dNTP binding to HIV-1 reverse transcriptase and mammalian DNA polymerase β as revealed by affinity labeling with a photoreactive dNTP analog

Olga I. Lavrik, Rajendra Prasad, William A. Beard, Igor V. Safronov, Mikhail I. Dobrikov, Deepak K. Srivastava, Gennadii V. Shishkin, Thomas Wood, Samuel H. Wilson

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Abstract

The dNTP binding pocket of human immunodeficiency virus type i reverse transcriptase (RT) and DNA polymerase β (β-pol) were labeled using a photoreactive analog of dCTP, exo-N-[β-(p-azidotetrafluorobenzamido)- ethyl]-deoxycytidine-5'-triphosphate (FABdCTP). Two approaches of photolabeling were utilized. In one approach, photoreactive FABdCTP and radiolabeled primer-template were UV-irradiated in the presence of each enzyme and resulted in polymerase radiolabeling. In an alternate approach, FABdCTP was first UV-cross-linked to enzyme; subsequently, radiolabeled primer-template was added, and the enzyme-linked dCTP analog was incorporated onto the 3'-end of the radiolabeled primer. The results showed strong labeling of the p66 subunit of RT, with only minor labeling of p51. No difference in the intensity of cross-linking was observed with either approach. FABdCTP cross-linking was increased in the presence of a dideoxyterminated primer-template with RT, but not with β-pol, suggesting a significant influence of prior primer-template binding on dNTP binding for RT. Mutagenesis of β-pol residues observed to interact with the incoming dNTP in the crystal structure of the ternary complex resulted in labeling consistent with kinetic characterization of these mutants and indicated specific labeling of the dNTP binding pocket.

Original languageEnglish (US)
Pages (from-to)21891-21897
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number36
DOIs
StatePublished - 1996

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RNA-Directed DNA Polymerase
DNA-Directed DNA Polymerase
Labeling
Enzymes
HIV Reverse Transcriptase
Deoxycytidine
Mutagenesis
Viruses
Crystal structure
Kinetics
exo-N-(2-(4-azidotetrafluorobenzamido)ethyl)-deoxycytidine-5'-triphosphate
Human immunodeficiency virus 1 reverse transcriptase
2'-deoxycytidine 5'-triphosphate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Lavrik, O. I., Prasad, R., Beard, W. A., Safronov, I. V., Dobrikov, M. I., Srivastava, D. K., ... Wilson, S. H. (1996). dNTP binding to HIV-1 reverse transcriptase and mammalian DNA polymerase β as revealed by affinity labeling with a photoreactive dNTP analog. Journal of Biological Chemistry, 271(36), 21891-21897. https://doi.org/10.1074/jbc.271.36.21891

dNTP binding to HIV-1 reverse transcriptase and mammalian DNA polymerase β as revealed by affinity labeling with a photoreactive dNTP analog. / Lavrik, Olga I.; Prasad, Rajendra; Beard, William A.; Safronov, Igor V.; Dobrikov, Mikhail I.; Srivastava, Deepak K.; Shishkin, Gennadii V.; Wood, Thomas; Wilson, Samuel H.

In: Journal of Biological Chemistry, Vol. 271, No. 36, 1996, p. 21891-21897.

Research output: Contribution to journalArticle

Lavrik, OI, Prasad, R, Beard, WA, Safronov, IV, Dobrikov, MI, Srivastava, DK, Shishkin, GV, Wood, T & Wilson, SH 1996, 'dNTP binding to HIV-1 reverse transcriptase and mammalian DNA polymerase β as revealed by affinity labeling with a photoreactive dNTP analog', Journal of Biological Chemistry, vol. 271, no. 36, pp. 21891-21897. https://doi.org/10.1074/jbc.271.36.21891
Lavrik, Olga I. ; Prasad, Rajendra ; Beard, William A. ; Safronov, Igor V. ; Dobrikov, Mikhail I. ; Srivastava, Deepak K. ; Shishkin, Gennadii V. ; Wood, Thomas ; Wilson, Samuel H. / dNTP binding to HIV-1 reverse transcriptase and mammalian DNA polymerase β as revealed by affinity labeling with a photoreactive dNTP analog. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 36. pp. 21891-21897.
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abstract = "The dNTP binding pocket of human immunodeficiency virus type i reverse transcriptase (RT) and DNA polymerase β (β-pol) were labeled using a photoreactive analog of dCTP, exo-N-[β-(p-azidotetrafluorobenzamido)- ethyl]-deoxycytidine-5'-triphosphate (FABdCTP). Two approaches of photolabeling were utilized. In one approach, photoreactive FABdCTP and radiolabeled primer-template were UV-irradiated in the presence of each enzyme and resulted in polymerase radiolabeling. In an alternate approach, FABdCTP was first UV-cross-linked to enzyme; subsequently, radiolabeled primer-template was added, and the enzyme-linked dCTP analog was incorporated onto the 3'-end of the radiolabeled primer. The results showed strong labeling of the p66 subunit of RT, with only minor labeling of p51. No difference in the intensity of cross-linking was observed with either approach. FABdCTP cross-linking was increased in the presence of a dideoxyterminated primer-template with RT, but not with β-pol, suggesting a significant influence of prior primer-template binding on dNTP binding for RT. Mutagenesis of β-pol residues observed to interact with the incoming dNTP in the crystal structure of the ternary complex resulted in labeling consistent with kinetic characterization of these mutants and indicated specific labeling of the dNTP binding pocket.",
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AU - Prasad, Rajendra

AU - Beard, William A.

AU - Safronov, Igor V.

AU - Dobrikov, Mikhail I.

AU - Srivastava, Deepak K.

AU - Shishkin, Gennadii V.

AU - Wood, Thomas

AU - Wilson, Samuel H.

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