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

---

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

The bacteriophage lambda O and P protein replication initiators, in conjunction with six purified Escherichia coli replication proteins, replicate the single-stranded chromosomes of phages M13 and phi X174 to a duplex form. Several discrete steps are involved in this DNA synthesis reaction. In an ATP-dependent step that precedes priming, the lambda O and P proteins interact with the Escherichia coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto DNA coated with single-stranded DNA binding protein. This creates a stable prepriming intermediate, isolable by gel filtration, that is rapidly primed and replicated upon the addition of primase and DNA polymerase III holoenzyme. Each of the eight proteins required for this nonspecific single strand replication reaction also have physiological roles in the replication of the bacteriophage lambda chromosome in vivo. We propose a scheme for the lambda O and P protein-dependent initiation of DNA synthesis that may be relevant to strand initiation events occurring during lambda DNA replication.
...
PMID:Initiation of DNA synthesis on single-stranded DNA templates in vitro promoted by the bacteriophage lambda O and P replication proteins. 624 Sep 27

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.
...
PMID:Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein. 629 50

Extracts of the yeast Saccharomyces cerevisiae support DNA replication on exogenous yeast 2-microns plasmid DNA templates. A crude extract from a S. cerevisiae cell division cycle mutant, cdc8-1, expressed the temperature-sensitive phenotype since it could be inactivated at 42 degrees C in vitro. This heat-inactivated extract was fully complemented by the addition of either wild-type or cdc8-1 single-stranded DNA binding protein (SSB). restoration by SSB of the activity of the mutant cell extract allowed replication like that of a wild-type crude extract, as shown by bidirectional DNA synthesis from the in vivo origin. The DNA binding protein specifically stimulates the reaction catalyzed by yeast DNA polymerase I, a true DNA replicase, using the hybrid of phi X174 single-stranded DNA and a restriction endonuclease fragment as a template. It also increases processivity of DNA polymerase I at least 10-fold. Escherichia coli SSB, but not T4 gene 32 protein, can substitute for yeast SSB. Both restoration of DNA synthesis in the heated mutant cell extract and stimulation of the DNA polymerase I reaction by SSB from cdc8-1 cells are inactivated at nonpermissive temperatures, suggesting that yeast SSB is the CDC8 gene product.
...
PMID:Yeast 2-microns plasmid DNA replication in vitro: purification of the CDC8 gene product by complementation assay. 633 94

A fraction (P1) which showed DNA polymerase III holoenzyme activity was obtained by partial purification including Polymin P fractionation from extracts of E. coli K-12 wild type (pol A+, pol B+) cells. The P1 fraction was composed of three macromolecular complexes, 11 S, 18 S and 24 S, all of which possessed holoenzyme activity. The activity of the P1 fraction was maximal at about 70 mM NaCl. The synthesis of long-chain poly(dT) with a poly(dA) oligo(dT)10 primer was dependent on the presence of ATP, but not on the presence of spermidine, suggesting that the single-stranded DNA binding protein (SSB) was present in the fraction. The intermediate lengths of the products in the absence of ATP and NaCl also suggest the functioning of DNA polymerase III'.
...
PMID:DNA replication by novel macromolecular complexes involving DNA polymerase III holoenzyme activity. 634 Jun 88

Sites on an fd DNA template which terminate synthesis catalyzed by each of four forms of Escherichia coli DNA polymerase III have been identified at single nucleotide resolution. Results were obtained by comparing the products made by forms of DNA polymerase III with products generated from the same 3'-terminus by using the dideoxynucleotide sequencing method, on high-resolution polyacrylamide gel electrophoresis. Each form of DNA polymerase III generates products of distinct lengths ending at a limited number of preferred sites of synthesis termination. The addition of auxiliary subunits to the DNA polymerase III core form of the enzyme has a distinct functional effect on primer elongation and specificity of polymerase pausing. Most sites (65%) can be correlated to positions of potential secondary structure in the template arising via local hydrogen-bonding interactions. The proximity of polymerase pausing to sites adjacent to hairpin stems was related to the size of the enzyme since the smaller core form of DNA polymerase III generally paused at sites which were closer to the base of these structures than the larger holoenzyme. The occurrence of termination sites is markedly affected by the inclusion of spermidine or Escherichia coli single-stranded DNA binding protein in the reaction mixtures. Additionally, a nucleotide composition specificity of pause sites has been observed.
...
PMID:Site-specific pausing of deoxyribonucleic acid synthesis catalyzed by four forms of Escherichia coli DNA polymerase III. 636 Feb 4

The 140,000-Da adenovirus-encoded DNA polymerase (Ad Pol) is required for viral DNA replication both in vitro and in vivo. The polymerase co-purifies in a complex with the 80,000-Da precursor (pTP) of the terminal protein (TP) found covalently attached to the 5' ends of adenovirus DNA. To better understand their function in DNA replication, we have examined the properties of the Ad Pol and the pTP X Ad Pol complex on natural and synthetic DNA templates. The pTP X Ad Pol complex utilizes a variety of homopolymer template-primer combinations including poly(dC) X oligo(dG), poly(dA) X oligo(dT), poly(dT) X oligo(dA), and poly(dT) X oligo(rA). With poly(dT) as template and oligo(rA) or oligo(dA) as primer, DNA synthesis by the pTP X Ad Pol complex is stimulated as much as 100-fold by the 59,000-Da adenovirus DNA-binding protein (Ad DBP). ATP (4 mM) can further increase the rate of DNA synthesis 3- to 10-fold. The Ad DBP does not stimulate the activity of host (HeLa cell) DNA polymerase alpha with poly(dT) X oligo(dA) (or oligo(rA)) as the template-primer, and Escherichia coli single-stranded DNA binding protein cannot substitute for the Ad DBP in the stimulation of the Ad Pol activity. Under optimal conditions, poly(dA) chains 30,000 nucleotides in length are formed indicating that the Ad Pol can be a highly processive enzyme. An exonuclease activity co-sediments with the pTP X Ad Pol complex during glycerol gradient centrifugation, and co-purifies with the 140,000-Da Ad Pol after dissociation of the pTP X Ad Pol complex with urea. The Ad Pol-associated nuclease hydrolyzes single-stranded DNA in a 3'----5' direction and is at least 10-fold more active on single-stranded DNA than on duplex DNA. The Ad Pol has no detectable endonuclease activity on single-stranded DNA or duplex circular DNA. Analysis of the products of the nuclease activity showed that 5'-deoxynucleoside monophosphates were released during the hydrolysis of single-stranded DNA. The Ad DBP inhibits the hydrolysis of DNA by the polymerase-associated nuclease activity.
...
PMID:Properties of the adenovirus DNA polymerase. 654 Feb 63

recA protein, in the presence of single-stranded DNA binding protein and ATP, promotes the complete exchange of strands between circular single-stranded DNA containing pyrimidine dimers and a homologous linear duplex, converting the pyrimidine dimer-containing single-stranded DNA to a circular duplex. Bypass of a pyrimidine dimer during the branch-migration phase of the reaction requires approximately 20 seconds, a rate 1/50th of that in the absence of the dimer. The circular duplex product is specifically incised by the pyrimidine dimer-specific T4 endonuclease V, and the resulting 3' hydroxyl termini can serve as primers for deoxynucleotide polymerization by DNA polymerase I. These findings indicate that recA protein serves a direct role in recombinational repair and demonstrate that the pyrimidine dimers that have been bypassed can be processed by enzymes of the excision-repair pathway.
...
PMID:Recombinational bypass of pyrimidine dimers promoted by the recA protein of Escherichia coli. 695 68

DNA polymerase epsilon (pol epsilon) from HeLa cells was purified to near homogeneity, utilizing Mono S fast protein liquid chromatography for complete separation from pol alpha. The purified pol epsilon preparation showed two polypeptides of > 200 and 55 kDa and a small amount of active 122-kDa proteolysis product on denaturing polyacrylamide gels. Pol epsilon (as well as pols alpha and delta) is optimally active in 100-150 mM potassium glutamate and 15 mM MgCl2. Replication factors RF-A and RF-C, proliferating cell nuclear antigen, and Escherichia coli single-stranded DNA binding protein showed no significant effect on this preparation's pol epsilon activity, processivity, or substrate specificity. The size of the pol epsilon transcript for the catalytic subunit (> 200 kDa) was investigated in both normal human fibroblasts and HeLa cells. A 7.7-kilobase transcript was detected which was 5-16-fold more prevalent in proliferating than in quiescent HeLa cells. No significant difference in the level of pol epsilon transcript in HeLa cells or fibroblasts was seen after ultraviolet irradiation. Mouse polyclonal antiserum was produced to a 144-amino acid fragment of pol epsilon fused to staphylococcal protein A. This non-neutralizing polyclonal antiserum specifically recognized the catalytic subunit of pol epsilon by immunoblotting, but not that of pol alpha, beta, or delta. In addition, mouse polyclonal antiserum raised against column-purified pol epsilon was able to recognize and to neutralize pol epsilon, and a mouse monoclonal antibody was raised which was able to recognize specifically the catalytic subunit of pol epsilon.
...
PMID:Further characterization of HeLa DNA polymerase epsilon. 753 91

Many prokaryotic and viral DNA helicases involved in DNA replication stimulate their cognate DNA primase activity. To assess the stimulation of DNA primase activity by mammalian DNA helicases, we analyzed the synthesis of oligoribonucleotides by mouse DNA polymerase alpha-primase complex on single-stranded circular M13 DNA in the presence of mouse DNA helicase B. DNA helicase B was purified by sequential chromatography through eight columns. When the purified DNA helicase B was applied to a Mono Q column, the stimulatory activity for DNA primase-catalyzed oligoribonucleotide synthesis and DNA helicase and DNA-dependent ATPase activities of DNA helicase B were co-eluted from the column. The synthesis of oligoribonucleotides 5-10 nt in length was markedly stimulated by DNA helicase B. The synthesis of longer species of oligoribonucleotides, which were synthesized at a low level in the absence of DNA helicase B, was inhibited by DNA helicase B. The stimulatory effect of DNA helicase B was marked at low template concentrations and little or no effect was observed at high concentrations. The mouse single-stranded DNA binding protein, replication protein A (RP-A), inhibited the primase activity of the DNA polymerase alpha-primase complex and DNA helicase B partially reversed the inhibition caused by RP-A.
...
PMID:Stimulation of mouse DNA primase-catalyzed oligoribonucleotide synthesis by mouse DNA helicase B. 759 31

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.
...
PMID:Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells. 762 1


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---