US EPA

8332680 

Journal Article 

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

Dezhurov, SV; Grin, IR; Safronov, IV; Shishkin, GV; Lavrik, OI; Khodyreva, SN 

2005 

1 

Russian Chemical Bulletin
ISSN: 1066-5285
EISSN: 1573-9171 

54 

5 

1311-1321 

English 

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 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. © 2005 Springer Science+Business Media, Inc. 

DNA polymerase β; Modification efficiency; Photoaffinity modification; Sensitized modification