N2-(p-n-Octylphenyl)dGTP: Synthesis and Inhibitory Activity Against DNA Polymerases

Abstract

N²-(p-n-Octylphenyl)-2′-deoxyguanosine 5′-triphosphate (OctPdGTP)has been synthesized chemically. OctPdGTP inhibited DNA polymerases (pol) α, δ and ε from calf thymus, with moderate selectivity for pol α. Mechanistic studies on pol α and bacteriophage T4 DNA polymerase revealed competitive and mixed kinetics of OctPdGTP with respect to the substrate dGTP when the enzymes were assayed on activated DNA and oligo dT:poly dA, respectively.

     

Reasons and Limits of Substrate Activity of Modified L-dNTP in DNA Biosynthesis

Abstract

Theoretical and experimental analysis of interaction of modified D- and L- dNTP as substrates for template-dependent and template-independent DNA polymerases was performed. It is shown that if the modified nucleoside 5′-triphosphates do not contain a substituent in position 3′ DNA chains can be extended by both strereoisomeric series with the same kinetic parameters. But the presence of even a 3′- hydroxy group in L-dNTP prevents their incorporation into the DNA chain.     

Carborane-linked 2′-deoxyuridine 5′-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA

5-[(p-Carborane-2-yl)ethynyl]-2′-deoxyuridine 5′-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases.     

Use of 5-Nitroindole-2′-deoxyribose-5′-triphosphate for Labelling and Detection of Oligonucleotides†

Abstract

The 5′-triphosphate of 5-nitroindole-2′-deoxyriboside has been shown to be a good substrate for terminal deoxynucleotidyl transferase (TdT). An antibody has been prepared for the detection of 5-nitroindole and has been used for the detection of 5-nitroindole tailed DNA both in single-stranded form and after hybridisation to a template. This is therefore a new method for the detection of nucleic acid probes.

     

Synthesis and Structural Analysis of Oxadiazole Carboxamide Deoxyribonucleoside Analogs

Two novel C-linked oxadiazole carboxamide nucleosides 5-(2′-deoxy-3′,5′-β-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-5-carboxamide (1) and 5-(2′-deoxy-3′,5′-β-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-3-carboxamide (2) were successfully synthesized and characterized by X-ray crystallography. The crystallographic analysis shows that both unnatural nucleoside analogs 1 and 2 adapt the C2′-endo (“south”) conformation. The orientation of the oxadiazole carboxamide nucleobase moiety was determined as anti (conformer A) and high anti (conformer B) in the case of the nucleoside analog 1 whereas the syn conformation is adapted by the unnatural nucleoside 2. Furthermore, nucleoside analogs 1 and 2 were converted with high efficiency to corresponding nucleoside triphosphates through the combination chemo-enzymatic approach. Oxadiazole carboxamide deoxyribonucleoside analogs represent valuable tools to study DNA polymerase recognition, fidelity of nucleotide incorporation, and extension.

     

Europium Cryptate Labeled Deoxyuridine-Triphosphate Analog: Synthesis and Enzymatic Incorporation

Abstract

The synthesis of an europium tris-bipyridine cryptate labeled 2′-deoxyuridine-5′-triphosphate analog (K-11-dUTP) is described. This labeled triphosphate was incorporated into DNA through enzymatic reactions with terminal transferase and DNA polymerases. The enzymatic reactions were monitored by TRACE (Time Resolved Amplification of Cryptate Emission), a homogeneous method using Fluorescence Resonance Energy Transfer (FRET) from an europium cryptate as donor to a modified allophycocyanine as acceptor.     

DNA Polymerase Incorporation of Universal Base Triphosphates†

Abstract

The 5′-triphosphates of various universal deoxynucleosides have been synthesised, and their ability to act as template directed substrates for various polymerases has been investigated.

     

Excision of β-L- and β-D-Nucleotide Analogs From DNA by p53 Protein

Abstract

The tumor suppressor p₅₃ protein plays a critical role in the cell-cycle progression. The role of the 3′-to-5′ exonuclease activity of p53 protein in the DNA repair process remains elusive. Using an in vitro exonuclease assay and defined oligonucleotides terminated with β-D- and β-L-nucleoside analogs at the 3′-terminus, we studied the ability of p53 protein to excise β-L- and β-D-nucleoside analogs which have anticancer or antiviral potential. p53 protein removes β-D-nucleoside analogs more efficiently compared to that of β-L-nucleoside analogs. The affinity of p53 protein for an β-L-nucleotide terminated primer was 5 fold lower compared to non-modified primer. The hypothesis on an important role of the 3′-to-5′ exonuclease activity of p53 protein in the action of nucleoside analogs was proposed.     

Possible involvement of DNA polymerases alpha and beta in bleomycin-induced unscheduled DNA synthesis in permeable HeLa cells

DNA polymerases involved in bleomycin-induced unscheduled DNA synthesis in some permeable human cells and rodent cells were studied by using selective inhibitors (aphidicolin, 2′,3′-dideoxythymidine-5′-triphosphate and N-ethylmaleimide) for DNA polymerases. The results suggest that both DNA polymerases α and β are involved in bleomycin-induced unscheduled DNA synthesis in permeable HeLa-S3 cells and probably in some other permeable human cells (HEp-2, KB and WI-38 VA-13 cells). Bleomycin-induced unscheduled DNA synthesis in some permeable rodent cells ($\text{SR-}\text{C3H}\text{He}$, $\text{Balb}\text{c}$ 3T3, 3Y1 and XC cells) is mostly attributed to DNA polymerase β.     

Rapid Synthesis of 2′ and 3′ Fluorinated Nucleotides and Their Use in 19F-NMR-Spectroscopy of Nucleotide-Binding Enzymes

Abstract

Improved methods have been developed for the syntheses of nucleoside analogs with fluorine atoms in the furanose moiety. 5′-O-mono and triphosphates of these analogs were used in stuctural studies on adenylate kinase using ¹⁹F-NMR.     

Acyclic 2′, 3′-Dideoxy-2′,3′-didehydronucleoside 5′-Triphosphates as Termination Substrates of Broad Set of DNA Polymerases

Abstract

O4′-Nor-2′,3′-dideoxy-2′,3′-didehydronucleoside 5′-triphosphates (acyclo-d₄NTP) have been shown to have the properties of effective termination substrates for DNA biosynthesis, catalyzed by several different DNA polymerases.     

Structurally Altered Substrates for DNA Topoisomerase. I. Effects of Inclusion of a Single 3′-Deoxynucleotide Within the Scissile Strand†

Abstract

A partial DNA duplex containing a high efficiency topoisomerase I cleavage site was substituted singly at each of three sites with 3′-deoxyadenosine. Depending on the site of substitution, the facility of the topoisomerase I-mediated cleavage or ligation reactions was altered. Inclusion of the modified nucleoside at the 5′-end of the acceptor oligonucleotide diminished the rate of religation following substrate cleavage by the enzyme.

     

Reinvestigation of 4-Thiothymidine-5′-triphosphate Synthesis

Abstract

The 4-Thiothymidine-5′-triphosphate 1 (S⁴TTP) was known to be a substrate for polymerase, however a commercial sample of this compound failed to be incorporated into DNA. Mass spectrometry combined to alkaline phosphatase digestion and ³¹P-NMR showed that this sample was in fact 5′-chloro-5′-deoxy-4-thiothymidine-3′-triphosphate 2 . The desired S⁴TTP was synthesized by two alternate routes, was fully characterized and was shown to be incorporated in a DNA polymerase assay.     

Kinetic Properties of Adenine Nucleotide Analogues Against Purified 5-Phosphoribosyl-1-pyrophosphate Synthetases from E. coli,Rat Liver and Human Erythrocytes

Abstract

The nucleoside analogue 2′,3′-dideoxyadenosine (ddA), the phosphonate isostere of 2′,3′-dideoxy-2′,3′-didehydro-adenosine (d4A) 5′-monophosphate (d4API), and the acyclic nucleoside phosphonates PMEoA, PMEA, FPMPA and PMPA are potent and selective antiretroviral agents. We found that these compounds are recognized as substrates by the PRPP synthetases from E. coli, rat liver and human erythrocytes, as their monophosphate and triphosphate form in the reverse and forward reaction, respectively. In particular, ddA-5′-monophosphate (ddAMP) and ddA-5′-triphosphate proved to be excellent substrates for the enzymes. D4API was a relatively good substrate of the rat liver and human erythrocyte PRPP synthetases. The acyclic nucleoside phosphonates were rather poor substrates, as evident from their low V_max values. None of the PRPP synthetases are found to act stereospecifically: they recognized both the S- and R-enantiomers of FPMPA and PMPA in a comparably efficient manner. Our data indicate that PRPP synthetase may recognize a much broader range of adenine nucleotide analogues than previously thought.     

Sensitivity of Bacteriophage RB69 DNA Polymerase to n2-(p-n-Butylphenyl)-2′-Deoxyguanosinenucleotides

Abstract

The response of bacteriophage RB69 DNA polymerase to N²-(p-n-butylphenyl)-2′-deoxyguanosine 5′-triphosphate (BuPdGTP), related nucleotides and non-nucleoside inhibitors was measured and compared to values obtained for the closely related DNA polymerase from bacteriophage T4. Both enzymes showed similar responses to inhibitors in terms of K_i values and the ability to utilize BuPdGTP as a substrate. These results provide the relevance of using the recent crystal structure of RB69 DNA polymerase for analysis of BuPdGTP/B family DNA polymerase interactions.