A photolabile 2',3'-dideoxyuridylate analog bearing an aryl(trifluoromethyl)diazirine moiety: photoaffinity labeling of HIV-1 reverse transcriptase.
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-trifluoromethyl-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 ([gamma-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.
Yamaguchi
T
T
Department of Biological Sciences, Teikyo University of Science and Technology, Yamanashi, Japan.
Saneyoshi
M
M
D000345:Affinity Labels; D001389:Azirines / Q000378:metabolism* / Q000528:radiation effects; D001665:Binding Sites; D003856:Deoxyuracil Nucleotides / Q000378:metabolism* / Q000528:radiation effects; D054306:Dideoxynucleotides; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D015497:HIV-1 / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism / Q000528:radiation effects; D014466:Ultraviolet Rays
C101663:2',3'-dideoxy-5-(4-(3-trifluoromethyl-3H-diazirin-3-yl)styryl)uridine triphosphate; D000345:Affinity Labels; D001389:Azirines; D003856:Deoxyuracil Nucleotides; D054306:Dideoxynucleotides; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase