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Volume 271, Number 36, Issue of September 6, 1996 pp. 21891-21897
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

dNTP Binding to HIV-1 Reverse Transcriptase and Mammalian DNA Polymerase beta  as Revealed by Affinity Labeling with a Photoreactive dNTP Analog

(Received for publication, May 21, 1996, and in revised form, June 27, 1996)

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

From the § Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1068 and the Dagger  Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, 630090 Novosobirsk, Russia

The dNTP binding pocket of human immunodeficiency virus type 1 reverse transcriptase (RT) and DNA polymerase beta  (beta -pol) were labeled using a photoreactive analog of dCTP, exo-N-[beta -(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 beta -pol, suggesting a significant influence of prior primer-template binding on dNTP binding for RT. Mutagenesis of beta -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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