases the affinity of the primase for its DNA template. DNA lacking a primase recognition site does not inhibit oligoribonucleotide synthesis, suggesting that the primase binds DNA in a sequence-specific manner. The affinity of the primase for templates is weak, ranging from 10 to 150 M. The tight DNA binding (<1 M) observed with the 63-kDa gene 4 protein occurs via interactions between DNA templates and the helicase domain.DNA polymerases require the presence of a 3Ј-hydroxyl terminated DNA or RNA primer to initiate polymerization. By far the most common mechanism for priming DNA synthesis involves the utilization of short oligoribonucleotides. Replicative DNA polymerases efficiently add deoxynucleotides to the 3Ј-end of RNA primers annealed to ssDNA. 1 In the cell, these RNA primers are synthesized by a specialized enzyme, DNA primase, a DNA-dependent RNA polymerase that catalyzes the synthesis of oligoribonucleotides at specific DNA sequences (1). The enzymatic analysis of oligoribonucleotide synthesis by DNA primases has been complicated primarily because DNA primases function as part of macromolecular complexes. These multiprotein complexes often catalyze additional reactions affecting NTP or DNA concentrations, confounding the examination of RNA synthesis alone. Moreover, when DNA primases have been separated from these complexes, their RNA synthesis activities are often profoundly decreased (2, 3).Gene 4 of Escherichia coli bacteriophage T7 encodes a multifunctional protein that provides both helicase and primase activities required for the replication of the phage DNA. The gene 4 product is a hexamer (4 -6) comprised of two co-linear proteins translated from separate in-frame start sites (7). The smaller 56-kDa protein translocates 5Ј-to-3Ј on ssDNA and unwinds duplex DNA (helicase activity) in a reaction coupled to the hydrolysis of nucleoside triphosphates (8 -10). The larger 63-kDa protein has all of the activities found in the 56-kDa protein and also acts as a primase by catalyzing the templatedirected synthesis of short RNA polymers (11-13). T7 DNA polymerase (14, 15) uses these oligoribonucleotides as primers.Although the presence of ...

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Section: Resultsmentioning

confidence: 83%

“…These multiprotein complexes often catalyze additional reactions affecting NTP or DNA concentrations, confounding the examination of RNA synthesis alone. Moreover, when DNA primases have been separated from these complexes, their RNA synthesis activities are often profoundly decreased (2,3).…”

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confidence: 99%

Interaction of Bacteriophage T7 Gene 4 Primase with Its Template Recognition Site

Frick

Richardson

1999

Journal of Biological Chemistry

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“…When nucleotides were omitted from reaction mixtures, we still detected a β-clamp complex on supercoiled plasmid DNA (Fig. S5 A-D), which is most likely being due to primase using dNTPs (40,41) or ATP (42) in the reaction mixture for primer synthesis. Due to DnaB and DnaC requirements for ATP, we could not omit ATP from reaction mixtures.…”

Section: Discussionmentioning

confidence: 99%

Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly

Wawrzycka

Gross

Wasaznik

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Although the molecular basis for replisome activity has been extensively investigated, it is not clear what the exact mechanism for de novo assembly of the replication complex at the replication origin is, or how the directionality of replication is determined. Here, using the plasmid RK2 replicon, we analyze the protein interactions required for Escherichia coli polymerase III (Pol III) holoenzyme association at the replication origin. Our investigations revealed that in E. coli, replisome formation at the plasmid origin involves interactions of the RK2 plasmid replication initiation protein (TrfA) with both the polymerase β-and α-subunits. In the presence of other replication proteins, including DnaA, helicase, primase and the clamp loader, TrfA interaction with the β-clamp contributes to the formation of the β-clamp nucleoprotein complex on origin DNA. By reconstituting in vitro the replication reaction on ssDNA templates, we demonstrate that TrfA interaction with the β-clamp and sequence-specific TrfA interaction with one strand of the plasmid origin DNA unwinding element (DUE) contribute to strand-specific replisome assembly. Wild-type TrfA, but not the TrfA QLSLF mutant (which does not interact with the β-clamp), in the presence of primase, helicase, Pol III core, clamp loader, and β-clamp initiates DNA synthesis on ssDNA template containing 13-mers of the bottom strand, but not the top strand, of DUE. Results presented in this work uncovered requirements for anchoring polymerase at the plasmid replication origin and bring insights of how the directionality of DNA replication is determined.DNA replication initiation | polymerase III | β-clamp | Rep | plasmid RK2

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“…How the slow enzymatic steps of priming and polymerase loading take place on the lagging strand without causing loss of coordination with continuous leading-strand synthesis is a long-standing question in the field of replication, and several divergent models have been proposed based on various strands of experimental evidence (Corn et al, 2005;Dixon, 2009;Frick et al, 1999;Lee et al, 2006;Li and Marians, 2000;Manosas et al, 2009;Pandey et al, 2009;Swart and Griep, 1995;Tanner et al, 2008;Yuzhakov et al, 1999). One model postulates that priming pauses leading-strand synthesis ( Figure 1A) to ensure that the events on each daughter strand remain synchronized (Lee et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes

et al. 2016

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. (2016). Simultaneous real-time imaging of leading and lagging strand synthesis reveals the coordination dynamics of single replisomes. Molecular Cell, 64 (6), 1035-1047.Simultaneous real-time imaging of leading and lagging strand synthesis reveals the coordination dynamics of single replisomes AbstractThe molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation. SummaryThe molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, andOkazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazakifragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.3

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Primer synthesis kinetics by Escherichia coli primase on single-stranded DNA templates

Cited by 59 publications

References 38 publications

Interaction of Bacteriophage T7 Gene 4 Primase with Its Template Recognition Site

Interaction of Bacteriophage T7 Gene 4 Primase with Its Template Recognition Site

Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly

Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes

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