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)

Initiation of bidirectional replication from the origin of the Escherichia coli chromosome (oriC) proceeds through stages in which the components of the two replication forks are assembled. From a complex containing proteins dnaA, dnaB, and dnaC bound at oriC, the dnaB helicase moves in both directions to unwind the duplex. In the absence of replication, this unwinding generates a bubble at oriC coated by single strand binding protein. Addition of gyrase allows unwinding to proceed extensively in both directions from oriC at 60 base pairs/s/fork at 37 degrees C. This rate is sharply dependent on temperature and also stimulated by both primase and DNA polymerase III holoenzyme, even in the absence of DNA synthesis. Primer and DNA synthesis are efficient when coupled to template unwinding. DNA synthesis proceeds bidirectionally from oriC at a rate limited by unwinding. With extensive unwinding preceding DNA synthesis, initiations are not limited to oriC.
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PMID:Helicase action of dnaB protein during replication from the Escherichia coli chromosomal origin in vitro. 303 79

The bacteriophage T4 61/41 protein primase-helicase is part of a seven T4 protein system needed for DNA synthesis in vitro. Although both 41 and 61 proteins are required for the synthesis and utilization of the normal pppApC(pN)3 pentanucleotide primer, we show in the accompanying paper (Hinton, D. M., and Nossal, N. G. (1987) J. Biol. Chem. 262, 10873-10878) that high concentrations of 61 protein alone carry out a limited, template-dependent oligonucleotide synthesis with the dimers pppApC and pppGpC as the major products labeled with [alpha-32P]CTP. At these high concentrations, 61 protein alone primes DNA synthesis by T4 DNA polymerase and the T4 genes 44/62 and 45 polymerase accessory proteins, or by Escherichia coli DNA polymerase I. The addition of T4 replication proteins other than 41 protein does not change the size distribution of oligonucleotides made by 61 protein. However, the primers used for DNA synthesis in the absence of 41 protein are not dimers, but rather trace quantities of longer oligonucleotides (5 to about 45 bases) which begin predominantly with pppGpC. These results show that 41 protein is required to prime with oligonucleotides beginning with pppApC and suggest that 41 protein, either alone or in conjunction with 61 protein, helps to stabilize the usual short pentamer primers on the template until they are elongated by the DNA polymerase. Moreover, since 61 protein by itself can only initiate DNA synthesis with primers beginning with pppGpC, but cannot make oligonucleotides starting with pppGpC on T4 DNA in which all the C is glucosylated and hydroxymethylated, both the T4 41 and 61 proteins are essential to prime DNA synthesis on their normal template. In our analysis of RNA-primed DNA, we demonstrate that although RNA primers at the 5' ends of DNA chains are relatively resistant to the 3' to 5' exonuclease of T4 DNA polymerase (Kurosawa, Y., and Okazaki, T. (1979) J. Mol. Biol. 135, 841-861), pppNpNpNpNpN oligomers are digested to a greater extent than the dephosphorylated pentamers NpNpNpNpN.
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PMID:Bacteriophage T4 DNA primase-helicase. Characterization of the DNA synthesis primed by T4 61 protein in the absence of T4 41 protein. 303 1

Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by an A + T-rich region. When the primary origin region is deleted replication initiates at secondary origins. We have analyzed the ability of plasmids containing cloned fragments of T7 to replicate after infection of Escherichia coli with bacteriophage T7. All cloned T7 fragments that support plasmid replication contain a T7 promoter but a T7 promoter alone is not sufficient for replication. Replication of plasmids containing the primary origin is dependent on T7 DNA polymerase and gene 4 protein (helicase/primase) and a portion of the A + T-rich region. The other T7 fragments that support plasmid replication after T7 infection are promoter regions phi OR, phi 13 and phi 6.5 (secondary origins). When both the primary and secondary origins are present simultaneously on compatible plasmids, replication of each is temporally regulated. Such regulation may play a role during T7 DNA replication.
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PMID:Initiation of DNA replication at cloned origins of bacteriophage T7. 306 20

Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.
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PMID:Repair of DNA-containing pyrimidine dimers. 329 78

More than ten proteins are known to participate in replication of plasmids bearing the unique origin of the Escherichia coli chromosome (oriC). Initiation of replication of oriC plasmids has been resolved into five separable stages. An initial complex formation (Stage I) requires an oriC plasmid, dnaA protein and HU protein. In the presence of ATP at a temperature of greater than 28 degrees C, a dnaB-C protein complex interacts to form a prepriming complex (Stage II). This is followed by extensive unwinding of the template that depends on the further addition of gyrase and single-strand binding protein (SSB) (Stage III). Hydrolysis of an rNTP by dnaB protein (a helicase action) and of ATP by gyrase (a swivelling action) drives the extreme unwinding of the template. This unwound template-protein complex is the substrate for priming by primase (Stage IV) and elongation by DNA polymerase III holoenzyme (Stage V). Priming of all DNA chains is done by primase; RNA polymerase functions in template activation rather than priming. DNA polymerase III holoenzyme, composed of at least seven subunits, synthesizes the DNA chains. The alpha subunit is the polymerase, the epsilon subunit is the 3'----5' exonuclease; alpha + epsilon is the proofreading activity. Following the synthesis of new DNA chains, DNA polymerase I and ribonuclease H remove the RNA primers, polymerase I fills the gaps, and ligase seals the daughter strands (Stage VI). Replication produces plasmids identical in structure and sequence to the initial template.
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PMID:Enzyme systems initiating replication at the origin of the Escherichia coli chromosome. 333 50

Three proteins catalyze RNA-primed DNA synthesis on the lagging strand side of the replication fork of bacteriophage T7. Oligoribonucleotides are synthesized by T7 gene 4 protein, which also provides helicase activity. DNA synthesis is catalyzed by gene 5 protein of the phage, and processivity of DNA synthesis is conferred by Escherichia coli thioredoxin, a protein that is tightly associated with gene 5 protein. T7 DNA polymerase and gene 4 protein associate to form a complex that can be isolated by filtration through a molecular sieve. The complex is stable in 50 mM NaCl but is dissociated by 100 mM NaCl, a salt concentration that does not inhibit RNA-primed DNA synthesis. T7 DNA polymerase forms a stable complex with single-stranded M13 DNA at 50 mM NaCl as measured by gel filtration, and this complex requires 200 mM NaCl for dissociation, a salt concentration that inhibits RNA-primed DNA synthesis. Gene 4 protein alone does not bind to single-stranded DNA. In the presence of MgCl2 and dTTP or beta, gamma-methylene dTTP, a gene 4 protein-M13 DNA complex that is stable at 200 mM NaCl is formed. The affinity of DNA polymerase for both gene 4 protein and single-stranded DNA leads to the formation of a gene 4 protein-DNA polymerase-M13 DNA complex even in the absence of nucleoside triphosphates. However, the binding of each protein to DNA plays an important role in mediating the interaction of the proteins with each other. High concentrations of single-stranded DNA inhibit RNA-primed DNA synthesis by diluting the amount of proteins bound to each template and reducing the frequency of protein-protein interactions. Preincubation of gene 4 protein, DNA polymerase, and M13 DNA in the presence of dTTP forms protein-DNA complexes that most efficiently catalyze RNA-primed DNA synthesis in the presence of excess single-stranded competitor DNA.
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PMID:Interactions of the DNA polymerase and gene 4 protein of bacteriophage T7. Protein-protein and protein-DNA interactions involved in RNA-primed DNA synthesis. 353 39

Rep protein as a helicase combines its actions with those of gene A protein and single-stranded DNA binding protein to separate the strands of phi X174 duplex DNA and thereby can generate and advance a replication fork (Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197). Tritium-labeled rep protein is bound in an active gene A protein. phi X174 closed circular duplex supercoiled DNA complex in a 1:1 ratio. Catalytic separation of the strands of the duplex by rep protein, as measured by incorporation of tritium-labeled single-stranded DNA binding protein, requires ATP at a Km value of 8 microM, and hydrolyzes two molecules of ATP for every base pair melted. When coupled to replication in the synthesis of single-strand viral circles, a "looped" rolling-circle intermediate is formed that can be isolated in an active form containing gene A protein, rep protein, single-stranded DNA binding protein, and DNA polymerase III holoenzyme. Unlike the binding of rep protein to single-stranded DNA, where its ATPase activity is distributive, binding to the replicating fork is not affected by ATP, further suggesting a processive action linked to gene A protein. Limited tryptic hydrolysis of rep protein abolishes its replicative activity without affecting significantly its binding of ATP and its ATPase action on single-stranded DNA. These results augment earlier findings by describing the larger role of rep proteins as a helicase, linked in a complex ith other proteins, at the replication fork of a duplex DNA.
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PMID:Rep protein as a helicase in an active, isolatable replication fork of duplex phi X174 DNA. 611 28

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

In a mixture of Escherichia coli DNA polymerase III holoenzyme, single-strand-binding protein, artificially forked lambda bacteriophage DNA with primer annealed to the leading side of the fork, dNTPs and ATP, DNA synthesis is enhanced by helicase II, less so by helicases, I, III or rep protein of E. coli or T4 phage helicase. The effect of helicase II depends on ATP, it is enhanced by helicase III, and it is not observed using DNA polymerase I or T4 DNA polymerase. In the absence of dNTPs helicase II is less active than helicase I or T4 helicase in unwinding the forked DNA. We believe that helicase II both shifts the forks and stimulates DNA polymerase III. The results support the conclusion derived from previous studies that helicase II is part of the DNA-synthesizing system of E. coli.
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PMID:DNA synthesis at a fork in the presence of DNA helicases. 612 89

The gene 4 protein of bacteriophage T7 is both a primase and a helicase. In this paper, we present a detailed description of a third activity, single-stranded DNA-dependent nucleoside 5'-triphosphate hydrolysis, and show that this activity is coupled to the unidirectional translocation of the gene 4 protein on single-stranded DNA (Tabor, S., and Richardson, C.C. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 205-209). The competitive inhibitor of NTP hydrolysis, beta, gamma-methylene dTTP, is also a potent inhibitor of gene 4 protein-dependent, RNA-primed DNA synthesis; inhibition is not due to a direct inhibition of T7 DNA polymerase or RNA primer synthesis. We conclude that the energy derived from the hydrolysis of NTPs by the gene 4 protein is required for translocation of the protein to primase recognition sites. Measurement of the rates of hydrolysis of NTPs using a variety of DNAs of known structure and length support the unidirectional translocation of the gene 4 protein on single-stranded DNA. Duplex DNA, RNA, and single-stranded DNA coated with single-stranded DNA-binding protein do not serve as effectors for the nucleoside triphosphatase of the gene 4 protein. Kinetic data suggest that the gene 4 protein does not remain bound to newly synthesized oligoribonucleotide primers but continues to search for other primase recognition sites. Although all the predominant naturally occurring NTPs except rCTP are hydrolyzed by the gene 4 protein, the enzyme shows specificity for dTTP with a Km of 0.4 mM. In the accompanying paper (Matson, S.W., Tabor, S., and Richardson, C.C. (1983) J. Biol. Chem. 258, 14017-14024), we show that the hydrolysis of NTPs is also required for the protein to function as a helicase in duplex regions of DNA.
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PMID:DNA-dependent nucleoside 5'-triphosphatase activity of the gene 4 protein of bacteriophage T7. 613 75


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