scholarly journals Kinetic Interaction of the Diphosphates of 9-(2-phosphonylmethoxyethyl)adenine and Other anti-HIV Active Purine Congeners with HIV Reverse Transcriptase and Human DNA Polymerases α, β and γ

1995 ◽  
Vol 6 (4) ◽  
pp. 217-221 ◽  
Author(s):  
J. M. Cherrington ◽  
S. J. W. Allen ◽  
N. Bischofberger ◽  
M. S. Chen
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Inhibitory Effects ◽  
Hiv Reverse Transcriptase ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Dna Template ◽  
Anti Hiv ◽  
Micromolar Range

The inhibitory effects of the diphosphates of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and its analogues on HIV reverse transcriptase and human DNA polymerases α, β, and γ have been studied. The analogues investigated are the diphosphates of 9-(2-phosphonylmethoxypropyl)adenine (PMPApp), 9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine (PMPDAPpp), and (2R,5R)-9-[2,5-dihydro-5-(phosphonyl methoxy)-2-furanyl]adenine (D4APpp). These four compounds are much more inhibitory to HIV reverse transcriptase when an RNA template rather than a DNA template is used. The Ki, values for the four compounds range from 11 to 22 nM with an RNA template. The Ki, values for ddCTP and AZTTP are 54 nM and 8 nM, respectively. PMEApp and its analogues show varying degrees of inhibition of the human DNA polymerases. The Ki, values for PMEApp, PMPApp and PMPDAPpp against DNA polymerase α are in the micromolar range, while D4APpp is a poor inhibitor of this enzyme with a Ki, value of 65.9 μM. The inhibition of DNA polymerase β by PMEApp, PMPApp and D4APpp is minimal, while PMPDAPpp shows higher inhibition of DNA polymerase β with a Ki, value of 9.71 μM. The Ki, values for PMEApp and D4APpp against DNA polymerase γ are submicromolar, while PMPApp and PMPDAPpp are much less inhibitory to this enzyme. For comparison, ddCTP was found to be a more potent inhibitor of DNA polymerases β and γ than the diphosphates of PMEA and its analogues.

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 Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase β

2019 ◽  
Vol 295 (6) ◽  
pp. 1613-1622
Author(s):  
Mallory R. Smith ◽  
Khadijeh S. Alnajjar ◽  
Nicole M. Hoitsma ◽  
Joann B. Sweasy ◽  
Bret D. Freudenthal
Keyword(s):  
Dna Polymerase ◽  
Conformational Changes ◽  
Dna Polymerases ◽  
Catalytic Efficiency ◽  
Dna Polymerase Β ◽  
Replication Errors ◽  
Human Dna ◽  
Oxidatively Modified ◽  
Dna Replication Errors

During oxidative stress, inflammation, or environmental exposure, ribo- and deoxyribonucleotides are oxidatively modified. 8-Oxo-7,8-dihydro-2′-guanosine (8-oxo-G) is a common oxidized nucleobase whose deoxyribonucleotide form, 8-oxo-dGTP, has been widely studied and demonstrated to be a mutagenic substrate for DNA polymerases. Guanine ribonucleotides are analogously oxidized to r8-oxo-GTP, which can constitute up to 5% of the rGTP pool. Because ribonucleotides are commonly misinserted into DNA, and 8-oxo-G causes replication errors, we were motivated to investigate how the oxidized ribonucleotide is utilized by DNA polymerases. To do this, here we employed human DNA polymerase β (pol β) and characterized r8-oxo-GTP insertion with DNA substrates containing either a templating cytosine (nonmutagenic) or adenine (mutagenic). Our results show that pol β has a diminished catalytic efficiency for r8-oxo-GTP compared with canonical deoxyribonucleotides but that r8-oxo-GTP is inserted mutagenically at a rate similar to those of other common DNA replication errors (i.e. ribonucleotide and mismatch insertions). Using FRET assays to monitor conformational changes of pol β with r8-oxo-GTP, we demonstrate impaired pol β closure that correlates with a reduced insertion efficiency. X-ray crystallographic analyses revealed that, similar to 8-oxo-dGTP, r8-oxo-GTP adopts an anti conformation opposite a templating cytosine and a syn conformation opposite adenine. However, unlike 8-oxo-dGTP, r8-oxo-GTP did not form a planar base pair with either templating base. These results suggest that r8-oxo-GTP is a potential mutagenic substrate for DNA polymerases and provide structural insights into how r8-oxo-GTP is processed by DNA polymerases.

Kinetic study of various binding modes between human DNA polymerase β and different DNA substrates by surface-plasmon-resonance biosensor

2002 ◽  
Vol 361 (2) ◽  
pp. 317-325 ◽  
Author(s):  
Pui Yan TSOI ◽  
Mengsu YANG
Keyword(s):  
Surface Plasmon Resonance ◽  
Dna Polymerase ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Single Strand ◽  
The Other ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Dna Template ◽  
Gapped Dna

The interaction of a series of DNA substrates with human DNA polymerase β has been studied in real time by using a surface-plasmon-resonance (SPR) biosensor technique. We have prepared the sensor surfaces comprising different DNA targets, including single-stranded DNA, blunt-end double-stranded DNA, gapped DNA and DNA template—primer duplexes containing various mismatches at different positions. The binding and dissociation of polymerase β at the DNA-modified surfaces was measured in real time, and the kinetics profiles of polymerase—DNA interaction were analysed using various physical models. The results showed that polymerase β binding to single-stranded DNA (KA = 1.25×108M−1; where KA is the equilibrium affinity constant) was thermodynamically more favourable than to blunt-end DNA duplex (KA = 7.56×107M−1) or gapped DNA (KA = 8.53×107M−1), with a single binding mode on each DNA substrate. However, polymerase β bound to DNA template—primer duplexes (15bp with a 35nt overhang) at two sites, presumably one at the single-strand overhang and the other at the 3′-end of the primer. When the DNA duplex was fully matched, most of the polymerase β (83%) bound to the template—primer duplex region. The introduction of different numbers of mismatches near the 3′-end of the primer caused the binding affinity and the fraction of polymerase β bound at the duplex region to decrease 8–58-fold and 15–40%, respectively. On the other hand, the affinity of polymerase β for the single-strand overhang remained unchanged while the fraction bound to the single-strand region increased by 15–40%. The destabilizing effect of the mismatches was due to both a decrease in the rate of binding and an increase in the rate of dissociation for polymerase β.

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.

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