scholarly journals Inhibition of deoxyribonucleic acid polymerases from human cells and from simian sarcoma virus by pyran

1978 ◽  
Vol 175 (2) ◽  
pp. 519-524 ◽  
Author(s):  
R A Dicioccio ◽  
B I S Srivastava
Keyword(s):  
Dna Polymerase ◽  
Lymphoblastic Leukemia ◽  
Dna Polymerases ◽  
Human Cell Line ◽  
Bivalent Cation ◽  
Molecular Weights ◽  
Bivalent Cations ◽  
Sarcoma Virus ◽  
Simian Sarcoma Virus ◽  
Alpha Beta

Pyrans are co-polymers of divinyl ether and maleic anhydride. Four pyrans of various molecular weights more potently inhibited terminal deoxyribonucleotidyltransferase (EC 2.7.7.31) from a human cell line of acute lymphoblastic leukemia origin (Molt-4) than they did DNA polymerases alpha, beta and gamma from these cells and DNA polymerase from simian sarcoma virus. For example, the concentrations of one pyran required for 50% inhibition of terminal deoxynucleotidyltransferase, DNA polymerases alpha, beta and gamma and viral DNA polymerase were 0.9, 110, 125, 35 and 47 microgram/ml respectively. Quantitatively similar results were obtained with the other pyrans. Inhibition of these enzymes by pyran was dependent on the concentrations of both the bivalent cation and template/primer or initiator in assay mixtures, but not on the concentrations of the substrate (deoxyribonucleoside 5′-triphosphate), enzyme, or bovine serum albumin. These results suggested that pyran inhibited these enzymes by complexing bivalent cations, which caused a decreased affinity of template/primer or initiator for each enzyme and a decrease in enzyme activity.

scholarly journals Evaluation of PCR-Generated Chimeras, Mutations, and Heteroduplexes with 16S rRNA Gene-Based Cloning

2001 ◽  
Vol 67 (2) ◽  
pp. 880-887 ◽  
Author(s):  
Xiaoyun Qiu ◽  
Liyou Wu ◽  
Heshu Huang ◽  
Patrick E. McDonel ◽  
Anthony V. Palumbo ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Rrna Gene ◽  
Community Studies ◽  
Positive Bacterium ◽  
16S Ribosomal Dna ◽  
Model Community ◽  
Pcr Products ◽  
Alpha Beta ◽  
Optimal Approach

ABSTRACT To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division Proteobacteriaas well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR artifacts were significantly different among the three TaqDNA polymerases examined: 20% for Z-Taq, with the highest processitivity; 15% for LA-Taq, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, AmpliTaq. In contrast to the theoretical prediction, the frequency of chimeras for both Z-Taq(8.7%) and LA-Taq (6.2%) was higher than that for AmpliTaq (2.5%). The frequencies of chimeras and of heteroduplexes for Z-Taq were almost three times higher than those of AmpliTaq. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using AmpliTaq and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.

scholarly journals DNA-metabolizing enzymes in normal human lymphoid cells. VI. Induction of DNA polymerases alpha, beta, and gamma following stimulation with phytohemagglutinin

Blood ◽  
1975 ◽  
Vol 46 (4) ◽  
pp. 509-518
Author(s):  
RJ Mayer ◽  
RG Smith ◽  
RC Gallo
Keyword(s):  
Dna Polymerase ◽  
Mammalian Cells ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Lymphoid Cells ◽  
Blood Lymphocytes ◽  
Polymerase Beta ◽  
Normal Human ◽  
Alpha Beta ◽  
Cellular Dna

At least three distinct DNA polymerases, named alpha, beta, and gamma, have been isolated from normal mammalian cells. The function of these enzymes in regard to DNA replication and repair remains unclear. Stimulation of blood lymphocytes with the plant mitogen phytohemagglutinin (PHA), is known to increase total DNA polymerase activity. In this study, we measured the change of each of these activities as lymphocytes intered a mitotic cycle. Aliquots of a pool of normal human blood lymphocytes were incubated with PHA for 0, 24, 48, and 72 hr, respectively, and the various DNA polymerase activities quantitated at each point. No significant DNA polymerase activity was detected in unstimulated cells. Low levels of polymerase beta were found at 24 hr. The average DNA content per cell doubled between 24 and 48 hr, and during this interval all three DNA polymerases increased to easily detectable levels. By far the greatest fractional increase in activity of all three polymerases was seen between 48 and 72 hr, after the average doubling of cellular DNA. In summary, these blood lymphocytes lack significant levels of DNA polymerases; stimulation with PHA induces all three of the major DNA polymerase species. In both these respects, these cells differ from other proliferating mammalian cell systems. The possible significance of this difference is discussed.

scholarly journals DNA-metabolizing enzymes in normal human lymphoid cells. VI. Induction of DNA polymerases alpha, beta, and gamma following stimulation with phytohemagglutinin

Blood ◽  
1975 ◽  
Vol 46 (4) ◽  
pp. 509-518 ◽  
Author(s):  
RJ Mayer ◽  
RG Smith ◽  
RC Gallo
Keyword(s):  
Dna Polymerase ◽  
Mammalian Cells ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Lymphoid Cells ◽  
Blood Lymphocytes ◽  
Polymerase Beta ◽  
Normal Human ◽  
Alpha Beta ◽  
Cellular Dna

Abstract At least three distinct DNA polymerases, named alpha, beta, and gamma, have been isolated from normal mammalian cells. The function of these enzymes in regard to DNA replication and repair remains unclear. Stimulation of blood lymphocytes with the plant mitogen phytohemagglutinin (PHA), is known to increase total DNA polymerase activity. In this study, we measured the change of each of these activities as lymphocytes intered a mitotic cycle. Aliquots of a pool of normal human blood lymphocytes were incubated with PHA for 0, 24, 48, and 72 hr, respectively, and the various DNA polymerase activities quantitated at each point. No significant DNA polymerase activity was detected in unstimulated cells. Low levels of polymerase beta were found at 24 hr. The average DNA content per cell doubled between 24 and 48 hr, and during this interval all three DNA polymerases increased to easily detectable levels. By far the greatest fractional increase in activity of all three polymerases was seen between 48 and 72 hr, after the average doubling of cellular DNA. In summary, these blood lymphocytes lack significant levels of DNA polymerases; stimulation with PHA induces all three of the major DNA polymerase species. In both these respects, these cells differ from other proliferating mammalian cell systems. The possible significance of this difference is discussed.

scholarly journals DNA polymerases from Chlamydomonas reinhardii. Purification and properties

1978 ◽  
Vol 171 (1) ◽  
pp. 231-240 ◽  
Author(s):  
C A Ross ◽  
W J Harris
Keyword(s):  
Dna Polymerase ◽  
Nuclear Dna ◽  
Dna Polymerases ◽  
Deae Cellulose ◽  
Single Species ◽  
Bivalent Cation ◽  
Chlamydomonas Reinhardii ◽  
Synchronous Cultures ◽  
Purification And Properties ◽  
Calf Thymus

Three DNA polymerase activities, A, B and C, were identified in extracts of exponentially growing synchronous cultures of Chlamydomonas reinhardii, and DNA polymerases A and B were characterized in detail. Both enzymes have the same binding affinity for DEAE-cellulose at pH 7.8, but can be distinguished from each other by their behaviour on phosphocellulose and DNA-agarose. ‘Activated’ calf thymus DNA was used as template, and the pH, K+ and bivalent-cation optima were measured. DNA polymerase A sediments at 5.3 S in glycerol gradients, with an apparent mol.wt. of 90000-100000. Polymerase B sediments between 8S and 10S in 100mM-KCl, the predominant species having an apparent mol.wt. of 200000. In 200mM-KCl, polymerase B dissociates to a single species, which sediments at 5.8S. A 3S species was found in aged preparations of both enzymes. The activity of polymerase B from cells harvested during nuclear DNA synthesis is twice that found in Chlamydomonas at other times during the cell cycle.

PrimPol (Primase/Polymerase), replicating enzyme – A mini review

2020 ◽  
Vol 2 (4) ◽  
pp. 89-92
Author(s):  
Muhammad Amir ◽  
Sabeera Afzal ◽  
Alia Ishaq
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Genetic Information ◽  
Nuclear Dna ◽  
De Novo ◽  
Dna Polymerases ◽  
Test Site ◽  
Mitochondrial Dna Replication ◽  
Dna Template ◽  
Preliminary Phase

Polymerases were revealed first in 1970s. Most important to the modest perception the enzyme responsible for nuclear DNA replication that was pol , for DNA repair pol and for mitochondrial DNA replication pol  DNA construction and renovation done by DNA polymerases, so directing both the constancy and discrepancy of genetic information. Replication of genome initiate with DNA template-dependent fusion of small primers of RNA. This preliminary phase in replication of DNA demarcated as de novo primer synthesis which is catalyzed by specified polymerases known as primases. Sixteen diverse DNA-synthesizing enzymes about human perspective are devoted to replication, reparation, mutilation lenience, and inconsistency of nuclear DNA. But in dissimilarity, merely one DNA polymerase has been called in mitochondria. It has been suggest that PrimPol is extremely acting the roles by re-priming DNA replication in mitochondria to permit an effective and appropriate way replication to be accomplished. Investigations from a numeral of test site have significantly amplified our appreciative of the role, recruitment and regulation of the enzyme during DNA replication. Though, we are simply just start to increase in value the versatile roles that play PrimPol in eukaryote.

DNA-Polymerases α, δ and ε from T-Cell Spontaneous Lymphoblastic Leukemia of Sprague-Dawley Inbred Rat: Isolation and Characterization

1995 ◽  
Vol 60 (9) ◽  
pp. 1555-1572 ◽  
Author(s):  
Pavel Kramata ◽  
Jaroslav Černý ◽  
Gabriel Birkuš ◽  
Ivan Votruba ◽  
Berta Otová ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Lymphoblastic Leukemia ◽  
Dna Polymerases ◽  
Sedimentation Coefficient ◽  
Nuclear Antigen ◽  
Exonuclease Activity ◽  
Sprague Dawley ◽  
Dna Primase ◽  
Isolation And Characterization ◽  
Inbred Rat

Using a single isolation procedure and selective assays for the determination of enzyme activity, highly purified DNA-polymerases α, δ and ε were isolated from the lymphoma of Sprague-Dawley inbred rats. For pol α the subunit composition was 170, 70, 57 and 53 kDa with sedimentation coefficient 8.7 S for the native molecule; pol delta consists of two polypeptides (133 and 46 kDa; sedimentation coefficient 8.2 S), while pol ε is a single polypeptide (140 kDa) and its sedimentation coefficient is 7.0 S. Comparison of the interaction of individual enzymes with known inhibitors and proliferating cell nuclear antigen (PCNA) using the template-primer poly dA-oligo dT12-18, gave the following data: (i) pol α is selectively inhibited by N2-(p-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and stimulated by dimethyl sulfoxide; (ii) all the enzymes are inhibited by N-ethylmaleimide and aphidicolin; (iii) PCNA stimulates pol δ approximately 50 times while pol ε is moderately inhibited; (iv) pol α exhibits considerably higher DNA-primase activity with poly dC as template than with poly dT, and negligible 3'-5'-exonuclease activity whereas pol δ and pol ε, which do not exert any DNA-primase activity have approximately the same 3'-5'-exonuclease activity. The ability of individual polymerases to utilize poly dT-oligo dA12-18 as a template-primer at different pH values, ionic strengths and Mg2+-concentrations was also investigated. In comparison to poly dA-oligo dT12-18 template-primer, pol α has 140% of enzyme activity on poly dT-oligo dA12-18 under optimal conditions, whereas the activity of pol ε and pol δ is 4 times and 10 times lower, respectively.

scholarly journals Antimutator mutations in the alpha subunit of Escherichia coli DNA polymerase III: identification of the responsible mutations and alignment with other DNA polymerases.

Genetics ◽  
1993 ◽  
Vol 134 (4) ◽  
pp. 1039-1044 ◽  
Author(s):  
I J Fijalkowska ◽  
R M Schaaper
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Alpha Subunit ◽  
Sequence Motifs ◽  
Dna Polymerase Iii ◽  
Conserved Sequence ◽  
Polymerase Iii ◽  
Dna Replication Errors

Abstract The dnaE gene of Escherichia coli encodes the DNA polymerase (alpha subunit) of the main replicative enzyme, DNA polymerase III holoenzyme. We have previously identified this gene as the site of a series of seven antimutator mutations that specifically decrease the level of DNA replication errors. Here we report the nucleotide sequence changes in each of the different antimutator dnaE alleles. For each a single, but different, amino acid substitution was found among the 1,160 amino acids of the protein. The observed substitutions are generally nonconservative. All affected residues are located in the central one-third of the protein. Some insight into the function of the regions of polymerase III containing the affected residues was obtained by amino acid alignment with other DNA polymerases. We followed the principles developed in 1990 by M. Delarue et al. who have identified in DNA polymerases from a large number of prokaryotic and eukaryotic sources three highly conserved sequence motifs, which are suggested to contain components of the polymerase active site. We succeeded in finding these three conserved motifs in polymerase III as well. However, none of the amino acid substitutions responsible for the antimutator phenotype occurred at these sites. This and other observations suggest that the effect of these mutations may be exerted indirectly through effects on polymerase conformation and/or DNA/polymerase interactions.

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