Abstract
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.
Highlights
From the Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892
We describe a novel assay for the DNA binding activity of HIV-11 integrase using modified DNA oligonucleotides containing amino-reactive abasic sites and sodium borohydride stabilization
Because abasic sites do not significantly perturb the structure of B-DNA [19, 20], we reasoned that introduction of one such lesion into a duplex U5 long terminal repeat (LTR) oligonucleotide may not alter its ability to act as a substrate for human immunodeficiency virus type 1 (HIV-1) integrase
Summary
From the Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892. 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. Two nucleotides are excised from each 3Ј end of the linear, blunt-ended, viral DNA This 3Ј-processing reaction exposes the 3Ј-hydroxyl of a CA dinucleotide which is conserved among all retroviruses. We examined the role of lysine 136 in DNA interactions both proximal and distal to the conserved CA dinucleotide
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