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)

We have studied spontaneous and UV mutagenesis of the glyU gene in Escherichia coli trpA461 (GAG) strains carrying the pIP11 plasmid, in which the dnaQ gene encoding the 3'-5' exonuclease subunit (epsilon) of DNA polymerase III is fused to the tac(trp-lac) promoter. We have used a pair of M13glyU phage in which the gene encoding the glycyl-tRNA is cloned in opposite orientations, consequently the phage present either GGG or CCC anticodon triplets for mutagenesis. The presence of IPTG, the inducer of the tac-dnaQ fusion, results in about 100-fold decrease in frequency of spontaneous Su+ (GAG) mutations arising in the CCC phage. The enhanced expression of tac-dnaQ reduces 10-fold the frequency of UV-induced Su+ (GAG) mutations in the CCC phage and nearly completely prevents generation by UV of Su+ (GAG) mutations in the GGG phage, in which UV-induced pyrimidine photo-products can be formed only in the vicinity of the target triplet. These results suggest that both locally and regionally targeted mutagenesis is affected by overproduction of the epsilon subunit. By delayed photoreversal mutagenesis we have shown that UV-induced chromosomal mutagenesis of the umuC36 trpA461 strain harboring pIP11 is completely abolished in the presence of IPTG. This result seems to indicate that the misinocorporation step of DNA translesion synthesis is affected by excess of the epsilon subunit. Finally, we have introduced the pIP13 plasmid carrying the dnaQ gene into the recA1207 strain, which is deficient in the recombinase activity of RecA but constitutive in the protease activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of enhanced synthesis of the epsilon subunit of DNA polymerase III on spontaneous and UV-induced mutagenesis of the Escherichia coli glyU gene. 219 32

DNA polymerase III holoenzyme (holoenzyme) is the 10-subunit replicase of the Escherichia coli chromosome. In this report, pure preparations of delta, delta', and a gamma chi psi complex are resolved from the five protein gamma complex subassembly. Using these subunits and other holoenzyme subunits isolated from overproducing plasmid strains of E. coli, the rapid and highly processive holoenzyme has been reconstituted from only five pure single subunits: alpha, epsilon, gamma, delta, and beta. The preceding report showed that of the three subunits in the core polymerase, only a complex of alpha (DNA polymerase) and epsilon (3'-5' exonuclease) are required to assemble a processive holoenzyme on a template containing a preinitiation complex (Studwell, P.S., and O'Donnell, M. (1990) J. Biol. Chem. 265, 1171-1178). This report shows that of the five proteins in the gamma complex only a heterodimer of gamma and delta is required with the beta subunit to form the ATP-activated preinitiation complex with a primed template. Surprisingly, the delta' subunit does not form an active complex with gamma but forms a fully active heterodimer complex with the tau subunit (as does delta). Hence, the tau delta' and gamma delta heterodimers are fully active in the preinitiation complex reaction with beta and primed DNA. Holoenzymes reconstituted using the alpha epsilon complex, beta subunit, and either gamma delta or tau delta' are fully processive in DNA synthesis, and upon completing the template they rapidly cycle to a new primed template endowed with a preinitiation complex clamp. Since the holoenzyme molecule contains all of these accessory subunits (gamma, delta, tau, delta', and beta) in all likelihood it has the capacity to form two preinitiation complex clamps simultaneously at two primer termini. Two primer binding components within one holoenzyme may mediate its rapid cycling to multiple primers on the lagging strand and also provides functional evidence for the hypothesis of holoenzyme as a dimeric polymerase capable of simultaneous replication of both leading and lagging strands of a replication fork.
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PMID:Total reconstitution of DNA polymerase III holoenzyme reveals dual accessory protein clamps. 240 6

We have purified yeast DNA polymerase II to near homogeneity as a 145-kDa polypeptide. During the course of this purification we have detected and purified a novel form of DNA polymerase II that we designate as DNA polymerase II. The most highly purified preparations of DNA polymerase II are composed of polypeptides with molecular masses of 200, 80, 34, 30, and 29 kDa. Immunological analysis and peptide mapping of DNA polymerase II and the 200-kDa subunit of DNA polymerase II indicate that the 145-kDa DNA polymerase II polypeptide is derived from the 200-kDa polypeptide of DNA polymerase II. Activity gel analysis shows that the 145- and the 200-kDa polypeptides have catalytic function. The polypeptides present in the DNA polymerase II preparation copurify with the polymerase activity with a constant relative stoichiometry during chromatography over five columns and co-sediment with the activity during glycerol gradient centrifugation, suggesting that this complex may be a holoenzyme form of DNA polymerase II. Both forms of DNA polymerase II possess a 3'-5' exonuclease activity that remains tightly associated with the polymerase activity during purification. DNA polymerase II is similar to the proliferating cell nuclear antigen (PCNA)-independent form of mammalian DNA polymerase delta in its resistance to butylpheny-dGTP, template specificity, stimulation of polymerase and exonuclease activity by KCl, and high processivity. Although calf thymus PCNA does not stimulate the activity of DNA polymerase II on poly(dA):oligo(dT), possibly due to the limited length of the template, the high processivity of yeast DNA polymerase II on this template can be further increased by the addition of PCNA, suggesting that conditions may exist for interactions between PCNA and yeast DNA polymerase II.
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PMID:Purification and characterization of DNA polymerase II from the yeast Saccharomyces cerevisiae. Identification of the catalytic core and a possible holoenzyme form of the enzyme. 240 68

The oligonucleotide [5'-32P]pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site [d(-)], yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination. The incubation of [5'-32P]pdT8d(-)dTn, hybridized with poly(dA), with E. coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination. The oligonucleotide pdT8d(-) is degraded by the 3'-5' exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3' end is not an obstacle for this activity. The radioactive products from [5'-32P]pdT8d(-)dTn cleaved by alkaline beta-elimination or by E. coli endonuclease III are not degraded by the 3'-5' exonuclease activity of T4 DNA polymerase. When DNA containing AP sites labelled with 32P 5' to the base-free deoxyribose labelled with 3H in the 1' and 2' positions is degraded by E. coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA. When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E. coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate. Treatment of the DNA containing doubly-labelled AP sites with E. coli endonuclease III, then with E. coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E. coli endonuclease VI (exonuclease III). From these data, we conclude that E. coli endonuclease III cleaves the phosphodiester bond 3' to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3' end as it has been so far assumed. The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys. Thus it would seem that E. coli 'endonuclease III' is, after all, not an endonuclease.
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PMID:Escherichia coli endonuclease III is not an endonuclease but a beta-elimination catalyst. 243 70

Cytosine arabinoside (araC) is a potent antileukemic agent that is misincorporated into DNA in the course of its action. We have developed a chemical synthetic method that allows site-specific introduction of araC into synthetic DNA oligomers. We describe here the utilization of these oligomers as primer/template substrates for in vitro DNA synthesis reactions and as fragments for DNA ligation. These studies were undertaken to investigate the manner in which sites of araC misincorporation constitute sites of DNA dysfunction. AraCMP at the primer terminus dramatically reduced the rate of next nucleotide addition for Escherichia coli polymerase I (Klenow fragment) (Pol I), T4 polymerase, HeLa cell polymerase alpha 2 (Pol alpha 2), and AMV reverse transcriptase. Polymerases with associated 3'-5' exonuclease activity preferentially excised araCMP from the primer terminus prior to chain elongation. AraCMP-terminated fragments were ligated more slowly than control fragments by T4 DNA ligase. AraCMP located at an internucleotide site in the template markedly slowed replicative bypass for Pol I, T4 polymerase, and Pol alpha 2, but not for reverse transcriptase. Synthesis was partially arrested after insertion of the correct nucleotide opposite the lesion site. These results suggest a complex mechanism for the inhibition of DNA replication by araC when it is misincorporated into DNA.
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PMID:Functional consequences of the arabinosylcytosine structural lesion in DNA. 245 56

DNA primase-DNA polymerase alpha, purified 53,000-fold from CV-1 cells, synthesized predominantly (p)ppA(pA)6-primed DNA on a poly(dT) template. About 80% of the RNA primers synthesized on an M13 DNA template were (p)ppA/G(pN)5-7, and 20% were (p)ppA/G(pN)0-4. RNA primer size was determined by gel electrophoresis after removing nascent DNA with phage T4 DNA polymerase 3'-5' exonuclease, leaving a single dNMP at the 3'-end of the RNA primer, and the terminal 5'-(p)ppN residue was determined by "capping" with [alpha-32P]GTP using vaccinia guanylyl-transferase. The processivity of DNA synthesis initiated by de novo synthesis of RNA primers was the same as that initiated on pre-existing RNA primers (10-15 dNMPs), although initiation on pre-existing primers was strongly preferred. Primers always began with A or G, even at high levels of CTP or UTP, although the ratio of A to G varied from 4:1 to 1:1 depending on the relative concentrations of ATP and GTP in the assay. ATP and GTP had no effect on primer length, but the fraction of shorter RNA primers increased 2-fold with higher concentrations of CTP or UTP. Nearest-neighbor analysis revealed a preference for purine ribonucleotides at RNA covalently linked to the 5'-end of DNA (RNA-p-DNA) junctions, and increasing the concentration of a single rNTP increased slightly its presence at RNA-p-DNA junctions. Thus, the base composition and size of RNA primers synthesized by DNA primase-DNA polymerase alpha is modulated by the relative concentrations of ribonucleoside triphosphates.
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PMID:DNA primase-DNA polymerase alpha from simian cells. Modulation of RNA primer synthesis by ribonucleoside triphosphates. 258 49

Saccharomyces cerevisiae cdc2 mutants arrest in the S-phase of the cell cycle when grown at the non-permissive temperature, implicating this gene product as essential for DNA synthesis. The CDC2 gene has been cloned from a yeast genomic library in vector YEp13 by complementation of a cdc2 mutation. An open reading frame coding for a 1093 amino acid long protein with a calculated mol. wt of 124,518 was determined from the sequence. This putative protein shows significant homology with a class of eukaryotic DNA polymerases exemplified by human DNA polymerase alpha and herpes simplex virus DNA polymerase. Fractionation of extracts from cdc2 strains showed that these mutants lacked both the polymerase and proofreading 3'-5' exonuclease activity of DNA polymerase III, the yeast analog of mammalian DNA polymerase delta. These studies indicate that DNA polymerase III is an essential component of the DNA replication machinery.
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PMID:Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III. 267 May 63

The DNA polymerase activity of different members of Mollicutes was studied. A single DNA polymerase was found in Mycoplasma mycoides and Ureaplasma urealyticum, type species of the genera Mycoplasma and Ureaplasma, and was compared with the previously described Mycoplasma orale enzyme. Most of their properties were comparable; an immunological relationship was demonstrated between M. orale and M. mycoides enzymes by immunoblotting. In contrast to these results, three different DNA polymerases were purified in Acholeplasma laidlawii, type species of the genus Acholeplasma which, in this aspect, resembles the genus Spiroplasma. A 3'-5' exonuclease activity was found in the different purified preparations. In M. mycoides, M. orale and one of the three A. laidlawii preparations, the 3'-5' exonuclease could be separated from the DNA polymerase by non-denaturing PAGE. The presence of a single DNA polymerase seems to be a typical feature of the Mycoplasmataceae, which include the genera Mycoplasma and Ureaplasma, in contrast to the occurrence of three enzymes within the Acholeplasmataceae and Spiroplasmataceae. These results are in agreement with the phylogenetic tree of Mollicutes proposed from their 5 S and 16 S rRNA sequence comparisons, in which the evolution of Acholeplasma and Spiroplasma branches led, by genome reductions, to Mycoplasma and Ureaplasma species.
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PMID:Mollicutes DNA polymerases: characterization of a single enzyme from Mycoplasma mycoides and Ureaplasma urealyticum and of three enzymes from Acholeplasma laidlawii. 269 45

cis-Thymine glycol, a product of ionizing radiation damage to DNA, has been introduced quantitatively at a single site into oligonucleotide templates. The ability of DNA polymerases to replicate templates containing thymine glycol was studied by a primer extension assay, and three factors that influence replicative bypass of this lesion in vitro have been identified. These factors include template length, sequence context, and 3'-5' exonuclease activity. Synthesis by the large fragment of DNA polymerase I (Klenow fragment) terminates quantitatively opposite thymine glycol when the template strand extends only two nucleotides beyond the lesion. Significant bypass is observed when the length of the template beyond the lesion is increased to six nucleotides. On the longer templates, the frequency of bypass of the Klenow fragment depends upon the identity of the base immediately 5' to thymine glycol. The extent of bypass is greatest with cytosine and least with adenine at this position. Bypass of thymine glycol lesions by DNA polymerase alpha 2 from HeLa cells shows a qualitatively similar dependence upon local sequence context. In contrast, synthesis by T4 DNA polymerase is quantitatively blocked opposite the lesion regardless of template length or DNA sequence context. Synthesis by a mutant Klenow fragment that is deficient in 3'-5' exonuclease activity, or by AMV reverse transcriptase, results in a significant increase in the frequency of bypass. Thus, increased nucleotide turnover at, or beyond, the site of the lesion is likely to contribute significantly to the arrest of synthesis provoked by cis-thymine glycol in vitro.
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PMID:Template length, sequence context, and 3'-5' exonuclease activity modulate replicative bypass of thymine glycol lesions in vitro. 271 44

As a step toward the molecular elucidation of the putative replicational apparatus associated with the nuclear matrix, we have investigated the possible matrix association of several replicational related enzymes. In addition to the previously identified DNA polymerase alpha, DNA primase, 3'-5' exonuclease, RNase H, and DNA methylase were all recovered at significant levels (20-30% of total nuclear activity) in nuclear matrix isolated from regenerating rat liver during maximal in vivo replication (22 h post-hepatectomy). In contrast, DNA ligase was not detected on the nuclear matrix even though significant activity was present in isolated nuclei. Examination of the replicative dependency of these enzyme activities following partial hepatectomy revealed pre-replicative elevations which were distinct for each matrix-bound enzyme. A second late-replicative peak in DNA methylase is consistent with a role of this matrix-bound enzyme in the maintenance of the inheritable methylation pattern. Mild sonication resulted in a significant release of all of these activities except RNase H. A major portion of the matrix-solubilized DNA polymerase alpha, DNA primase, 3'-5' exonuclease, and DNA methylase activities cosedimented on sucrose gradients between approximately 8-12 S. Our results are consistent with the organization of at least a portion of these replicative enzymes into nuclear matrix-bound replicational complexes. We also propose a novel pre-replicative assembly model of the matrix-bound replicational apparatus in which DNA primase plays an initial and critical role.
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PMID:Pre-replicative association of multiple replicative enzyme activities with the nuclear matrix during rat liver regeneration. 302 82


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