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)

Quasi-homogeneous fractions of male mouse germ cells at definite stages of meiosis and spermiogenesis were obtained by using a separation method based on sedimentation velocity in an albumin gradient. In the various cell types, the total DNA-dependent DNA polymerase activity was determined, and the major enzymatic forms were characterized. The DNA polymerase species present in premeiotic, meiotic and post-meiotic cells were analyzed by glycerol gradient sedimentation. Two types of DNA polymerase were identified in fractions enriched in spermatogonia and preleptotene spermatocytes. One showed a sedimentation coefficient of about 7.5 S and was sensitive to N-ethylmaleimide (NEM); the other exhibited a sedimentation coefficient between 3 and 4 S and was resistant to NEM. On the basis of their sedimentation coefficients, their sensitivity to NEM and their template specificities, these 2 enzymes were identified respectively as alpha and beta DNA polymerases as reported in mammals. The gradient analysis performed on fractions enriched in meiotic and post-meiotic cells revealed the presence of DNA polymerase beta only. A quantitative analysis showed that the activity of the DNA polymerase beta reaches a maximum at middle-late pachytene stage and then drops gradually during spermiogenesis. Although any conclusion as to the biological role of this high level of DNA polymerase activity in pachytene spermatocytes is premature, it is tempting to suggest that this enzyme is involved in meiotic recombination.
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PMID:DNA-dependent DNA polymerase species in male germ cells of the mouse. 69 53

A low molecular weight DNA polymerase which sediments at 3.3 S on sucrose gradients has been purified from total cell homogenates of rapidly dividing embryos of the sea urchin Strongylocentrotus purpuratus. In the presence of 2 mM N-ethylmaleimide, it is the major polymerase activity in whole cell homogenates when assayed with an oligo(dT)10.poly(dA)200 template; a template which it uses about 200 times more efficiently than activated DNA. The requirement for N-ethylmaleimide exists only in crude cell fractions where it acts to inhibit a template digesting nuclease activity. The polymerase is highly stable if maintained in the presence of 20% glycerol, is completely dependent on added template, and shows no end addition activity. The physical and enzymatic properties of this enzyme clearly distinguish it from the DNA polymerase previously described by Loeb (Loeb, L. A. (1969) J. Biol. Chem. 244, 1672-1681) which sediments as a high moeluclar weight (5.6 to 6.6 S) enzyme and prefers the activated DNA template. In addition, these two DNA polymerase enzymes show distinctive chromatographic properties using DEAE-cellulose and phosphocellulose columns as well as their sensitivity to N-ethylmaleimide. The properties of the low molecular weight polymerase indicate close similarity to the beta-polymerase isolated from mammalian cells. These low molecular weight enzymes are both sensitive to phosphate salt and able to utilize the artificial ribohomopolymer template oligo(dT)10.poly(rA)200. A quantitative analysis of the low molecular weight DNA polymerase during early embryonic development indicates that the activity of this enzyme increases at least 2-fold immediately following fertilization and again during early blastula stage (hatching). Such quantitative changes in a beta enzyme activity are in contrast to findings with the alpha-polymerase which remains constant during early development.
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PMID:A low molecular weight DNA polymerase beta in the sea urchin Strongylocentrotus purpurantus. Partial purification, properties, and changes in development. 71 48

An E. coli lysate after being gently washed to remove soluble components, supports replicative DNA synthesis, if soluble proteins and the deoxyribonucleotide triphosphates are added. This DNA synthesis is dependent on ATP and on the presence of the gene products of the dnaB, dnaG, and polC (DNA polymerase III) genes. It continues at the replication forks preformed in vivo and "Okazaki fragments" are intermediate products of the reaction. Two different methods were used to prepare the washed DNA containing fraction. The one method involves washing of a cell lysate situated on a dialysis membrane. The other method involves DNAase treatment of a lysate and sedimentation of the degraded DNA through a glycerol gradient. Both washed preparation contain not only the DNA and the replication forks but also functional amounts of DNA polymerase III and of the dnaB gene product. Other factors, that are essential for replicative DNA synthesis, including the dnaG gene product, are washed out of the DNA containing preparations and the system is reconstituted by readdition of the soluble proteins.
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PMID:Replication of E. coli duplex DNA in vitro. The separation of the DNA containing fractions of a lysate from the soluble enzymes and their complementation properties. 77 84

Sedimentation analysis of glycerol-density gradients has shown that freshly purified DNA polymerases A and B (pol A and pol B) of Euglena gracilis have molecular weights of 185,000 (8.7S) and 240,000 (10.3S) respectively. They can aggregate in fresh preparations to give forms of higher molecular weight as shown by gel filtration through Sepharose 6B, but on ageing pol B progressively generates species with sedimentation coefficients of 7.4-7.7S, 6.3-6.5S, 4.8S and finally 3.0S. Pol A apparently behaves in a similar fashion though it is unstable. Exposure of pol A and pol B to high ionic strengths can also cause their breakdown to species with lower sedimentation coefficients. The mitochondrial DNA polymerase is distinct, having a molecular weight of 170,000. It is proposed that pol A and pol B are oligomers of the 3.0S subunit and possibly other dissimilar subunits, with pol B having additional factors conferring upon it its extra catalytic functions.
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PMID:DNA polymerases of Euglena gracilis: heterogeneity of molecular weight and subunit structure. 80 92

A DNA-dependent DNA polymerase from rat liver mitochondria was partially purified and characterized. Mitochondrial DNA polymerase has been found to be quite different from other DNA-dependent DNA polymerases alpha and beta present in the rat liver in the following points: elution patterns in a DEAE-cellulose column chromatography, sedimentation coefficients determined by the glycerol gradient centrifugation in the presence of high salt, and sensitivities to N-ethylmaleimide, ethidium bromide and KCl.
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PMID:Mitochondrial DNA polymerase from rat liver. 85 77

Chromatin isolated from adult rat liver was fractionated into template active and inactive components by controlled shearing and glycerol gradient centrifugation. The fractionated chromatin was assayed for DNA-dependent DNA polymerase (DNA mucleotidyl transferase EC 2.7.7.7) activity with and without exogenous activated DNA serving as template. With endogenous chromatin as template, it was found that 90% of the endogenous chromatin bound DNA polymerase activity was located in the transcriptionally active fraction of chromatin, while the distribution of DNA polymerase assayed with exogenous activated DNA was found to be 65% in the transcriptionally active and 35% in the inactive fractions. However, when DNA polymerase was solubilized from these fractions by salt extraction, enzyme activity was found to be equally distributed, suggesting that the difference in endogenous DNA polymerase activity observed between eu- and heterochromatin is due to the restricted template found in repressed fractions.
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PMID:Endogenous DNA polymerase activity in fractionated rat lever chromatin. 90 88

Mitochondrial DNA polymerase was purified 2300-fold over isolated mitochondria from rat liver. Template-primer specificities of this enzyme were investigated. Activated DNA was satisfactorily used as an active template-primer, but both native and denatured DNAs showed a slight activity. Synthetic polynucleotide, poly(dA) - oligo(dT)10 was found to have a high efficiency under the same condition for activated DNA. When the closed-circular, nicked and gapped Co1E1 DNAs were employed as a template-primer, the enzyme could only utilize the gapped DNA, indicating that the displacement synthesis was not catalyzed by the enzyme itself. The enzyme also copied poly(A) - oligo(dT)10 in high efficiency at pH 7.5 in the presence of MnCl2. Such RNA-directed DNA polymerase activity of the enzyme was further characterized. Cofractionated endouclease activity was completely separated from the enzyme by glycerol gradient centrifugation.
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PMID:Template specificity of rat mitochondrial DNA polymerase. 92

We have isolated a nuclear membrane fraction from KB cells infected with human adenovirus 2 that synthesizes exclusively small viral DNA chains (approx. 9 S) in vitro (Yamashita, T., Arens, M. and Green, M. (1975) J. Biol. Chem. 250, 3273-3279). The DNA polymerase activity present in the adenovirus 2 DNA-nuclear membrane complex was purified through chromatography on phosphocellulose and DEAE-cellulose, glycerol gradient centrifuation and DNA-cellulose chromatography. A single peak of enzymatic activity sedimented in glycerol gradients at about 6.7 S which corresponds to a molecular weight of 125000. The enzyme preparation in the step of glycerol gradient centrifugation utilized activated calf thymus, KB cell and adenovirus 2 DNA as template-primer in the presence of Mg2+; Km values for these DNAs were 5.5, 4.0, and 0.8 mug/ml, respectively. The partially purified enzyme preparation was characterized by several criteria which were compared to the properties of the three major mammalian DNA polymerases, alpha, beta, and psi. On the basis of template-primer preference, effect of salt, inhibition by N-ethylmaleimide and Km for dTTP, the DNA polymerase activity from the membrane complex can be distinguished from the alpha and beta DNA polymerases. The elution profile from DNA cellulose revealed a minor peak (I) and a major peak (II) of DNA polymerase activity utilizing poly(A) -(dT)10 as template-primer in the presence of Mn2+ - Peak II from DNA cellulose, which contained about 90% of the total DNA polymerase activity eluted from the column, was 2-3 times as active with poly(A) - (dT)10 as template-primer in the presence of Mn2+ than with activated calf thymus DNA in the presence of Mg2+. On the other hand, peak I had a low ratio of poly(A) - (dT)10 to activated calf thymus DNA activity. DNA polymerase was also purified from the nuclear membrane fraction of uninfected KB cells by the same procedures as those used in enzyme purification from the adenovirus 2 DNA-nuclear membrane complex. A minor peak and a major peak of DNA polymerase activity utilizing poly(A) - (dT)10 as template primer in the presence of Mn2+ were again observed that eluted from DNA cellulose at the same KCl concentrations as peak I and II from adenovirus 2-infected cells. The enzymes of the nuclear membrane fraction of uninfected KB cells could not be differentiated from the enzymes of the adenovirus 2 DNA-nuclear membrane complex through any of the purification steps nor by their template specificities. These results indicate that the predominant enzyme in the adenovirus 2 DNA-nuclear membrane complex and in the KB cell nuclear membrane complex belongs to the class of DNA polymerase psi.
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PMID:Characterization of DNA polymerase associated with nuclear membrane fractions from uninfected and adenovirus 2-infected KB cells. 97 29

Activity of DNA alpha-polymerase in extracts from MOPC-104E was not associated with a single protein molecule, but with several molecular species that differed in isoelectric point. The three most abundant of these enzyme species were first separated from other DNA polymerases and then resolved from each other by repeated chromatography on diethylaminoethylcellulose columns. Next, with the use of glycerol gradient centrifugation and DNA-cellulose column chromatography, the three species were further purified to a state representing more than 5000-fold purification over the crude extract. These three highly purified enzyme species exhibited very similar catalytic properties, and the main activity of each species sedimented at the same rate (6-7S) in glycerol gradients containing 0.5 M KCl. Analysis of the polypeptide content of each species revealed that polypeptides of about 150 000 and 60 000 daltons cofractionated with the DNA polymerase activity. The multiple alpha-polymerase species did not appear to result from in vitro proteolytic cleavage, since multiple species were observed in extracts prepared under several different types of conditions, including the presence of the protease inhibitors, phenylmethanesulfonyl fluoride, or trasylol. The three species were recovered in about the same relative amounts from both the nuclear and cytoplasmic fractions of MOPC-104E, and it appeared that multiple species of alpha-polymerase were also present in extracts from fetal bovine liver.
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PMID:Studies on DNA alpha-polymerase of mouse myeloma: partial purification and comparison of three molecular forms of the enzyme. 99 8

Nuclei were isolated from monolayer cultures of mouse and human cells using a nonaqueous procedure of cell fractionation in which lyophilized cells were homogenized and centrifuged in 100% glycerol. In previous work we have shown that the nuclear pellet and cytoplasmic supernatant fraction contained 10% or less of the nucleic acids characteristic of the other cell fraction. Aqueous extracts made from fresh cultures and from nonaqueous material at each step of the fractionation procedure were assayed fro DNA polymerase activity. Activities were normalized to DNA contents of extracted material. Specific activity was preserved quantitatively through freezing and drying the cells, but was found to be unstable in glycerol suspensions with approximate half-lives and 1 h at 23 degrees and 4 h at 0-4 degrees. Activities were relatively stable at -25 degrees, however, so that by homogenizing only 15 min at 4 degrees and centrifuging at -25 degrees we preserved approximately 85% of the specific activity of fresh cultures in the nonaqueous nuclear fraction. Sedimentation analyses showed that the nuclear fraction contained both DNA polymerase-alpha and-beta in approximately the proportions expected if all polymerase activities were confined to the nucleus in living cells. DNA polymerase-alpha was found to be more unstable in glycerol suspensions than DNA polymerase-beta. Nuclear location of both activities was found in exponential cultures and in 3T3 mouse cultures synchronized in the G1 and S phases of the cell division cycle. We found no evidence for cytoplasmic factors affecting nuclear polymerase activities. We have concluded that the two major DNA polymerases are nuclear although one, DNA polymerase-alpha, frequently is present as a weakly bound nuclear protein.
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PMID:Nuclear location of mammalian DNA polymerase activities. 100 14


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