Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used the T7 DNA replication system to examine coordination of leading and lagging strand synthesis at a replication fork. The 63 kd gene 4 protein provides both helicase and primase activities; we demonstrate that primer synthesis inhibits helicase activity on a synthetic replication fork. Lagging strand DNA synthesis by a complex of gene 4 protein and T7 DNA polymerase decreases the rate of leading strand synthesis. Both leading and lagging strand synthesis are resistant to dilution of the replication proteins, and to challenge with heparin. Furthermore, dilution does not increase the average length of Okazaki fragments. We propose that leading and lagging strand synthesis at a T7 replication fork are coupled and that the replication proteins are recycled.
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PMID:Coordination of leading and lagging strand DNA synthesis at the replication fork of bacteriophage T7. 815 91

The product of gene 2.5 of bacteriophage T7, a single-stranded DNA binding protein, physically interacts with the phage-encoded gene 5 protein (DNA polymerase) and gene 4 proteins (helicase and primase) and stimulates their activities. Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth. T7 phages that contain null mutants of gene 2.5 were constructed by homologous recombination. These gene 2.5 null mutants contain either a deletion of gene 2.5 (T7 delta 2.5) or an insertion into gene 2.5 and cannot grow in Escherichia coli (efficiency of plating, < 10(-8)). After infection of E. coli with T7 delta 2.5, host DNA synthesis is shut off, and phage DNA synthesis is reduced to < 1% of phage DNA synthesis in wild-type T7-infected E. coli cells as measured by incorporation of [3H]thymidine. In contrast, RNA synthesis is essentially normal in T7 delta 2.5-infected cells. The defects in growth and DNA replication are overcome by wild-type gene 2.5 protein expressed from a plasmid harboring the T7 gene 2.5.
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PMID:Bacteriophage T7 gene 2.5 protein: an essential protein for DNA replication. 823 73

Bacteriophage T7 gene 2.5 single-stranded DNA-binding protein and gene 4 DNA helicase together promote pairing of two homologous DNA molecules and subsequent polar branch migration (Kong, D., and Richardson, C. C. (1996) EMBO J. 15, 2010-2019). In this report, we show that gene 2.5 protein is not required for the initiation or propagation of strand transfer once a joint molecule has been formed between the two DNA partners, a reaction that is mediated by the gene 2.5 protein alone. A mutant gene 2.5 protein, gene 2.5-Delta21C protein, lacking 21 amino acid residues at its C terminus, cannot physically interact with gene 4 protein. Although it does bind to single-stranded DNA and promote the formation of joint molecule via homologous base pairing, subsequent strand transfer by gene 4 helicase is inhibited by the presence of the gene 2.5-Delta21C protein. Bacteriophage T4 gene 32 protein likewise inhibits T7 gene 4 protein-mediated strand transfer, whereas Escherichia coli single-stranded DNA-binding protein does not. The 63-kDa gene 4 protein of phage T7 is also a DNA primase in that it catalyzes the synthesis of oligonucleotides at specific sequences during translocation on single-stranded DNA. We find that neither the rate nor extent of strand transfer is significantly affected by concurrent primer synthesis. The bacteriophage T4 gene 41 helicase has been shown to catalyze polar branch migration after the T4 gene 59 helicase assembly protein loads the helicase onto joint molecules formed by the T4 UvsX and gene 32 proteins (Salinas, F., and Kodadek, T. (1995) Cell 82, 111-119). We find that gene 32 protein alone forms joint molecules between partially single-stranded homologous DNA partners and that subsequent branch migration requires this single-stranded DNA-binding protein in addition to the gene 41 helicase and the gene 59 helicase assembly protein. Similar to the strand transfer reaction, strand displacement DNA synthesis catalyzed by T4 DNA polymerase also requires the presence of gene 32 protein in addition to the gene 41 and 59 proteins.
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PMID:Role of the bacteriophage T7 and T4 single-stranded DNA-binding proteins in the formation of joint molecules and DNA helicase-catalyzed polar branch migration. 907 62

The 63-kDa gene 4 primase of bacteriophage T7 recognizes a core trinucleotide sequence, 5'-GTC-3', on single-stranded DNA at which it catalyzes the synthesis of the ribodinucleotide pppAC. The dinucleotide is extended to a tetranucleotide primer at the sites 5'-(G/T)GGTC-3' and 5'-GTGTC-3'. In the presence of T7 primase, T7 DNA polymerase extends the synthetic ribotetranucleotide pACCA (1 microM), but not pCACA, on M13 DNA templates. The reaction is specific for T7 DNA polymerase and depends on dTTP and translocation of the gene 4 protein. T7 primase extends the dinucleotide AC and trinucleotide ACC to ACCC in the presence of CTP and an appropriate template, whereas other dinucleotides are extended less efficiently; the deoxyribodinucleotide dAC is not extended. The Cys4 zinc motif of the primase is essential for extension of the dinucleotides. The 5'-cryptic cytidine of the recognition sequence is essential for extension of the dinucleotide AC to tri- and tetranucleotides. At a preformed replication fork, the dinucleotide AC provides for primer synthesis on the lagging strand. The synthesis of all Okazaki fragments is initiated by primers arising from the recognition sequence 5'-GGGTC-3'; none arise at an adjacent 5'-GGGTT-3' sequence. If ADP or AMP replaces ATP in the primase reaction, primers terminating in di- or monophosphate, respectively, are synthesized.
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PMID:Gene 4 DNA primase of bacteriophage T7 mediates the annealing and extension of ribo-oligonucleotides at primase recognition sites. 913 92

The gene 4 proteins of bacteriophage T7 provide both primase and helicase activities at the replication fork. Efficient DNA replication requires that the functions of the gene 4 protein be coordinated with the movement of the T7 DNA polymerase. We show that a carboxyl-terminal domain of the gene 4 protein is required for interaction with T7 DNA polymerase during leading strand DNA synthesis. The carboxyl terminus of the gene 4 protein is highly acidic: of the 17 carboxyl-terminal amino acids 7 are negatively charged. Deletion of the coding region for these 17 residues results in a gene 4 protein that cannot support the growth of T7 phage. The purified mutant gene 4 protein has wild-type levels of both helicase and primase activities; however, DNA synthesis catalyzed by T7 DNA polymerase on a duplex DNA substrate is stimulated by this mutant protein to only about 5% of the level of synthesis obtained with wild-type protein. The mutant gene 4 protein can form hexamers and bind single-stranded DNA, but as determined by native PAGE analysis, the protein cannot form a stable complex with the DNA polymerase. The mutant gene 4 protein can prime DNA synthesis normally, indicating that for lagging strand synthesis a different set of helicase/primase-DNA polymerase interactions are involved. These findings have implications for the mechanisms coupling leading and lagging strand DNA synthesis at the T7 replication fork.
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PMID:The acidic carboxyl terminus of the bacteriophage T7 gene 4 helicase/primase interacts with T7 DNA polymerase. 921 86

Intermediates in the replication of circular and linear M13 double-stranded DNA by bacteriophage T7 proteins have been examined by electron microscopy. Synthesis generated double-stranded DNA molecules containing a single replication fork with a linear duplex tail. A complex presumably consisting of T7 DNA polymerase and gene 4 helicase/primase molecules was present at the fork together with a variable amount of single-stranded DNA sequestered by gene 2.5 single-stranded DNA binding protein. Analysis of the length distribution of Okazaki fragments formed at different helicase/primase concentrations was consistent with coupling of leading and lagging strand replication. Fifteen to forty percent of the templates engaged in replication have a DNA loop at the replication fork. The loops are fully double-stranded with an average length of approximately 1 kilobase. Labeling with biotinylated dCTP showed that the loops consist of newly synthesized DNA, and synchronization experiments using a linear template with a G-less cassette demonstrated that the loops are formed by active displacement of the lagging strand. A long standing feature of models for coupled leading/lagging strand replication has been the presence of a DNA loop at the replication fork. This study provides the first direct demonstration of such loops.
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PMID:Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins. 947 83

Primase and helicase activities of bacteriophage T7 are present in a single polypeptide coded by gene 4. Because the amino terminal region of the gene 4 protein contributes to primase activity, we constructed a truncated gene 4 encoding the N-terminal 271-aa residues. The truncated protein, purified from cells overexpressing the protein, is a dimer in solution; the full-length protein is a hexamer. Although the fragment is devoid of dTTPase and helicase activities, it catalyzes template-directed synthesis of di-, tri-, and tetranucleotides. The rates for tetraribonucleotide synthesis and for dinucleotide extension on a 20-nucleotide template are similar for the full-length and truncated proteins. However, the activity of the primase fragment is unaffected by dTTP whereas the primase activity of the full-length protein is stimulated >14-fold. The primase fragment is defective in the interaction with T7 DNA polymerase in that primer synthesis cannot be coupled to DNA synthesis.
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PMID:An N-terminal fragment of the gene 4 helicase/primase of bacteriophage T7 retains primase activity in the absence of helicase activity. 965 22

The gene 4 protein of bacteriophage T7, a functional hexamer, comprises DNA helicase and primase activities. Both activities depend on the unidirectional movement of the protein along single-stranded DNA in a reaction coupled to the hydrolysis of dTTP. We have characterized dTTPase activity and hexamer formation for the full-length gene 4 protein (gp4) as well as for three carboxyl-terminal fragments starting at residues 219 (gp4-C219), 241 (gp4-C241), and 272 (gp4-C272). The region between residues 242 and 271, residing between the primase and helicase domains, is critical for oligomerization of the gene 4 protein. A functional TPase active site is dependent on oligomerization. During native gel electrophoresis, gp4, gp4-C219, and gp4-C241 migrate as oligomers, whereas gp4-C272 is monomeric. The steady-state k(cat) for dTTPase activity of gp4-C272 increases sharply with protein concentration, indicating that it forms oligomers only at high concentrations. gp4-C219 and gp4-C241 both form a stable complex with gp4, whereas gp4-C272 interacts only weakly with gp4. Measurements of surface plasmon resonance indicate that a monomer of T7 DNA polymerase binds to a dimer of gp4, gp4-C219, or gp4-C241 but to a monomer of gp4-C272. Like the homologous RecA and F(1)-ATPase proteins, the oligomerization domain of the gene 4 protein is adjacent to the amino terminus of the NTP-binding domain.
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PMID:The linker region between the helicase and primase domains of the bacteriophage T7 gene 4 protein is critical for hexamer formation. 1051 25

The three-dimensional structure of bacteriophage T7 DNA polymerase reveals the presence of a loop of 4 aa (residues 401-404) within the DNA-binding groove; this loop is not present in other members of the DNA polymerase I family. A genetically altered T7 DNA polymerase, T7 polDelta401-404, lacking these residues, has been characterized biochemically. The polymerase activity of T7 polDelta401-404 on primed M13 single-stranded DNA template is one-third of the wild-type enzyme and has a 3'-to-5' exonuclease activity indistinguishable from that of wild-type T7 DNA polymerase. T7 polDelta401-404 polymerizes nucleotides processively on a primed M13 single-stranded DNA template. T7 DNA polymerase cannot initiate de novo DNA synthesis; it requires tetraribonucleotides synthesized by the primase activity of the T7 gene 4 protein to serve as primers. T7 primase-dependent DNA synthesis on single-stranded DNA is 3- to 6-fold less with T7 polDelta401-404 compared with the wild-type enzyme. Furthermore, the altered polymerase is defective (10-fold) in its ability to use preformed tetraribonucleotides to initiate DNA synthesis in the presence of gene 4 protein. The location of the loop places it in precisely the position to interact with the tetraribonucleotide primer and, presumably, with the T7 gene 4 primase. Gene 4 protein also provides helicase activity for the replication of duplex DNA. T7 polDelta401-404 and T7 gene 4 protein catalyze strand-displacement DNA synthesis at nearly the same rate as does wild-type polymerase and T7 gene 4 protein, suggesting that the coupling of helicase and polymerase activities is unaffected.
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PMID:A unique loop in the DNA-binding crevice of bacteriophage T7 DNA polymerase influences primer utilization. 1105 Jan 88

The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them. Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase.
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PMID:A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization. 1127 45


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