BIOMAT Database Logo BIOMAT Database Logo

dNTP binding to HIV-1 reverse transcriptase and mammalian DNA polymerase beta as revealed by affinity labeling with a photoreactive dNTP analog. 1996

O I Lavrik, and R Prasad, and W A Beard, and I V Safronov, and M I Dobrikov, and D K Srivastava, and G V Shishkin, and T G Wood, and S H Wilson
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.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D003843 Deoxycytidine Monophosphate Deoxycytidine (dihydrogen phosphate). A deoxycytosine nucleotide containing one phosphate group esterified to the deoxyribose moiety in the 2'-,3'- or 5- positions. DCMP,Deoxycytidylic Acid,Deoxycytidylic Acids,Acid, Deoxycytidylic,Acids, Deoxycytidylic,Monophosphate, Deoxycytidine
D003845 Deoxycytosine Nucleotides Cytosine nucleotides which contain deoxyribose as the sugar moiety. Deoxycytidine Phosphates,Nucleotides, Deoxycytosine,Phosphates, Deoxycytidine
D004256 DNA Polymerase I A DNA-dependent DNA polymerase characterized in prokaryotes and may be present in higher organisms. It has both 3'-5' and 5'-3' exonuclease activity, but cannot use native double-stranded DNA as template-primer. It is not inhibited by sulfhydryl reagents and is active in both DNA synthesis and repair. DNA Polymerase alpha,DNA-Dependent DNA Polymerase I,Klenow Fragment,DNA Pol I,DNA Dependent DNA Polymerase I,Polymerase alpha, DNA
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000345 Affinity Labels Analogs of those substrates or compounds which bind naturally at the active sites of proteins, enzymes, antibodies, steroids, or physiological receptors. These analogs form a stable covalent bond at the binding site, thereby acting as inhibitors of the proteins or steroids. Affinity Labeling Reagents,Labeling Reagents, Affinity,Labels, Affinity,Reagents, Affinity Labeling
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D001665 Binding Sites The parts of a macromolecule that directly participate in its specific combination with another molecule. Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D012194 RNA-Directed DNA Polymerase An enzyme that synthesizes DNA on an RNA template. It is encoded by the pol gene of retroviruses and by certain retrovirus-like elements. EC 2.7.7.49. DNA Polymerase, RNA-Directed,RNA-Dependent DNA Polymerase,Reverse Transcriptase,RNA Transcriptase,Revertase,DNA Polymerase, RNA Directed,DNA Polymerase, RNA-Dependent,RNA Dependent DNA Polymerase,RNA Directed DNA Polymerase

Related Publications

O I Lavrik, and R Prasad, and W A Beard, and I V Safronov, and M I Dobrikov, and D K Srivastava, and G V Shishkin, and T G Wood, and S H Wilson
Investigation of the dNTP-binding site of HIV-1 reverse transcriptase using photoreactive analogs of dNTP.
September 2001, Biochemistry. Biokhimiia,
O I Lavrik, and R Prasad, and W A Beard, and I V Safronov, and M I Dobrikov, and D K Srivastava, and G V Shishkin, and T G Wood, and S H Wilson
Affinity labeling and functional analysis of the primer binding domain of HIV-1 reverse transcriptase.
April 1993, Biochemistry,
O I Lavrik, and R Prasad, and W A Beard, and I V Safronov, and M I Dobrikov, and D K Srivastava, and G V Shishkin, and T G Wood, and S H Wilson
Exploring the dNTP -binding site of HIV-1 reverse transcriptase for inhibitor design.
December 2021, European journal of medicinal chemistry,
O I Lavrik, and R Prasad, and W A Beard, and I V Safronov, and M I Dobrikov, and D K Srivastava, and G V Shishkin, and T G Wood, and S H Wilson
HIV-1 Reverse Transcriptase-Based Assay to Determine Cellular dNTP Concentrations.
January 2016, Methods in molecular biology (Clifton, N.J.),
O I Lavrik, and R Prasad, and W A Beard, and I V Safronov, and M I Dobrikov, and D K Srivastava, and G V Shishkin, and T G Wood, and S H Wilson
Integrative structural biology studies of HIV-1 reverse transcriptase binding to a high-affinity DNA aptamer.
January 2020, Current research in structural biology,
O I Lavrik, and R Prasad, and W A Beard, and I V Safronov, and M I Dobrikov, and D K Srivastava, and G V Shishkin, and T G Wood, and S H Wilson
Glutamine 151 participates in the substrate dNTP binding function of HIV-1 reverse transcriptase.
May 1995, Biochemistry,
O I Lavrik, and R Prasad, and W A Beard, and I V Safronov, and M I Dobrikov, and D K Srivastava, and G V Shishkin, and T G Wood, and S H Wilson
Reduced dNTP binding affinity of 3TC-resistant M184I HIV-1 reverse transcriptase variants responsible for viral infection failure in macrophage.
April 2008, The Journal of biological chemistry,
O I Lavrik, and R Prasad, and W A Beard, and I V Safronov, and M I Dobrikov, and D K Srivastava, and G V Shishkin, and T G Wood, and S H Wilson
Affinity modification of human immunodeficiency virus reverse transcriptase and DNA template by photoreactive dCTP analogs.
November 1994, FEBS letters,
O I Lavrik, and R Prasad, and W A Beard, and I V Safronov, and M I Dobrikov, and D K Srivastava, and G V Shishkin, and T G Wood, and S H Wilson
dNTP binding site in rat DNA polymerase beta revealed by controlled proteolysis and azido photoprobe cross-linking.
March 1996, Biochemistry,
O I Lavrik, and R Prasad, and W A Beard, and I V Safronov, and M I Dobrikov, and D K Srivastava, and G V Shishkin, and T G Wood, and S H Wilson
Increased dNTP binding affinity reveals a nonprocessive role for Escherichia coli beta clamp with DNA polymerase IV.
August 2004, The Journal of biological chemistry,
Copied contents to your clipboard!