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)

At a replication fork DNA primase synthesizes oligoribonucleotides that serve as primers for the lagging strand DNA polymerase. In the bacteriophage T7 replication system, DNA primase is encoded by gene 4 of the phage. The 63-kDa gene 4 protein is composed of two major domains, a helicase domain and a primase domain located in the C- and N-terminal halves of the protein, respectively. T7 DNA primase recognizes the sequence 5'-NNGTC-3' via a zinc motif and catalyzes the template-directed synthesis of tetraribonucleotides pppACNN. T7 DNA primase, like other primases, shares limited homology with DNA-dependent RNA polymerases. To identify the catalytic core of the T7 DNA primase, single-point mutations were introduced into a basic region that shares sequence homology with RNA polymerases. The genetically altered gene 4 proteins were examined for their ability to support phage growth, to synthesize functional primers, and to recognize primase recognition sites. Two lysine residues, Lys-122 and Lys-128, are essential for phage growth. The two residues play a key role in the synthesis of phosphodiester bonds but are not involved in other activities mediated by the protein. The altered primases are unable to either synthesize or extend an oligoribonucleotide. However, the altered primases do recognize the primase recognition sequence, anneal an exogenous primer 5'-ACCC-3' at the site, and transfer the primer to T7 DNA polymerase. Other lysines in the vicinity are not essential for the synthesis of primers.
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PMID:Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7. 1167 65

The proteins of bacteriophage T7 DNA replication mediate coordinated leading and lagging strand synthesis on a minicircle template. A distinguishing feature of the coordinated synthesis is the presence of a replication loop containing double and single-stranded DNA with a combined average length of 2600 nucleotides. Lagging strands consist of multiple Okazaki fragments, with an average length of 3000 nucleotides, suggesting that the replication loop dictates the frequency of initiation of Okazaki fragments. The size of Okazaki fragments is not affected by varying the components (T7 DNA polymerase, gene 4 helicase-primase, gene 2.5 single-stranded DNA binding protein, and rNTPs) of the reaction over a relatively wide range. Changes in the size of Okazaki fragments occurs only when leading and lagging strand synthesis is no longer coordinated. The synthesis of each Okazaki fragment is initiated by the synthesis of an RNA primer by the gene 4 primase at specific recognition sites. In the absence of a primase recognition site on the minicircle template no lagging strand synthesis occurs. The size of the Okazaki fragments is not affected by the number of recognition sites on the template.
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PMID:Lagging strand synthesis in coordinated DNA synthesis by bacteriophage t7 replication proteins. 1182

DNA primases catalyze the synthesis of oligoribonucleotides to initiate lagging strand DNA synthesis during DNA replication. Like other prokaryotic homologs, the primase domain of the gene 4 helicase-primase of bacteriophage T7 contains a zinc motif and a catalytic core. Upon recognition of the sequence, 5'-GTC-3' by the zinc motif, the catalytic site condenses the cognate nucleotides to produce a primer. The TOPRIM domain in the catalytic site contains several charged residues presumably involved in catalysis. Each of eight acidic residues in this region was replaced with alanine, and the properties of the altered primases were examined. Six of the eight residues (Glu-157, Glu-159, Asp-161, Asp-207, Asp-209, and Asp-237) are essential in that altered gene 4 proteins containing these mutations cannot complement T7 phage lacking gene 4 for T7 growth. These six altered gene 4 proteins can neither synthesize primers de novo nor extend an oligoribonucleotide. Despite the inability to catalyze phosphodiester bond formation, the altered proteins recognize the sequence 5'-GTC-3' in the template and deliver preformed primer to T7 DNA polymerase. The alterations in the TOPRIM domain result in the loss of binding affinity for ATP as measured by surface plasmon resonance assay together with ATP-agarose affinity chromatography.
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PMID:Acidic residues in the nucleotide-binding site of the bacteriophage T7 DNA primase. 1591 41

The replication and repair of organellar genomes in the malaria parasite Plasmodium falciparum is poorly understood. We have assessed the properties of an open reading frame Pfprex (formerly known as pom1) and confirm that it specifies a multi-domain polypeptide with DNA primase, DNA helicase, DNA polymerase and 3'-5' exonuclease activities. The sequence of the primase/helicase domain is phylogenetically related to the T7-bacteriophage gene 4 product and mammalian mitochondrial helicase, Twinkle. Despite that, the N-terminal sequence of this multi-domain polypeptide directs a green fluorescent protein reporter specifically to the P. falciparum apicoplast and not to the mitochondrion. Phylogenetic analysis placed the DNA polymerase sequence with the family A bacterial polymerases, most closely to those of the thermophilic Aquifex species. Notably, the malarial enzyme was optimally active at 75 degrees C. Pfprex is the first example of a gene encoding contiguous DNA polymerase, DNA primase and DNA helicase components. We propose it has a key role in replication of the malarial plastid genome, a validated drug target.
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PMID:The plastidic DNA replication enzyme complex of Plasmodium falciparum. 1592 3

The gene 4 protein of bacteriophage T7 plays a central role in DNA replication by providing both helicase and primase activities. The C-terminal helicase domain is not only responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding, but it also interacts with T7 DNA polymerase to coordinate helicase and polymerase activities. The C-terminal 17 residues of gene 4 protein are critical for its interaction with the T7 DNA polymerase/thioredoxin complex. This C terminus is highly acidic; replacement of these residues with uncharged residues leads to a loss of interaction with T7 DNA polymerase/thioredoxin and an increase in oligomerization of the gene 4 protein. Such an alteration on the C terminus results in a reduced efficiency in strand displacement DNA synthesis catalyzed by gene 4 protein and T7 DNA polymerase/thioredoxin. Replacement of the C-terminal amino acid, phenylalanine, with non-aromatic residues also leads to a loss of interaction of gene 4 protein with T7 DNA polymerase/thioredoxin. However, neither of these modifications of the C terminus affects helicase and primase activities. A chimeric gene 4 protein containing the acidic C terminus of the T7 gene 2.5 single-stranded DNA-binding protein is more active in strand displacement synthesis. Gene 4 hexamers containing even one subunit of a defective C terminus are defective in their interaction with T7 DNA polymerase.
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PMID:The C-terminal residues of bacteriophage T7 gene 4 helicase-primase coordinate helicase and DNA polymerase activities. 1680 31

Bacteriophage T7 helicase (T7 gene 4 helicase-primase) is a prototypical member of the ring-shaped family of helicases, whose structure and biochemical mechanisms have been studied in detail. T7 helicase assembles into a homohexameric ring that binds single-stranded DNA in its central channel. Using RecA-type nucleotide binding and sensing motifs, T7 helicase binds and hydrolyzes several NTPs, among which dTTP supports optimal protein assembly, DNA binding and unwinding activities. During translocation along single stranded DNA, the subunits of the ring go through dTTP hydrolysis cycles one at a time, and this probably occurs also during DNA unwinding. Interestingly, the unwinding speed of T7 helicase is an order of magnitude slower than its translocation rate along single stranded DNA. The slow unwinding rate is greatly stimulated when DNA synthesis by T7 DNA polymerase is coupled to DNA unwinding. Using the T7 helicase as an example, we highlight critical findings and discuss possible mechanisms of helicase action.
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PMID:Mechanisms of a ring shaped helicase. 1693 79

T7 gene 5 DNA polymerase (gp5) and its processivity factor, Escherichia coli thioredoxin, together with the T7 gene 4 DNA helicase, catalyze strand displacement synthesis on duplex DNA processively (>17,000 nucleotides per binding event). The processive DNA synthesis is resistant to the addition of a DNA trap. However, when the polymerase-thioredoxin complex actively synthesizing DNA is challenged with excess DNA polymerase-thioredoxin exchange occurs readily. The exchange can be monitored by the use of a genetically altered T7 DNA polymerase (gp5-Y526F) in which tyrosine-526 is replaced with phenylalanine. DNA synthesis catalyzed by gp5-Y526F is resistant to inhibition by chain-terminating dideoxynucleotides because gp5-Y526F is deficient in the incorporation of these analogs relative to the wild-type enzyme. The exchange also occurs during coordinated DNA synthesis in which leading- and lagging-strand synthesis occur at the same rate. On ssDNA templates with the T7 DNA polymerase alone, such exchange is not evident, suggesting that free polymerase is first recruited to the replisome by means of T7 gene 4 helicase. The ability to exchange DNA polymerases within the replisome without affecting processivity provides advantages for fidelity as well as the cycling of the polymerase from a completed Okazaki fragment to a new primer on the lagging strand.
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PMID:Exchange of DNA polymerases at the replication fork of bacteriophage T7. 1736 50

Escherichia coli thioredoxin binds to a unique flexible loop of 71 amino acid residues, designated the thioredoxin binding domain (TBD), located in the thumb subdomain of bacteriophage T7 gene 5 DNA polymerase. The initial designation of thioredoxin as a processivity factor was premature. Rather it remodels the TBD for interaction with DNA and the other replication proteins. The binding of thioredoxin exposes a number of basic residues on the TBD that lie over the duplex region of the primer-template and increases the processivity of nucleotide polymerization. Two small solvent-exposed loops (loops A and B) located within TBD electrostatically interact with the acidic C-terminal tail of T7 gene 4 helicase-primase, an interaction that is enhanced by the binding of thioredoxin. Several basic residues on the surface of thioredoxin in the polymerase-thioredoxin complex lie in close proximity to the TBD. One of these residues, lysine 36, is located proximal to loop A. The substitution of glutamate for lysine has a dramatic effect on the binding of gene 4 helicase to a DNA polymerase-thioredoxin complex lacking charges on loop B; binding is decreased 15-fold relative to that observed with wild-type thioredoxin. This defective interaction impairs the ability of T7 DNA polymerase-thioredoxin together with T7 helicase to mediate strand displacement synthesis. This is the first demonstration that thioredoxin interacts with replication proteins other than T7 DNA polymerase.
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PMID:Interactions of Escherichia coli thioredoxin, the processivity factor, with bacteriophage T7 DNA polymerase and helicase. 1875 58

DNA helicase and primase are essential for DNA replication. The helicase unwinds the DNA to provide single-stranded templates for DNA polymerase. The primase catalyzes the synthesis of oligoribonucleotides for the initiation of lagging strand synthesis. The two activities reside in a single polypeptide encoded by gene 4 of bacteriophage T7. Their coexistence within the same polypeptide facilitates their coordination during DNA replication. One surface of helix E within the helicase domain is positioned to interact with the primase domain and the linker connecting the two domains within the functional hexamer. The interaction occurs in trans such that helix E interacts with the primase domain and the linker of the adjacent subunit. Most alterations of residues on the surface of helix E (Arg(404), Lys(408), Tyr(411), and Gly(415)) eliminate the ability of the altered proteins to complement growth of T7 phage lacking gene 4. Both Tyr(411) and Gly(415) are important in oligomerization of the protein. Alterations G415V and K408A simultaneously influence helicase and primase activities in opposite manners that mimic events observed during coordinated DNA synthesis. The results suggest that Asp(263) located in the linker of one subunit can interact with Tyr(411), Lys(408), or Arg(404) in helix E of the adjacent subunit depending on the oligomerization state. Thus the switch in contacts between Asp(263) and its three interacting residues in helix E of the adjacent subunit results in conformational changes that modulate helicase and primase activity.
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PMID:An in trans interaction at the interface of the helicase and primase domains of the hexameric gene 4 protein of bacteriophage T7 modulates their activities. 1957 19

The ring-shaped helicase of bacteriophage T7 (gp4), the product of gene 4, has basic beta-hairpin loops lining its central core where they are postulated to be the major sites of DNA interaction. We have altered multiple residues within the beta-hairpin loop to determine their role during dTTPase-driven DNA unwinding. Residues His-465, Leu-466, and Asn-468 are essential for both DNA unwinding and DNA synthesis mediated by T7 DNA polymerase during leading-strand DNA synthesis. Gp4-K467A, gp4-K471A, and gp4-K473A form fewer hexamers than heptamers compared to wild-type helicase and alone are deficient in DNA unwinding. However, they complement for the growth of T7 bacteriophage lacking gene 4. Single-molecule studies show that these three altered helicases support rates of leading-strand DNA synthesis comparable to that observed with wild-type gp4. Gp4-K467A, devoid of unwinding activity alone, supports leading-strand synthesis in the presence of T7 DNA polymerase. We propose that DNA polymerase limits the backward movement of the helicase during unwinding as well as assisting the forward movement necessary for strand separation.
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PMID:Residues in the central beta-hairpin of the DNA helicase of bacteriophage T7 are important in DNA unwinding. 2035 Dec 55


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