5-[3-(<i>E</i>)-(4-Azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-2‘-deoxy- uridine-5‘-triphosphate Substitutes for Thymidine-5‘-triphosphate in the Polymerase Chain Reaction

Godovikova, Tatyana S.; Kolpashchikov, Dmitry M.; Orlova, Tetiana; Richter, Vladimír; Иванова, Т. В.; Grochovsky, Sergey L.; Наседкина, Т. В.; Victorova, Lubov S.; Poletaev, A. I.

doi:10.1021/bc980144r

Cited by 20 publications

(21 citation statements)

References 10 publications

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“…Analogs of deoxythymidine-5′-triphosphate (dTTP) were synthesized from dUTP (Sigma-Aldrich, Missouri) and characterized as described previously [10–12]. For inactivation experiments, samples containing 1 nM T4 DNA polymerase and biotin-labeled oligonucleotide substrate (HS) (5′ bio-CCT TCG T TCG TTG TTC CCT A GGC TGT ATA GCC CCT ACC TTT TTG GTA GGG GCT ATA CAG CC) were incubated for fifteen minutes at 37°C in the presence of 10 μM dTTP analog I.…”

Section: Methodsmentioning

confidence: 99%

Two-component covalent inhibitor

Cornett

Gerasimova

Kolpashchikov

2013

Bioorganic & Medicinal Chemistry

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Inhibitors that covalently damage proteins or nucleic acids offer great potency, but are difficult to rationally design and suffer from poor specificity. Here we outline a general concept for constructing covalent inhibitors, called the two-component covalent inhibitor (TCCI). The approach takes advantage of two ligand analogs equipped with pre-reactive groups. Binding of the analogs to the adjacent sites of a target biopolymer brings the pre-reactive groups in close proximity and causes their interaction followed by covalent damage of the target. In the present study we used light-activated pre-reactive groups to inactivate a DNA polymerase. It was found that the efficiency of a traditional single-component inhibitor was greatly reduced in the presence of a non-target protein, while the TCCI was not significantly affected. Our findings suggest that TCCI approach has advantages in inactivation of biopolymers in complex multi-component systems.

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Section: Methodsmentioning

confidence: 99%

Two-component covalent inhibitor

Cornett

Gerasimova

Kolpashchikov

2013

Bioorganic & Medicinal Chemistry

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“…1‐Pyrenebutyric acid was from Fluka. FAB‐4‐dUTP was synthesized earlier [12]. 5‐(amino‐ trans ‐propenyl‐1)‐2′‐deoxyuridine‐5′‐triphosphate (I) was synthesized according to [13].…”

Section: Methodsmentioning

confidence: 99%

Sensitized photomodification of mammalian DNA polymerase β. A new approach for highly selective affinity labeling of polymerases

et al. 1999

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To enhance the specificity of polymerase photoaffinity labeling, a novel approach based on sensitized photomodification has been developed. A base-substituted analog of TTP containing a pyrene group (PyrdUTP) was synthesized and used as an active site-bound photosensitizer for photoaffinityL with a photoreactive analog of TTP (FAB-4-dUTP). The pyrene sensitizer (PyrdUTP), excited by light (365^450 nm), can activate the photoreagent, crosslinking it to pol L L as a result of fluorescence resonance energy transfer. The initial rate of pol L L photomodification was shown to increase by a factor of ten. The selectivity of pol L L photosensitized modification was proved by adding human replication protein A.z 1999 Federation of European Biochemical Societies.

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“…The lower modification efficiency in the case of FAP 8 dUTP is also consistent with this scheme. The spectroscopic characteristics of FAB 4 dUTP (λ max = 255 nm) 14 and FAP 8 dUTP (λ max = 300 nm) 3 suggest that on irradiation with light at 365-450 nm in the pres ence of FAP containing reagents, the difference between the rates of photolysis in solution and in the photoreactive DNA-DNA polymerase-photosensitizer ternary com plex would be much lower than for the FAB containing reagents. This would result in a pronounced photolysis of the free reagent outside the complex and a decrease in the efficiency of modification of DNA polymerase due to both nonproductive consumption of the reagent and the competition of the photolyzed and intact primer-tem plate complexes for binding to the enzyme.…”

Section: Highly Efficient Sensitized Modification Of Dna Polymerase βmentioning

confidence: 99%

Highly efficient modification of DNA polymerase β under conditions of direct and sensitized activation of photoreactive DNAs. Modification of cell extract proteins

et al. 2005

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dUTP and dCTP derivatives containing a 4 azido 2,3,5,6 tetrafluorobenzylideneaminooxy group were incorporated into the 3´ end of the DNA primer within complexes with the DNA matrix as analogs of natural dTTP by virtue of catalytic activity of DNA polymerase β or endogenous DNA polymerases of the cell extract. The photoreactive DNAs synthesized in situ were used for affinity modification of DNA polymerase β and DNA binding proteins of the cell extract. For the photoreactive DNA based on these analogs, the efficiency of formation of covalent adducts with DNA polymerase β under the highest degree of DNA complexation with the enzyme was determined. The yield of covalent DNA adducts with the enzyme was 28-47%, depending on the type of the analog. The effect of the sequence of the DNA template near the localization of the photoreactive group on the redistribution of covalent cross links between the possible targets was demonstrated. A possibility of increasing the efficiency of DNA poly merase β modification in the presence of a substantial excess of photoreactive DNA using a sensitizer, a dUTP derivative containing a pyrene residue, was studied. When photoreactive DNA containing a 2,3,5,6 tetrafluoro 4 azidobenzoyl (FAB) group was used, about 60% of DNA polymerase β was covalently attached to DNA. Photoreactive dNTP analogs ensuring a high level of protein modification in the cell extract were found.Previously, 1-4 we performed a number of compara tive studies of the efficiency of formation of photo induced adducts in primer-template complexes of DNA polymerase β and DNA polymerase of Thermus thermophilus. As the photoaffinity reagents, we used DNA primers containing various arylazido groups incorporated into heterocyclic bases at the 3´ end nucleotide. The high est efficiency (~30%) of covalent cross linking of pho tolysis products of this type of primers with other compo nents of the complexes was attained with the 4 (4 azido 2,3,5,6 tetrafluorobenzylideneaminooxy)butoxy (FABO) and 4 azido 2,5 difluoro 3 chloropyridin 6 yl 1 (FAP) groups. 2 A comparative study of the visible light sensi tized photomodification of the primer-template com plex with DNA polymerase β in the presence of АТР derivatives bearing sensitizing groups attached to the γ phosphate was also carried out. 4 It was found that the total efficiency of cross linking and the distribution of the cross links among components of the complexes de pend crucially on their structures (the sequence of the primer-template duplex, the structure of the sensi tizer, etc.) and irradiation conditions. A number of studies 5-7 demonstrated the possibility of conducting sensitized photomodification using 5 substituted dUTP-pyrene derivatives as the sensitizers.This work aims at further development of the design of affinity photoreagents to study the composition and struc ture of macromolecular replication and repair protein complexes in reconstructed systems and cell extracts and, in the future, in vivo. To this end, we performed a number of studies aimed at increasing ...

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5-[3-(E)-(4-Azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-2‘-deoxy- uridine-5‘-triphosphate Substitutes for Thymidine-5‘-triphosphate in the Polymerase Chain Reaction

Cited by 20 publications

References 10 publications

Two-component covalent inhibitor

Two-component covalent inhibitor

Sensitized photomodification of mammalian DNA polymerase β. A new approach for highly selective affinity labeling of polymerases

Highly efficient modification of DNA polymerase β under conditions of direct and sensitized activation of photoreactive DNAs. Modification of cell extract proteins

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