Sequence-specific actinomycin D binding to single-stranded DNA inhibits HIV reverse transcriptase and other polymerases.
Primer extension assays using recombinant templates constructed to contain all 256 possible base quartets in a minimum length sequence were used to examine binding of the anticancer drug actinomycin D to single-stranded DNA. Single-stranded templates were generated by digestion of linearized plasmid with the double-strand-specific T7 gene 6 exonuclease. Actinomycin D formed high-affinity, kinetically stable complexes that paused primer elongation at specific sites by HIV-1 reverse transcriptase, Sequenase (modified T4 DNA polymerase), the Klenow fragment of Escherichia coli DNA polymerase, and Vent (exo-) DNA polymerase. Pauses occurred most commonly near G+C-rich nucleotide clusters, including GpC steps, the preferred sites of double-stranded DNA binding. Complexes were stable for several minutes at temperatures over 50 degrees C as determined by their abilities to pause Vent polymerase at elevated temperatures. Significant variations were noted in pause patterns of different polymerases, demonstrating differential responses of polymerases to a bound actinomycin. Covalent adducts formed on template DNA by a photoaffinity analog of actinomycin D completely stopped primer extension. These results support the possibility that actinomycin D inhibits transcription elongation by complexing single-stranded DNA in the open transcription complex. Single-stranded DNA binding by actinomycin D or analogs may also provide routes for combating HIV or other viruses which replicate through single-stranded intermediates.
Rill
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RL
Department of Chemistry, Institute of Molecular Biophysics, Florida State University, Tallahassee, 32306-3006, USA.
Hecker
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KH
D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis; D018736:DNA Adducts; D004277:DNA, Single-Stranded / Q000737:chemistry*; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry; D054303:HIV Reverse Transcriptase; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D018736:DNA Adducts; D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D018894:Reverse Transcriptase Inhibitors; C059346:7-azidoactinomycin D; D003609:Dactinomycin; D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase