Detection and characterization of DNA polymerase activity in Toxoplasma gondii

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011569 ◽

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.

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

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029700800302 ◽

1997 ◽

Vol 8 (3) ◽

pp. 187-195 ◽

Cited By ~ 33

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%).

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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

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029500600403 ◽

1995 ◽

Vol 6 (4) ◽

pp. 217-221 ◽

Cited By ~ 52

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.

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