Year 2005, Month 5 (Section Full Articles, Page 1311)
Highly efficient modification of DNA polymerase β under conditions of
direct and sensitized activation of photoreactive DNAs.
Modification of cell extract proteins
S. V. Dezhurov, I. R. Grin, I. V. Safronov, G. V. Shishkin,† O. I. Lavrik, and S. N. Khodyreva*
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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