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.
Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA. pommiery@box-P.nci.nih.gov
A
A
Mazumder
N
N
Neamati
A A
AA
Pilon
S
S
Sunder
Y
Y
Pommier
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