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

• Published in: Russian chemical bulletin Vol. 54; no. 5; pp. 1311 - 1321
• Main Authors: Dezhurov, S. V., Grin, I. R., Safronov, I. V., Shishkin, G. V., Lavrik, O. I., Khodyreva, S. N.
• Format: Journal Article
• Language: English
• Published: 01-05-2005
• ISSN: 1066-5285; 1573-9171
• DOI: 10.1007/s11172-005-0400-7

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 beta or endogenous DNA polymerases of the cell extract. The photoreactive DNAs synthesized in situ were used for affinity modification of DNA polymerase beta 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 beta 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 beta 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 beta was covalently attached to DNA. Photoreactive dNTP analogs ensuring a high level of protein modification in the cell extract were found.