Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Multiple forms of nuclear deoxyribonucleic acid polymerases and their relationship with the soluble enzyme.

ABSTRACT: 1. DNA polymerase from nuclear and supernatant fractions of cultured mouse L929 cells was fractionated on columns of Sephadex G-200, Sepharose 4B and of DEAE-cellulose. Several peaks of activity are found on Sephadex chromatography and the distribution of activity between these depends on: (a) the source of the enzyme, i.e. nuclear or supernatant fraction; (b) the mode of extraction of the enzyme from the nucleus; (c) the amount of enzyme applied to the column. 2. The DNA polymerase activity in the lower-molecular-weight peaks (approximate molecular weights are 35000, 70000 and 140000) is firmly bound within the cell nucleus and shows a preference for native DNA as template, whereas the high-molecular-weight peak (peak I, molecular weight 250000 or greater) is found in supernatant fractions and shows greater activity with a denatured DNA template. 3. During periods of DNA synthesis the high-molecular-weight enzyme becomes more firmly bound within the nucleus. 4. Peak I enzymic activity is relatively unstable and is inhibited by thiol-blocking reagents and deoxycholate, but it is stimulated by univalent cations. 5. Very little endonuclease is present in the polymerase preparations, but a very active exonuclease and nucleoside diphosphokinase are present. On Sephadex chromatography, however, it was shown that the immediate precursors for DNA synthesis, at least by peak I enzyme, are the deoxyribonucleoside triphosphates. 6. Attempts to decrease the molecular weight of the peak I enzyme while still retaining activity failed.

SUBMITTER: Adams RL

PROVIDER: S-EPMC1177462 | biostudies-other | 1973 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

The relationship between the deoxyribonucleic acid-bound and low-molecular-weight soluble forms of Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polymerase.

Project description:1. Slow, spontaneous lysis of Halobacterium cutirubrum in 3 M-KCl yields DNA-dependent RNA polymerase as a complex with DNA that sediments completely at 45 000g. 2. Controlled deoxyribonuclease digestion of the complex, with or without subsequent sonication, releases the enzyme quantitatively in a soluble form that passes through ultrafilters with a molecular-weight exclusion limit of 50 000. 3. Purification of the active ultrafiltrate by gel filtration and hydroxyapatite chromatography gives a high yield of the purified alpha and beta subunits. 4. The low mol.wt. (17 800-19 000) of the soluble enzyme was confirmed by gel filtration and is unchanged by sonication of the DNA-enzyme complex. 5. A new assay applicable to both forms of the enzyme was developed. 6. The bivalent-cation requirement of the soluble form depends on the buffer concentration. 7. Both the DNA-enzyme complex and the low-molecular-weight soluble forms of the polymerase catalyse formation of short RNA chains only.

| S-EPMC1165679 | biostudies-other

Deoxyribonucleic acid polymerases of Euglena gracilis. Primer-template utilization of and enzyme activities associated with the two deoxyribonucleic acid polymerases of high molecular weight.

Project description:The two high-molecular-weight DNA polymerases from Euglena gracilis, pol A (mol. wt. 190 000) and pol B (mol. wt. 240 000), were differentiated on the basis of associated enzymic activities and primer-template utilization. Neither enzyme had endodeoxyribonuclease activity, but pol B, like pol B of yeast and the corresponding enzyme from Tetrahymena pyriformis, exhibited at least one other nuclease activity directed against denatured DNA and the RNA of an RNA-DNA hybrid. These nuclease functions preferred an alkaline pH and Mg2+. Pol B also exhibited nucleoside diphosphokinase activity. Both enzymes were active with 'activated' DNA and poly[d(A-T)] as primer-templates and were sensitive, especially pol B, to inhibition by excess of native or heat-denatured DNA. Pol B also utilized oligo[d(T)] and poly(A) templates under certain conditions, whereas pol A exhibited only slight activity with poly[d(A)]. (U)6 was not used as a primer by either enzyme.

| S-EPMC1172352 | biostudies-other

Multiple deoxyribonucleic acid polymerases from quiescent wheat embryos. Purification and characterization of three enzymes from the soluble cytoplasm and one from purified mitochondria.

Project description:Three DNA polymerases (A, B and C) have been purified from the soluble cytoplasm of ungerminated embryos. Mainly on the basis of chromatographic, template-specificity and salt-inhibition evidence, we have characterized the three enzymes. Other physico-chemical and enzymic properties are described. From purified mitochondria we have purified a DNA polymerase that behaves like DNA polymerase B on chromatographic and template-specificity criteria. Only highly purified enzyme B from the soluble cytoplasm showed an exonuclease activity able to degrade 3'- or 5'-labelled polydeoxyribonucleotides, as well as a 'proof-reading' capacity.

| S-EPMC1161140 | biostudies-other

Deoxyribonucleic acid polymerases of Euglena gracilis. Purification and properties of two distinct deoxyribonucleic acid polymerases of high molecular weight.

Project description:Two DNA polymerases of high molecular weight, pol A (mol.wt. 190 000) and pol B (mol.wt. 240 ooo), have been purified 6300-fold and 1600-fold respectively from an extramitochondrial supernatant of a bleached strain of Euglena gracilis. They have very similar requirements when assayed with an 'activated'-DNA primer-template [the optimum conditions of pH and ionic (K+ and Mn2+) composition being 7.2, 25 mM and 0.2 mM respectively]. 0.2 mM-Mn2+ was about 1.5-2-fold as effective as 2 mM-Mg2+, owing to substrate activation by deoxyribonucleoside 5'-triphosphates in the presence of Mn2+. Km values for the triphosphates in the absence of activation were about 10(-6)M with Mn2+ and 8 X 10(-6) M with Mg2+ for both enzymes. They were inhibited to the same extent by N-ethylmaleimide, novobiocin and o-phenanthroline, but differed in their chromatographic behaviour on DEAE-cellulose and in their electrophoretic mobilities on polyacrylamide gel. No evidence was found for the existence in these cells of a DNA polymerase of low molecular weight, but there were indications that a third enzyme of high molecular weight might exist.

| S-EPMC1172351 | biostudies-other

Presence of two deoxyribonucleic acid polymerases in bull spermatozoa.

Project description:A DNA polymerase-endogenous template complex was isolated from nuclear heads of bull spermatozoa. The buoyant density of the complex was 1.15 g/cm 3. The sedimentation coefficient of the nuclear DNA polymerase isolated from the complex was higher at low ionic strength, but approached 3.4S when centrifuged in a medium containing 2M-KCl. Activated exogenous DNA increased polymerase activity. Only very low activities were detected with synthetic templates such as poly(A).(dT)12-18 and poly(dT).poly(A). The nuclear reaction was stimulated by 150mM-KCl and was slightly inhibited by N-ethylmaleimide; it was resistant to actinomycin D, netropsin and ethidium bromide. Another DNA polymerase, highly sensitive to ethidium bromide, was extracted from the mitochondira-rich middle-piece fraction. Its sedimentation coefficient was close to 9S, but fell to approx. 4S in high-ionic-strength medium.

| S-EPMC1186108 | biostudies-other

Multiple forms of poly(A) polymerases in human cells.

Project description:We have cloned human poly(A) polymerase (PAP) mRNA as cDNA in Escherichia coli. The primary structure of the mRNA was determined and compared to the bovine PAP mRNA sequence. The two sequences were 97% identical at the nucleotide level, which translated into 99% similarity at the amino acid level. Polypeptides representing recombinant PAP were expressed in E. coli, purified, and used as antigens to generate monoclonal antibodies. Western blot analysis using these monoclonal antibodies as probes revealed three PAPs, having estimated molecular masses of 90, 100, and 106 kDa in HeLa cell extracts. Fractionation of HeLa cells showed that the 90-kDa polypeptide was nuclear while the 100- and 106-kDa species were present in both nuclear and cytoplasmic fractions. The 106-kDa PAP was most likely a phosphorylated derivative of the 100-kDa species. PAP activity was recovered in vitro by using purified recombinant human PAP. Subsequent mutational analysis revealed that both the N- and C-terminal regions of PAP were important for activity and suggested that cleavage and polyadenylylation specificity factor (CPSF) interacted with the C-terminal region of PAP. Interestingly, tentative phosphorylation sites have been identified in this region, suggesting that phosphorylation/dephosphorylation may regulate the interaction between the two polyadenylylation factors PAP and CPSF.

| S-EPMC521437 | biostudies-other

Changes in Deoxyribonucleic Acid Methylation Contribute to the Pathophysiology of Multiple Sclerosis.

Project description:Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by loss of coordination, weakness, dysfunctions in bladder capacity, bowel movement, and cognitive impairment. Thus, the disease leads to a significant socioeconomic burden. In the pathophysiology of the disease, both genetic and environmental risk factors are involved. Gene x environment interaction is modulated by epigenetic mechanisms. Epigenetics refers to a sophisticated system that regulates gene expression with no changes in the DNA sequence. The most studied epigenetic mechanism is the DNA methylation. In this review, we summarize the data available from the current literature by grouping sets of differentially methylated genes in distinct biological categories: the immune system including innate and adaptive response, the DNA damage, and the central nervous system.

| S-EPMC6874160 | biostudies-literature

Involvement of deoxyribonucleic acid polymerase beta in nuclear deoxyribonucleic acid synthesis.

Project description:The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.

| S-EPMC1185775 | biostudies-other

Effect of drugs on deoxyribonucleic acid synthesis in isolated mammalian cell nuclei. Comparison with partially purified deoxyribonucleic acid polymerases.

Project description:In order to ascertain the identity of the DNA-dependent DNA polymerase responsible for the observed DNA synthesis in nuclei isolated from baby-hamster kidney (BHK-21/C13) cells a comparative study was carried out on the effects of some drugs, reported to influence DNA synthesis, on DNA synthesis catalysed by these nuclei and by partially purified DNA polymerase-alpha and -beta. In all cases DNA synthesis by isolated nuclei and polymerase-alpha was inhibited to similar extents by N-ethylmaleimide, p-hydroxymercuribenzoate, novobiocin, heparin and phosphonoacetic acid; polymerase-beta was much less affected by these compounds. Ethidium bromide inhibited all DNA synthesis to similar extents, although at low concentrations (about 2 microgram/ml) synthesis in isolated nuclei was stimulated. The results are discussed in relation to the proposal that DNA polymerase-alpha catalyses the covalent extension of Okazaki fragments that these nuclei carry out in vitro.

| S-EPMC1186562 | biostudies-other

Multiple forms of mammalian deoxyribonucleic acid polymerase. An attempt to relate their interactions with nuclei and free deoxyribonucleic acid in vitro with their possible functions in vivo.

Project description:THE DNA POLYMERASES OF THE FOLLOWING EUKARYOTIC TISSUES WERE STUDIED: regenerating rat liver, normal rat liver, rat thymus, normal mouse liver and Ehrlich ascites-tumour cells. In all cases two main polymerase forms are observed, one of mol.wt. 200000, preferring denatured DNA to native calf thymus DNA primer, designated type I, and the other, designated type II, of mol.wt. 100000, showing a variable and slight preference for native calf thymus DNA primer. Some catalytic properties of these polymerases are described. Nuclei have been isolated from some of these tissues by using two different buffer systems. The ionic composition of the isolation medium is found to affect greatly the amounts and types of polymerase that bind to the nuclei, and also affects the kinetic properties of the polymerases. The way the polymerases and nuclei change properties as the ionic composition of the buffers is changed suggests that ionic effects may be a significant factor in the control of DNA synthesis in vivo. These ionic effects also explain much of the previous confusion over the localization of specific DNA polymerases.

| S-EPMC1178024 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data