scholarly journals Characterization of a Catalytically Slow AP Lyase Activity in DNA Polymerase γ and Other Family A DNA Polymerases

2000 ◽  
Vol 275 (17) ◽  
pp. 12509-12514 ◽  
Author(s):  
Kevin G. Pinz ◽  
Daniel F. Bogenhagen
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Lyase Activity ◽  
Ap Lyase ◽  
Dna Polymerase Γ

Detection and characterization of DNA polymerase activity in Toxoplasma gondii

Parasitology ◽  
1993 ◽  
Vol 107 (2) ◽  
pp. 135-139 ◽  
Author(s):  
A. Makioka ◽  
B. Stavros ◽  
J. T. Ellis ◽  
A. M. Johnson
Keyword(s):  
Toxoplasma Gondii ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Sedimentation Coefficient ◽  
Polymerase Activity ◽  
Magnesium Ions ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Approximate Molecular Weight

SUMMARYA DNA polymerase activity has been detected and characterized in crude extracts from tachzoites of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6·4 S, corresponding to an approximate molecular weight of 150000 assuming a globular shape. Like mammalian DNA polymerase α, the DNA polymerase of T. gondii was sensitive to N-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, δ and ε and also cytosine-β-D-arabinofuranoside-5′-triphosphate which is an inhibitor of α polymerase. The activity was inhibited by 2′,3′-dideoxythymidine-5′-triphosphate which is an inhibitor of mammalian DNA polymerase β and γ. Magnesium ions (Mg2+) were absolutely required for activity and its optimal concentration was 6 mM. The optimum potassium (K+) concentration was 50 mM and a higher concentration of K+ markedly inhibited the activity. Activity was optimal at pH 8. Monoclonal antibodies against human DNA polymerase did not bind to DNA polymerase of T. gondii. Thus the T. gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs.

Incorporation of Selected Nucleoside Phosphonates and Anti-Human Immunodeficiency Virus Nucleotide Analogues into DNA by Human DNA Polymerases α, β and γ

1997 ◽  
Vol 8 (3) ◽  
pp. 187-195 ◽  
Author(s):  
T Cihlar ◽  
MS Chen
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Immunodeficiency Virus ◽  
Deoxynucleoside Triphosphate ◽  
Template Dna ◽  
Dna Polymerase Γ ◽  
Incorporation Efficiency

Incorporation of selected diphosphates of nucleoside phosphonates and triphosphates of currently approved anti-human immunodeficiency virus nucleoside analogues into DNA by human DNA polymerases α, β and γ was studied. All three polymerases were able to incorporate diphosphates of 9-(2-phosphonomethoxyethyl)adenine (PMEApp), 9-(2-phosphonomethoxyethyl)guanine (PMEGpp), ( R)-9-(2-phosphonomethoxypropyl)adenine (PMPApp), ( R)-9-(2-phosphononomethoxypropyl)-2,6-diaminopurine (PMPDAPpp) and ( 2R,5R)-9-[2,5-dihydro-5-(phosphonomethoxy)-2-furanyl]adenine (D4APpp) into primer/template DNA of defined sequence. After incorporation, these nucleoside phosphonates acted as terminators of primer extension. Kinetic constants of their incorporation were determined and compared with those for incorporation of ddATP, ddCTP, (-)-2′-deoxy-3′-thiacytidine triphosphate (3TC-TP), 2′,3′-didehydro-3′-deoxythymidine triphosphate (d4T-TP) and 3′-azido-3′-deoxythymidine triphosphate (AZT-TP). Relative efficiencies of incorporation (percentage of the incorporation efficiency for the corresponding natural deoxynucleoside triphosphate) by DNA polymerase a ranged from 0.05% for 3TC-TP to 51% for PMEGpp. DNA polymerase β catalysed the incorporation with relative efficiencies ranging from 0.014% for AZT-TP to 125% for ddCTP, and efficiencies of incorporation by DNA polymerase γ varied between 0.13% for 3TC-TP and 25% for ddCTP. Generally, the lowest incorporation efficiencies with all three polymerases were found for PMPApp (0.06–1.4%) and PMPDAPpp (0.075–2.2%).

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

1979 ◽  
Vol 181 (1) ◽  
pp. 183-191 ◽  
Author(s):  
M Castroviejo ◽  
D Tharaud ◽  
L Tarrago-Litvak ◽  
S Litvak
Keyword(s):  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Dna Polymerases ◽  
Exonuclease Activity ◽  
Purification And Characterization ◽  
Physico Chemical ◽  
Proof Reading ◽  
Wheat Embryos ◽  
Template Specificity

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.

scholarly journals Characterization of Novel Reverse Transcriptase and Other RNA-associated Catalytic Activities by Human DNA Polymerase γ

2003 ◽  
Vol 278 (38) ◽  
pp. 36403-36409 ◽  
Author(s):  
Eisuke Murakami ◽  
Joy Y. Feng ◽  
Harold Lee ◽  
Jeremiah Hanes ◽  
Kenneth A. Johnson ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Catalytic Activities ◽  
Human Dna ◽  
Dna Polymerase Γ

scholarly journals Cloning and Characterization of a Family B DNA Polymerase from the Hyperthermophilic Crenarchaeon Pyrobaculum islandicum

2000 ◽  
Vol 182 (3) ◽  
pp. 655-663 ◽  
Author(s):  
Markus Kähler ◽  
Garabed Antranikian
Keyword(s):  
Dna Polymerase ◽  
Recombinant Dna ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dna Polymerases ◽  
Sodium Dodecyl ◽  
Binding Motif ◽  
Gene Encoding ◽  
Calf Thymus ◽  
Pyrobaculum Islandicum

ABSTRACT In order to extend the limited knowledge about crenarchaeal DNA polymerases, we cloned a gene encoding a family B DNA polymerase from the hyperthermophilic crenarchaeon Pyrobaculum islandicum. The enzyme shared highest sequence identities with a group of phylogenetically related DNA polymerases, designated B3 DNA polymerases, from members of the kingdom Crenarchaeota,Pyrodictium occultum and Aeropyrum pernix, and several members of the kingdom Euryarchaeota. Six highly conserved regions as well as a DNA-binding motif, indicative of family B DNA polymerases, were identified within the sequence. Furthermore, three highly conserved 3′-5′ exonuclease motifs were also found. The gene was expressed in Escherichia coli, and the DNA polymerase was purified to homogeneity by heat treatment and affinity chromatography. Activity staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an active polypeptide of approximately 90 kDa. For the recombinant DNA polymerase from P. islandicum, activated calf thymus DNA was used as a substrate rather than primed single-stranded DNA. The enzyme was strongly inhibited by monovalent cations andN-ethylmaleimide; it is moderately sensitive to aphidicolin and dideoxyribonucleoside triphosphates. The half-life of the enzyme at 100 and 90°C was 35 min and >5 h, respectively. Interestingly, the pH of the assay buffer had a significant influence on the 3′-5′ exonuclease activity of the recombinant enzyme. Under suitable assay conditions for PCR, the enzyme was able to amplify λ DNA fragments of up to 1,500 bp.

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