Nucleotide binding by the HIV-1 integrase protein in vitro.
Recombinant human immunodeficiency virus type 1 (HIV-1) integrase was shown to bind ATP and other nucleoside triphosphates and nucleotide analogs in vitro. Cross-linking of ATP and the photoaffinity analog 8-azido-ATP to integrase occurred in a UV dose-dependent manner. Covalent binding of ATP to integrase was also achieved without UV irradiation when the nucleotide was oxidized to the 2',3'-dialdehyde derivative (oxidized ATP) prior to incubation with the protein, indicating the presence of a reactive lysine residue in the nucleotide binding region of the protein. A number of experimental observations indicate that nucleotides and DNA substrates bind at the same or overlapping site(s) on the integrase protein. For example, the binding of nucleotides or nucleotide analogs to integrase was blocked by prior incubation with DNA substrates, and the covalent cross-linking of 8-azido-ATP to integrase inhibited the DNA binding and oligonucleotide cleavage activities of the protein. Oxidized ATP inhibited the oligonucleotide cleavage activity of integrase at concentrations that had no effect on DNA binding, suggesting that oxidized nucleotides may specifically target the catalytic center of the enzyme. These studies indicate that nucleotide analogs may serve as probes for the DNA binding and catalytic sites of the enzyme and may serve as models for the design of active site inhibitors of retroviral integrase.
Dana-Farber Cancer Institute, Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115.
J R
JR
Lipford
S T
ST
Worland
C M
CM
Farnet
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000528:radiation effects; D001386:Azides / Q000378:metabolism / Q000528:radiation effects; D001483:Base Sequence; D001665:Binding Sites / Q000528:radiation effects; D003432:Cross-Linking Reagents; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D015342:DNA Probes / Q000737:chemistry; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D019426:Integrases; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D009841:Oligonucleotides / Q000378:metabolism; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D012545:Schiff Bases / Q000737:chemistry; D014466:Ultraviolet Rays
D001386:Azides; D003432:Cross-Linking Reagents; D015342:DNA Probes; D004251:DNA Transposable Elements; D004279:DNA, Viral; D009711:Nucleotides; D009841:Oligonucleotides; D011994:Recombinant Proteins; D012545:Schiff Bases; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D004254:DNA Nucleotidyltransferases; D019426:Integrases