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)

The E. coli proteins that catalyze the conversion of varphiX174 single-stranded DNA to duplex DNA have now been purified extensively. The reaction depends on dnaB, dnaC(D), dnaE, and dnaG gene products, DNA elongation factors I and II, E. coli DNA binding protein, and two additional E. coli proteins, replication factors X and Y. DNA synthesis by these proteins requires varphiX174 viral DNA, dNTPs, Mg(+2), and ATP. The product synthesized is full-length linear varphiX174 DNA. The reaction has been resolved into two steps. The first step involves the interaction of ATP and varphiX174 DNA with dnaB and dnaC(D) gene products, E. coli DNA binding protein, and replication factors X and Y in the absence of dNTPs. Subsequent dNMP incorporation requires the addition of DNA polymerase III, DNA elongation factors I and II, dnaG gene product, and dNTPs.
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PMID:Conversion of phiX174 viral DNA to double-stranded form by purified Escherichia coli proteins. 461 May 69

Crystals suitable for X-ray crystallographic investigation have been grown of several nucleic acid binding proteins and their analysis is in progress. These include E. coli catabolite gene activator protein (CAP), the large fragment of DNA polymerase I (Pol I fragment), rec A, single strand DNA binding protein, resolvase, lac repressor and lac repressor 'Core', 5S RNA fragment and its complex with L25. Calculation of the electrostatic charge potential of CAP, using coordinates refined at 2.6 A resolution, suggests an orientation for B DNA on this repressor and activator of transcription. Both the electrostatic calculations and detailed model building suggests that the DNA must be bent or kinked on the protein in this orientation in order to make sufficient protein contacts. From a 3.5 A resolution map of Pol I fragment we have been able to obtain a preliminary trace through the polypeptide backbone. The large fragment consists of two domains. The smaller domain binds nucleoside monophosphate at the edge of a mostly parallel beta-pleated sheet, a structure that is reminiscent of kinase and dehydrogenase nucleotide binding domains. The larger domain contains about two thirds of the fragment and is mostly alpha-helical but with at least one four stranded antiparallel beta-sheet. The nucleoside monophosphate binds with its 5' phosphate on the Mg and is apparently in the conformation of nucleotides in B DNA.
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PMID:Crystallographic studies of protein-nucleic acid interaction: catabolite gene activator protein and the large fragment of DNA polymerase I. 610 Oct 86

The gene A protein of bacteriophage phi X 174 initiates replication of super-twisted RFI DNA by cleaving the viral (+) strand at the origin of replication and binding to the 5' end. Upon addition of E. coli rep protein (single-stranded DNA dependent ATPase), E. coli single-stranded DNA binding protein and ATP, complete unwinding of the two strands occurs. Electron microscopic analyses of intermediates in the reaction reveal that the unwinding occurs by movement of the 5' end into the duplex, displacing the viral strand in the form of a single-stranded loop. Since unwinding will not occur in the absence of either gene A protein or rep protein, it is presumed that the rep protein interacts to form a complex with the bound gene A protein. Single-stranded DNA binding protein facilitates the unwinding by binding to the exposed single-stranded DNA. Further addition of the four deoxyribotriphosphates and DNA polymerase III holoenzyme to the reaction results in synthesis of viral (+) single-stranded circles in amounts exceeding that of the input template. A model describing the role of gene A protein and rep protein in duplex DNA replication is presented and other properties of gene A protein discussed.
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PMID:The role of gene A protein and E. coli rep protein in the replication of phi X 174 replicative form DNA. 610 97

Using purified enzymes, double strand replication of phage fd DNA has been dissected into several intermediate steps. (i) Phage fd gene 2 protein cleaves supercoiled phage fd replicative form at a specific site in the viral strand (Meyer, T. F., Geider, K., Kurz, C., and Schaller, H. (1979) Nature 278, 365-367). (ii) Relaxed covalently closed circular replicative form DNA which is also formed by gene 2 protein as a side product in the initiation reaction preceding replication is converted into supercoils by DNA gyrase. (iii) The enzyme forms a noncovalent complex at the generated nick that is necessary for initiation of subsequent unwinding. (iv) The Escherichia coli rep helicase (rep protein) and E. coli DNA binding protein I unwind the double-stranded DNA. (v) Concomitant DNA replication by E. coli DNA polymerase III holoenzyme results in the formation of rolling circle intermediates. The double-stranded core of the rolling circle remains in an open form, thus allowing continued synthesis during several rounds of replication. (vi) Processing of replicated viral DNA can be subdivided into the cleavage and the circularization of viral single strands. Comparative studies of fd and phi X174 replication in vitro have revealed differences in the kinetics of individual steps besides an apparent contrast in the conformation of rolling circle intermediates in the electron microscopy where fd DNA features extended tails rather than looped-back structures observed for phi X174 DNA.
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PMID:Intermediate stages in enzymatic replication of bacteriophage fd duplex DNA. 612 86

The gene for Escherichia coli rep helicase (rep protein) was subcloned in a pBR plasmid and the protein overproduced in cells transformed with the hybrid DNA. The effect of purified enzyme on strand unwinding and DNA replication was investigated by electron microscopy. The templates used were partial duplexes of viral DNA from bacteriophage fd::Tn5 and reannealed DNA from bacteriophage Mu. The experiments with the two DNA species show DNA unwinding uncoupled from replication. The single-stranded phage fd::Tn5 DNA with the inverted repeat of transposon Tn5 could be completely replicated in the presence of the E. coli enzymes rep helicase, DNA binding protein I, RNA polymerase and DNA polymerase III holoenzyme. A block in the unwinding step increases secondary initiation events in single-stranded parts of the template, as DNA polymerase III holoenzyme cannot switch across the stem structure of the transposon.
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PMID:Functional aspects of Escherichia coli rep helicase in unwinding and replication of DNA. 614 40

A protein factor that participates in the formation of a covalent complex between the 80,000-dalton precursor of the adenovirus (Ad) terminal protein (pTP) and 5'-dCMP has been isolated and characterized. This 47,000-dalton protein, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor I. It is free of detectable DNA polymerase alpha, beta, and gamma activities. In the presence of Ad DNA-prot, the Ad-protein fraction (containing the pTP and the Ad-associated DNA polymerase), ATP, Mg2+, and dCTP, nuclear factor I stimulates formation of the pTP-dCMP complex. Addition of the Ad DNA binding protein (Ad DBP) renders the formation of the pTP-dCMP complex completely dependent on the addition of nuclear factor I. When Ad DNA-prot is replaced with phi X174 single-stranded circular DNA, pTP-dCMP complex formation requires only the Ad-protein fraction; Ad DBP and ATP are inhibitory and nuclear factor I has no effect on this reaction. This suggests that the initiation reaction observed with Ad DNA-prot in the absence of Ad DBP occurs at single-stranded DNA sites. In the presence of Ad DBP, these sites are blocked thus creating a requirement for nuclear factor I in pTP-dCMP complex formation.
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PMID:Adenovirus DNA replication in vitro: identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex. 621 80

Four T7 products, DNA polymerase, gene 4 protein, RNA polymerase, and DNA binding protein, have been purified from phage-infected cells. It has been previously shown (Hinkle, D. C., and Richardson, C. C. (1975) J. Biol. Chem. 250, 5523-5529; Kolodner, R., and Richardson, C. C. (1978) J. Biol. Chem. 253, 574-584) that two T7 products, DNA polymerase and gene 4 protein, catalyze extensive synthesis on duplex T7 DNA containing single strand breaks. However, the T7 DNA polymerase purified by our procedure does not efficiently contribute in this reaction, although the preliminary evidence suggests that this enzyme may be the native form of the DNA polymerase. Such inefficient T7 DNA synthesis is greatly augmented by adding the third T7 product, namely T7 RNA polymerase. This DNA synthesis apparently requires transcription, since each of the four rNTPs must be present. The rate of synthesis is increased about 2-fold by the addition of T7 DNA binding protein. In contrast to the results obtained when DNA synthesis is initiated at single strand breaks in a duplex DNA molecule, essentially none of the DNA synthesized in the presence of T7 RNA polymerase is covalently attached to the T7 DNA template. We postulate that in this in vitro system, T7 DNA replication is initiated using an RNA primer synthesized by the T7 RNA polymerase.
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PMID:Bacteriophage T7 DNA replication in vitro. Stimulation of DNA synthesis by T7 RNA polymerase. 624 22

Escherichia coli ribonuclease H was purified to near-homogeneity and identified as the only additional factor required for initiation of in vitro Co1E1 DNA replication from the unique origin by RNA polymerase and DNA polymerase I. Both ribonuclease H activity and stimulating activity for Co1E1 DNA synthesis comigrate with the single protein band in gel electrophoresis. These two activities coincide throughout the process of purification. Some DNA synthesis takes place on covalently closed-circular DNA molecules other than Co1E1 DNA with the three purified enzymes. This DNA synthesis is suppressed by an Escherichia coli single-strand DNA binding protein and/or a high concentration of ribonuclease H. Negative superhelicity of template DNA is required for efficient primer formation. No evidence that supports involvement of ribonuclease III in initiation of Co1E1 DNA replication or its regulation was found.
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PMID:Purification of ribonuclease H as a factor required for initiation of in vitro Co1E1 DNA replication. 629 61

Bacteriophage T7 codes for a single-stranded DNA binding protein. This protein is the product of gene 2.5 and has been found previously to stimulate specifically the activity of the phage-coded DNA polymerase. We report here that the T7 DNA binding protein also stimulates the activity of the phage-coded exonuclease. The gene 6 exonuclease is a double-stranded DNA specific 5'-exonuclease that has been implicated in destruction of bacterial DNA, removal of RNA primers during DNA replication, genetic recombination, and DNA maturation. The enzyme is markedly inhibited by physiological concentrations of NaCl. This inhibition, which is due to a marked reduction in the Vmax of the enzyme, can be largely overcome by the phage-coded DNA binding protein. This stimulation is specific since the Escherichia coli DNA binding protein is without effect. The stimulation by the binding protein is apparently not due to its coating of the 3' single-stranded tails generated during the digestion. Kinetic studies show that the stimulation is due to a combined effect on both the Km and Vmax of the exonuclease. These studies are consistent with a loose binding of the binding protein to either the DNA or the exonuclease.
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PMID:Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein. 629 50

Single-strand DNA binding protein (SSB) from Escherichia coli abolishes transfection of E.coli by viral M13mp2 DNA at levels that inhibit transfection by M13mp2 replicative form (RF) DNA by approx. 25%. Synthesis of M13mp2 RF DNA (SS leads to DS) has been carried out using DNA polymerase I (Klenow fragment) and a unique 15-nucleotide primer. A time course for in vitro synthesis showed that the increase in transfection in the presence of SSB paralleled DNA synthesis after an initial lag period for transfection. Digestion of replication products with restriction endonucleases and S1 endonuclease indicates that only those molecules that are fully or almost fully duplex transfect competent cells in the presence of SSB.
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PMID:Preferential transfection with M13mp2 RF DNA synthesized in vitro. 630 39


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