Investigation of the dNTP-binding site of HIV-1 reverse transcriptase using photoreactive analogs of dNTP

The interaction of dNTPs with the active site of HIV-1 reverse transcriptas e (HIV RT) has been investigated. The kinetic parameters of primer elongati on catalyzed by wild-type HIV-1 RT and two of its mutants with substitution s for Tyr115 using dTTP and two of its photoreactive analogs were determine d. The substitution for Tyr115 with alanine or tryptophan resulted in an in crease in Km values of dTTP and its analogs, Wild-type RT and its mutants w ere photoaffinity modified using photoreactive primer synthesized in situ. The modification was made in two variants: direct photo cross-linking under UV irradiation and photosensitized modification using Pyr-dUTP as a sensit izer. The use of the sensitizer decreased the number of modification produc ts and increased selective labeling of the catalytic subunit of both the mu tant and wild-type RT.