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)

Studies with a rolling-circle DNA replication system reconstituted in vitro with a tailed form II DNA template, the DNA polymerase III holoenzyme (Pol III HE), the Escherichia coli single-stranded DNA binding protein, and the primosome, showed that within the context of a replication fork, the oligoribonucleotide primers that were formed were limited to a length in the range of 9 to 14 nucleotides, regardless of whether they were subsequently elongated by the lagging-strand DNA polymerase. This is in contrast to the 8-60-nucleotide-long primers synthesized by the primosome in the absence of DNA replication on a bacteriophage phi X174 DNA template, although when primer synthesis and DNA replication were catalyzed concurrently in this system, the extent of RNA polymerization decreased. As described in this report, we therefore examined the effect of the DNA Pol III HE on the length of primers synthesized by primase in vitro in the absence of DNA replication. When primer synthesis was catalyzed either: i) by the primosome on a phi X174 DNA template, ii) by primase on naked DNA with the aid of the DnaB protein (general priming), or iii) by primase alone at the bacteriophage G4 origin, the presence of the DNA Pol III HE in the reaction mixtures resulted in a universal reduction in the length of the heterogeneous RNA products to a uniform size of approximately 10 nucleotides. dNTPs were not required, and the addition of dGMP, an inhibitor of the 3'----5' exonuclease of the DNA Pol III HE, did not alter the effect; therefore, neither the 5'----3' DNA polymerase activity nor the 3'----5' exonuclease activity of the DNA Pol III HE was involved. E. coli DNA polymerase I, and the DNA polymerases of bacteriophages T4 and T7 could not substitute for the DNA Pol III HE. The Pol III core plays a crucial role in mediating this effect, although other subunits of the DNA Pol III HE are also required. These observations suggest that the association of primase with the DNA Pol III HE during primer synthesis regulates its catalytic activity and that this regulatory interaction occurs independently of, and prior to, formation of a preinitiation complex of the DNA Pol III HE on the primer terminus.
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PMID:Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. III. A polymerase-primase interaction governs primer size. 153 80

The bacteriophage PRD1 DNA polymerase gene (gene I) has been cloned into the expression vector pPLH101 under the control of the lambda pL promoter. Tailoring of an efficient ribosome binding site in front of the gene by polymerase chain reaction led to a high level heat-inducible expression of the corresponding gene product (P1) in Escherichia coli cells. Expression was confirmed in vivo by complementation of phage PRD1 DNA polymerase gene mutants and in vitro by formation of the genome terminal protein P8-dGMP replication initiation complex. Expressed PRD1 DNA polymerase was purified to apparent homogeneity in an active form. DNA polymerase, 3'-5'-exonuclease, and P8-dGMP replication initiation complex formation activities cosedimented in glycerol gradient with a protein of 65 kDa, the size expected for PRD1 DNA polymerase. The DNA polymerase was active on DNase I-activated calf thymus DNA, poly(dA).oligo(dT) and poly(dA-dT) primer/templates as well as on native phage PRD1 genome. The 3'-5'-exonuclease activity was specific for single-stranded DNA and released mononucleotides. No 5'-3'-exonuclease activity was detected. The inhibitor/activator spectrum of the PRD1 DNA polymerase was also studied. An in vitro replication system with purified components for bacteriophage PRD1 was established. Formation of the P8-dGMP replication initiation complex was a prerequisite for phage DNA replication, which proceeded from the initiation complex and yielded genome length replication products.
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PMID:Overexpression, purification, and characterization of Escherichia coli bacteriophage PRD1 DNA polymerase. In vitro synthesis of full-length PRD1 DNA with purified proteins. 165 59

DNA polymerase III holoenzyme is responsible for chromosomal DNA synthesis in Escherichia coli and seems to be a major determinant of the fidelity of replication of this organism. Among ten different subunits of the holoenzyme, the alpha subunit, encoded by the dnaE gene, has a polymerase activity, while the epsilon subunit, encoded by the dnaQ gene, is a proofreader with a 3'-5' exonuclease activity. Using poly(dA)/oligo(dT)20 as a template-primer, misincorporation of dGMP, dCMP, and dAMP by the alpha subunit and exonucleolytic editing of those mispairs by the epsilon subunit were investigated. When the polymerization reaction was performed with the alpha subunit, dCMP and dGMP but not dAMP were misincorporated. This would suggest that the polymerase might have a base-selecting function to avoid dA:dA mispairing. A subassembly of the DNA polymerase III consisting of alpha, epsilon, and theta subunits misincorporated only dGMP. This would imply that the proofreading function of the epsilon subunit may correct the dC:dA but not the dG:dA mispair. Addition of a protein encoded by the mutT gene, defects of which cause AT to CG transversions in vivo, diminished the misincorporation of dGMP onto poly(dA) template by the alpha subunit. A dGTPase activity was associated with the MutT protein. The significance of the dGTPase activity in the prevention of dG:dA mispairing is discussed.
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PMID:Molecular mechanisms of replicational fidelity in Escherichia coli. 215 94

[3H]dGMP-3'-labelled, activated salmon testis DNA and [32P]dGMP-5'-labelled open circular M13 DNA were reacted with cis-diamminedichloroplatinum(II), cis-diamminechloroaquaplatinum(II), cis-diamminediaquaplatinum(II) or trans-diamminechloroaquaplatinum(II). The reaction was arrested after arbitrary times by adjustment to slightly alkaline solution conditions. The platinum-containing DNA was digested with Escherichia coli DNA polymerase I. The progress of nucleotide release was measured by acid precipitation of undigested DNA. Solubilized nucleotides and adducts were analyzed by HPLC. The 3'-5'-exonuclease activity liberated single-coordinated dGMP-platinum(II) adducts from both cis- and trans-platinum(II) treated salmon testis DNA and a small fraction of adducts of cis-platinum(II) that coordinated two molecules of dGMP. The bisadduct was derived from non-neighboring guanine residues probably located at or close to 3'-termini. This nuclease activity neither cut between nor after neighboring guanine residues crosslinked by cis-platinum(II). No bisadduct was liberated for trans-platinum(II). The 5'-3'-exonuclease activity did not liberate any nucleotide adducts from cis-platinum(II)-treated DNa. However, it removed single-coordinated guanine adducts of trans-diamminedichloroplatinum(II). From the kinetics of the appearance of dGMP monoadducts and the inhibition of digestion, a reaction scheme is formulated for the reaction of platinum(II) complexes with DNA that confirms and extends the previously published one [W. Schaller, H. Reisner & E. Holler (1987) Biochemistry 26, 943-950]. The longevity of the dGMP monoadduct intermediate is discussed in the context of the efficiency of cis-diamminedichloroplatinum(II) as an antitumor drug.
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PMID:Escherichia coli DNA polymerase I: inherent exonuclease activities differentiate between monofunctional and bifunctional adducts of DNA and cis- or trans-diamminedichloroplatinum(II). An exonuclease investigation of the kinetics of the adduct formation. 216 53

A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.
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PMID:Ligation-independent cloning of PCR products (LIC-PCR). 223 90

A group of proteins that act as primers for initiation of linear DNA replication are called DNA-terminal proteins (terminal proteins). We have found a short stretch of conserved amino acid sequence among the terminal proteins from six different sources. The location of this sequence motif is also similar among the different terminal-proteins. To determine the functional role of this terminal-protein domain in DNA replication, we have studied the bacteriophage PRD1 system. The PRD1 terminal protein and DNA polymerase genes were cloned into expression vectors, and the recombinant plasmids were used for constructing PRD1 terminal protein mutants. Site-directed mutagenesis and functional analysis showed that one of the two arginines (Arg-174) in the conserved sequence is critical for the initiation complex-forming activity of the PRD1 terminal protein. Replacement of Arg-174 by noncharged amino acids resulted in nonfunctional terminal protein. Phenylglyoxal, an alpha-dicarbonyl compound that reacts with the guanidino group of arginine, inhibits initiation complex formation between PRD1 terminal protein and dGMP. On the basis of these results, we propose that Arg-174 represents, at least in part, the binding site for phosphate groups of dGTP.
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PMID:An essential arginine residue for initiation of protein-primed DNA replication. 223 78

Reverse transcriptase from the human immunodeficiency virus type I (HIV-1) was expressed in E. coli and purified to near homogeneity. The enzyme was shown to contain reverse transcriptase, DNA polymerase and ribonuclease H activities. The DNA polymerase activity converted singly-primed phi X174 (+) DNA into the double-stranded form. Two third of the replication product is ligatable to covalently closed circular DNA (RFIV-form DNA) indicating that DNA synthesis by HIV reverse transcriptase can proceed until the enzyme matches the 5'-end of a pre-existing primer molecule. The in vitro accuracy of HIV reverse transcriptase was measured with the phi X174am16 reversion assay to be 1/7,400. Reversion rates for the individual mispairs were determined from pool bias studies to be 1/8,000 for the dGMP:T template mismatch, 1/35,000 for the dGMP:A template mismatch, 1/45,000 for the dAMP:G template mismatch, 1/73,000 for the dCMP:T template mispair, 1/140,000 for the dCMP:A template mispair, and 1/180,000 for the dGMP:G template mismatch. The dTMP:T template mispair was below the detection limit of the assay indicating a reversion rate of less than 1/300,000 for this particular mispair.
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PMID:Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural DNA. 246 38

DNA polymerase-primase complex, isolated with an apparently undegraded alpha-subunit, was immunoaffinity-purified to near homogeneity from the human lymphoblast line HSC93. The undegraded state of the alpha-subunit was monitored by Western-blot analysis of crude cellular extracts and all active fractions obtained during purification. The human polymerase-primase consists of four subunits with molecular weights of 195, 68, 55 and 48 kd. The fidelity of the polymerase-primase in copying bacteriophage phi X174am16 DNA in vitro was determined by measuring the frequency of production of different revertent phages. The overall accuracy was between 4 x 10(-6) and 10 x 10(-6). This value reflects the spontaneous mutation frequency of phi X174am16 phages in Escherichia coli, and is 10- to 20-fold higher than the accuracy of a conventionally purified enzyme from calf thymus. The frequencies of base pairing mismatches, estimated from pool bias measurements, were 3.5 x 10(-7) (1/2 880,000) for dGMP:Ttemplate mispairs, between 10(-7) and 10(-8) for dCMP:Ttemplate (1/35,000,000), dCMP:Atemplate (1/18,200,000) and dAMP:Gtemplate mispairs (1/16,500,000), and below 10(-8) (1/100,000,000) for dTMP:Ttemplate, dGMP:Atemplate and dGMP:Gtemplate mispairs. In contrast to previous preparations, the intact polymerase-primase possesses a 3'----5' exonuclease activity. This exonuclease removes both matched and mismatched 3'-OH ends, with a preference for mismatched bases. Fidelity was reduced 8-fold by increasing the concentration of the next nucleotide following the incorporated mismatch nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Exonucleolytic proofreading increases the accuracy of DNA synthesis by human lymphocyte DNA polymerase alpha-DNA primase. 252 47

Acyclovir triphosphate (ACVTP) was a substrate for herpes simplex virus type 1 (HSV-1) DNA polymerase and was rapidly incorporated into a synthetic template-primer designed to accept either dGTP or ACVTP followed by dCTP. HSV-1 DNA polymerase was not inactivated by ACVTP, nor was the template-primer with a 3'-terminal acyclovir monophosphate moiety a potent inhibitor. Potent inhibition of HSV-1 DNA polymerase was observed upon binding of the next deoxynucleoside 5'-triphosphate coded by the template subsequent to the incorporation of acyclovir monophosphate into the 3'-end of the primer. The Ki for the dissociation of dCTP (the "next nucleotide") from this dead-end complex was 76 nM. In contrast, the Km for dCTP as a substrate for incorporation into a template-primer containing dGMP in place of acyclovir monophosphate at the 3'-primer terminus was 2.6 microM. The structural requirements for effective binding of the next nucleotide revealed that the order of potency of inhibition of a series of analogs was: dCTP much greater than arabinosyl-CTP greater than 2'-3'-dideoxy-CTP much greater than CTP, dCMP, dCMP + PPi. In the presence of the next required deoxynucleotide (dCTP), high concentrations of dGTP compete with ACVTP for binding and thus retard the formation of the dead-end complex. This results in a first-order loss of enzyme activity indistinguishable from that expected for a mechanism-based inactivator. The reversibility of the dead-end complex was demonstrated by steady-state kinetic analysis, analytical gel filtration, and by rapid gel filtration through Sephadex G-25. Studies indicated that potent, reversible inhibition by ACVTP and the next required deoxynucleoside 5'-triphosphate also occurred when poly(dC)-oligo(dG) or activated calf thymus DNA were used as the template-primer.
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PMID:Herpes simplex virus type 1 DNA polymerase. Mechanism of inhibition by acyclovir triphosphate. 254 Jan 93

Occurrence of the transversion mutation A.T to C.G is specifically enhanced in Escherichia coli mutT mutants. With the aid of the cloned mutT gene, the MutT protein, which has a molecular mass of 15 kilodaltons, was overproduced and purified to near homogeneity. The protein catalyzes hydrolysis of dGTP to dGMP. dGDP and GTP were also hydrolyzed by the protein, but at a lower rate than seen with dGTP. No other deoxynucleoside triphosphates were hydrolyzed. Using poly(dA).(dT)20 as a template-primer, we investigated the misincorporation of dGMP, dCMP, and dAMP by the alpha subunit and the core of E. coli DNA polymerase III. When the polymerization reaction was performed with the alpha subunit, both dCMP and dGMP were misincorporated. The core, composed of alpha, epsilon, and theta subunits, misincorporated only dGMP. This would imply that the proofreading function of the epsilon subunit of DNA polymerase III may correct the dC.dA mispair but not the dG.dA mispair. Misincorporation of dAMP was not observed in reactions with the alpha subunit or core. The misincorporation of dGMP, but not dCMP, was almost completely suppressed by adding purified MutT protein to the reaction mixture. Under these conditions, only a portion of dGTP present in the reaction mixture was degraded. It is therefore likely that the MutT protein may prevent dGMP misincorporation by degrading a specific form of dGTP, probably the syn form, which can pair with deoxyadenosine.
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PMID:A specific role of MutT protein: to prevent dG.dA mispairing in DNA replication. 265 30


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