Roles of bacteriophage T7 gene 4 proteins in providing primase and helicase functions in vivo.

Mendelman, Lynn V.; Notarnicola, Stephen M.; Richardson, Charles C.

doi:10.1073/pnas.89.22.10638

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“…In addition to the full-length primase-helicase, a 56-kDa isoform lacking the ZBD is produced from an in-frame translation initiation site within T7 gene 4 during phage growth (35). The 56-kDa isoform of the primase-helicase has helicase activity but no primer synthesis activity (30,36).A second important activity of T7 primase is to promote the utilization of oligoribonucleotide primers by T7 DNA polymerase (37). The primase remains associated with its tetraribo-* This work was supported by National Institutes of Health Grants GM55390 (to T. E.) and GM47467 (to G. W.) and the resources of the Harvard-Armenise Center for Structural Biology.…”

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A Molecular Handoff between Bacteriophage T7 DNA Primase and T7 DNA Polymerase Initiates DNA Synthesis

Kato

Ito

Wagner

et al. 2004

Journal of Biological Chemistry

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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 singlestranded 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.DNA synthesis is initiated on the lagging strand of the replication fork by a site-specific RNA polymerase known as a DNA primase (1). The primase synthesizes a short RNA primer that is extended by DNA polymerase to create an Okazaki fragment (2, 3). During replication, interactions between the primase and other replicative proteins such as DNA polymerase, DNA helicase, and single-stranded DNA (ssDNA) 1 -binding protein are required to initiate DNA synthesis. It has been difficult to identify these essential interactions because they occur transiently and involve weak interactions between multiple partners.We have been studying the physical and functional interactions between proteins of the comparatively simple bacteriophage T7 replication system. Phage T7 replicates DNA using four proteins, T7 gene 4 primase-helicase protein (gp4), T7 gene 2.5 ssDNA-binding protein (gp2.5), and T7 DNA polymerase (gp5) complexed with its processivity factor, Escherichia coli thioredoxin (4). The reactions catalyzed by the individual proteins are coordinated during replication by the assembly of a multiprotein complex that moves with the replication fork (5, 6). During replication, the primase-helicase directly contacts both the DNA polymerase and gp2.5 (7-10). A C-terminal acidic segment of the primase-helicase is required for its interaction with the DNA polymerase (10). An interaction between gp2.5 and T7 DNA polymerase is required for the coordinated synthesis of both leading and lagging strands of the replication fork (5, 6). A C-terminal 21-residue region of gp2.5 is essential not only for the interactions with the DNA polymerase and the primase-helicase but also for dimerization of gp2.5 (9). Although many pairwise interactions between T7 replication proteins have been identified, the overall physical arrangement of subunits and their stoichiometries in the replication complex are unknown.Crystal structures of all the individual T7 replication proteins (11-16) have been determined, and their enzymatic ac...

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A Molecular Handoff between Bacteriophage T7 DNA Primase and T7 DNA Polymerase Initiates DNA Synthesis

Kato

Ito

Wagner

et al. 2004

Journal of Biological Chemistry

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“…In conjunction with Dr. Ellenberger we are analyzing these proteins and DNAs for the optimum conditions for crystallization of a gene 5 protein-thioredoxin/primer-template complex, which will be described under Method of Study. D..T7 DNA P01ym_r_lse.for DNA Sequence Analysis 1..E_ff¢ct of pyrophosphorolysis on DNA .sequence analysis (1,13) Pyrophosphorolysis is the reversal of polymerization whereby there is a nucleophilic attack on the 3'-temaimd internucleotide linkage by inorganic pyrophosphate to release a dNTP. With T7 DNA polymerase, specific dideoxy-tenninated fragments are sensitive to this reaction, resulting in their selective degradation and the loss of the corresponding b_mdon the gel.…”

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“…Structural Analysis of the Binding of T7 DNA Polymerase tq a Primer-Template 1. Binding to primers with medified 3'-termini (13,16) We have analyzed the static binding of T7 DNA polymerase to primer-template complexes by (i) measuring the resistance of the DNA to challenge with exo-and endonucleases, (ii) nitrocellulose filter binding, and (iii) fluorescence spectroscopy. The gene 5 protein alone binds to an unmodified primer-template with a half-life of less than one sec.…”

Section: Ldentif_ofmentioning

confidence: 99%

“…Crystallization ofT7 DNA Polymerase (13,14) One goal of this project is to obtain the three dimensional structure of T7 DNA polymerase. During the past two years we have collaborated with Dr. William E. Royer of the University of Massachusetts Medical School and Dr. Alex Rich at the Massachusetts Institute of Technology in attempts to crystallize T7 gene 5 protein, the T7 gene 5 protein-thioredoxin complex, and the T7 gene 5 protein-thioredoxin complex in the presence of a primer-template.…”

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Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing; Progress report, June 1, 1990--May 31, 1993

Richardson¹

1993

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Requirement for a zinc motif for template recognition by the bacteriophage T7 primase.

1994

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Gene 4 of bacteriophage T7 encodes two proteins, a 63 kDa and a colinear 56 kDa protein. The coding sequence of the 56 kDa protein begins at the residues encoding an internal methionine located 64 amino acids from the N‐terminus of the 63 kDa protein. The 56 kDa gene 4 protein is a helicase and the 63 kDa gene 4 protein is a helicase and a primase. The unique 7 kDa N‐terminus of the 63 kDa gene 4 protein is essential for primer synthesis and contains sequences with homology to a Cys4 metal binding motif, Cys‐X2‐Cys‐X17‐Cys‐X2‐Cys. The zinc content of the 63 kDa gene 4 protein is 1.1 g‐atom/mol protein, while the zinc content of the 56 kDa gene 4 protein is < 0.01, as determined by atomic absorption spectrometry. A bacteriophage deleted for gene 4, T7 delta 4‐1, is incapable of growing on Escherichia coli strains that contain plasmids expressing gene 4 proteins with single amino acid substitutions of Ser at each of the four conserved Cys residues (efficiency of plating, 10(‐7)). Primase containing a substitution of the third Cys for Ser has been overexpressed in E. coli and purified to homogeneity. This mutant primase cannot catalyze template‐directed synthesis of oligoribonucleotides although it is able to catalyze the synthesis of random diribonucleotides in a template‐independent fashion. The mutant primase has reduced helicase activity although it catalyzes single‐stranded DNA‐dependent hydrolysis of dTTP at rates comparable with wild type primase. The zinc content of the mutant primase is 0.5 g‐atom/mol protein.

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Roles of bacteriophage T7 gene 4 proteins in providing primase and helicase functions in vivo.

Cited by 50 publications

References 43 publications

A Molecular Handoff between Bacteriophage T7 DNA Primase and T7 DNA Polymerase Initiates DNA Synthesis

A Molecular Handoff between Bacteriophage T7 DNA Primase and T7 DNA Polymerase Initiates DNA Synthesis

Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing; Progress report, June 1, 1990--May 31, 1993

Requirement for a zinc motif for template recognition by the bacteriophage T7 primase.

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