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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Authors: | S V Dezhurov I R Grin I V Safronov G V Shishkin O I Lavrik S N Khodyreva |
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Institution: | (1) Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch, Russian Academy of Sciences, 8 prosp. Akad. Lavrent'eva, 630090 Novosibirsk, Russian Federation |
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Abstract: | 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 polymerase 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.
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Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1273–1283, May, 2005. |
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Keywords: | DNA polymerase β photoaffinity modification sensitized modification modification efficiency |
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