Chemical trapping of ternary complexes of human immunodeficiency virus type 1 integrase, divalent metal, and DNA substrates containing an abasic site. Implications for the role of lysine 136 in DNA binding.
We report a novel assay for monitoring the DNA binding of human immunodeficiency virus type 1 (HIV-1) integrase and the effect of cofactors and inhibitors. The assay uses depurinated oligonucleotides that can form a Schiff base between the aldehydic abasic site and a nearby enzyme lysine epsilon-amino group which can subsequently be trapped by reduction with sodium borohydride. Chemically depurinated duplex substrates representing the U5 end of the HIV-1 DNA were initially used. We next substituted an enzymatically generated abasic site for each of 10 nucleotides normally present in a 21-mer duplex oligonucleotide representing the U5 end of the HIV-1 DNA. Using HIV-1, HIV-2, or simian immunodeficiency virus integrases, the amount of covalent enzyme-DNA complex trapped decreased as the abasic site was moved away from the conserved CA dinucleotide. The enzyme-DNA complexes formed in the presence of manganese were not reversed by subsequent addition of EDTA, indicating that the divalent metal required for integrase catalysis is tightly bound in a ternary enzyme-metal-DNA complex. Both the N- and C-terminal domains of integrase contributed to efficient DNA binding, and mutation of Lys-136 significantly reduced Schiff base formation, implicating this residue in viral DNA binding.
Mazumder
A
A
Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA. pommiery@box-P.nci.nih.gov
Neamati
N
N
Pilon
A A
AA
Sunder
S
S
Pommier
Y
Y
D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D019427:HIV Integrase / Q000302:isolation & purification / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012545:Schiff Bases; D017384:Sequence Deletion; D013379:Substrate Specificity
D002413:Cations, Divalent; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012545:Schiff Bases; D004247:DNA; D019427:HIV Integrase; D008239:Lysine