Photoaffinity labeling of DNA polymerase from Thermus thermophilus and DNA template by photoreactive analogs of dCTP.

Abstract:

Substrate properties of dCTP analogs N4-[2-(4-azidotetrafluorobenzoylamino)-ethyl]-2;-deoxycytidine-5; -triphosphate (FABdCTP), 5-[N-(4-azidotetrafluorobenzoyl)-3-amino-trans-propen-1-yl] -2;-deoxycytidine-5;-triphosphate (AlFABdCTP), and N4-[2-(2-nitro-5-azidobenzoylamino)-ethyl]-2;-deoxycytidine-5; -triphosphate (NABdCTP) were studied in the reaction of DNA synthesis catalyzed by DNA polymerase from the extremely thermophilic bacterium Thermus thermophilus B 35 (Tte DNA polymerase). The enzyme was photoaffinity labeled with the mentioned derivatives, NABdCTP being used for the first time. The photoreactive primers containing FABdCTP and AlFABdCTP were synthesized in situ by Tte DNA polymerase and used in the complementary addressed labeling of DNA template. The efficiency of DNA template labeling is shown to be a function of the structure of the photoactive group.

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