Synaptic Complex Formation of Two Retrovirus DNA Attachment Sites by Integrase: A Fluorescence Energy Transfer Study

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Abstract
The integration of retroviral DNA by the viral integrase (IN) into the host genome occurs via assembled preintegration complexes (PIC). We investigated this assembly process using purified IN and viral DNA oligodeoxynucleotide (ODN) substrates (93 bp in length) that were labeled with donor (Cy3) and acceptor fluorophores (Cy5). The fluorophores were attached to the 5‘ 2 bp overhangs of the terminal attachment (att) sites recognized by IN. Addition of IN to the assay mixture containing the fluorophore-labeled ODN resulted in synaptic complex formation at 14 °C with significant fluorescence resonance energy transfer (FRET) occurring between the fluorophores in close juxtaposition (from ∼15 to 100 Å). Subsequent integration assays at 37 °C with the same ODN (32P-labeled) demonstrated a direct association of a significant FRET signal with concerted insertion of the two ODNs into the circular DNA target, here termed full-site integration. FRET measurements (ΔF) show that IN binds to a particular set of 3‘ OH recessed substrates (type I) generating synaptic complexes capable of full-site integration that, as shown previously, exhibit IN mediated protection from DNaseI digestion up to ∼20 bp from the ODN att ends. In contrast, IN also formed complexes with nonspecific DNA ends and loss-of-function att end substrates (type II) that had significantly lower ΔF values and were not capable of full-site integration, and lacked the DNaseI protection properties. The type II category may exemplify what is commonly understood as “nonspecific” binding by IN to DNA ends. Two IN mutants that exhibited little or no integration activity gave rise to the lower ΔF signals. Our FRET analysis provided the first direct physical evidence that IN forms synaptic complexes with two DNA att sites in vitro, yielding a complex that exhibits properties comparable to that of the PIC.

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