Investigation of the dNTP-Binding Site of HIV-1 Reverse Transcriptase Using Photoreactive Analogs of dNTP

A. L. Zakharenko^1*, D. M. Kolpashchikov¹, S. N. Khodyreva¹, O. I. Lavrik¹, and L. Menéndez-Arias²

¹Novosibirsk Institute of Bioorganic Chemistry, pr. Lavrent'eva 8, Novosibirsk-90, 630090 Russia; fax: (3832) 33-3677; E-mail: sashaz@niboch.nsc.ru

²Centro de Biología Molecular “Severo Ochoa”, CSIC-Universidad Autonoma de Madrid, 28049 Cantoblanco (Madrid), España; fax: (3491) 397-4799; E-mail: lmenendez@cbm.uam.es

^* To whom correspondence should be addressed.

Received February 6, 2001; Revision received April 10, 2001

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The interaction of dNTPs with the active site of HIV-1 reverse transcriptase (HIV RT) has been investigated. The kinetic parameters of primer elongation catalyzed by wild-type HIV-1 RT and two of its mutants with substitutions for Tyr115 using dTTP and two of its photoreactive analogs were determined. The substitution for Tyr115 with alanine or tryptophan resulted in an increase in K_m values of dTTP and its analogs. Wild-type RT and its mutants were photoaffinity modified using photoreactive primer synthesized in situ. The modification was made in two variants: direct photocross-linking under UV irradiation and photosensitized modification using Pyr-dUTP as a sensitizer. The use of the sensitizer decreased the number of modification products and increased selective labeling of the catalytic subunit of both the mutant and wild-type RT.

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KEY WORDS: HIV-1 reverse transcriptase, dNTP binding, photosensitizing modification

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