Journal Article

A Photolabile 2′, 3′-Dideoxyuridylate Analog Bearing an Aryl(trifluoromethyl)diazirine Moiety: Photoaffinity Labeling of HIV-1 Reverse Transcriptase

Toyofumi Yamaguchi and Mineo Saneyoshi

in Nucleic Acids Research

Volume 24, issue 17, pages 3364-3369
Published in print September 1996 | ISSN: 0305-1048
Published online September 1996 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/24.17.3364
A Photolabile 2′, 3′-Dideoxyuridylate Analog Bearing an Aryl(trifluoromethyl)diazirine Moiety: Photoaffinity Labeling of HIV-1 Reverse Transcriptase

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In order to develop a photoaffinity labeling reagent for DNA polymerases, including retroviral reverse transcriptase (RT), we utilized 2′,3′-dideoxy-E-5-[4-(3-trifluoro-methyl-3H-diazirin-3-yl)styryl]UTP (TDSddUTP) as a substrate dTTP analog. Photoaffinity labeling experiments with human immunodeficiency virus type-1 (HIV-1) RT using a radioactive labeling reagent ([γ-32P]TDSddUTP) and poly(A)·oligo(dT) as the template/primer yielded different results depending on the concentration of Mg2+. In the presence of 0.025 mM Mg2+, photoaffinity labeling showed that TDSddUTP bound selectively to the dTTP binding site in the 66 kDa subunit of the p66/p51 heterodimeric enzyme protein when irradiated by near-UV light (365 nm). In the presence of 4 mM Mg2+ or 0.05 mM Mn2+, TDSddUTP was incorporated into the 3′-end of the primer strand due to RT activity and the resulting photolabile primer bound to the 66 kDa subunit of HIV-1 RT on photoirradiation. These results suggest that TDSddUTP could be a useful tool for studying the substrate binding site(s) of DNA polymerases, including HIV-1 RT, which show affinity for this compound.

Journal Article.  3947 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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