Photoaffinity modification of HIV reverse transcriptase with a deoxyuridine 5′-triphosphate analog containing an arylazido group

A photoactivatable dNTP analog, 5-{γ-[N-(2″-nitro-5″-azidobenzoyl)amino]-trans-propenyl}- 2′-deoxyuridine-5′-triphosphate (NAB-dUTP), generating highly reactive intermediates on exposure to near UV light (>300 nm) was used as a photoaffinity-labeling reagent for modification of reverse transcriptase from human immunodeficiency virus (HIV RT) and in studies on its structural topography. The NAB-dNTP analog was shown to be a substrate in DNA polymerization catalyzed by HIV RT. Modification of HIV RT proceeded in the specific enzyme-analog complex and resulted in enzyme inactivation. Estimates were obtained for the Kd of the NAB-dUTP·RT complex and the k_app of inactivation, as well as for the K_d of the ternary complex NAB-dUTP-RT-(poly(rA) or poly(dA)) and k_app of the enzyme inactivation by NAB-dUTP in the presence of poly(rA) or poly(dA). Photoaffinity labeling of HIV RT by NAB-dUTP was achieved in two ways. In the first approach, the enzyme was exposed to UV light together with the analog and then the covalently bound dUTP analog was used for primer elongation in the presence of the complementary template. In this case, photolabeling of both subunits in the enzyme suggested that the dNTP-binding site of HIV RT was in the p66 subunit near its contact region with the p51 subunit. In the second approach, the NAB-dUMP residue was introduced into the primer enzymatically with HIV RT and then the primer was used as the photoaffinity probe. Upon exposure of the specific complex RT-template-NAB-dUMP-containing primer to UV light, both subunits of the enzyme were modified while no modification was observed for the DNA template.