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Interplay Between DNA-Binding/Catalytic Functions and Oligomerization of Retroviral Integrases Studied by a Combination of Time-Resolved Fluorescence Anisotropy, Fluorescence Correlation Spectroscopy and Resonance Energy Transfer

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Reviews in Fluorescence 2015

Part of the book series: Reviews in Fluorescence ((RFLU,volume 8))

Abstract

Fluorescence based technologies are widely used to study enzyme properties and to understand their mode of action. In particular, fluorescence anisotropy (including both steady-state and time-resolved approaches), fluorescence correlation spectroscopy (FCS) and resonance energy transfer (FRET) were used for monitoring the DNA-binding and enzymatic activities of retroviral integrases. This protein is involved in the integration step of the viral genome that is crucial for retroviral replication. Thanks to these methodologies, important information has been obtained regarding the oligomeric status responsible for integrase catalytic activities as well as the positioning of integrase onto its DNA substrate. We present in this chapter a steady-state fluorescent anisotropy-based assay which monitors, in the same sample, both the DNA-binding step and the subsequent reaction catalysed by integrase allowing the separation of binding and catalytic parameters. The combination of this approach with the analysis of time-resolved fluorescence anisotropy decays is also particularly suitable for studying the relationship between the overall size of integrase-DNA complexes and the catalytic activity. The characterization of catalytically competent complexes by time-resolved fluorescence anisotropy led to the identification of the dimeric form of HIV-1 integrase as the optimal active form for catalytic activity. The protein:DNA ratio determines the aggregative properties of HIV-1 integrase, and high ratios led to aggregative and inactive forms. The better solubility of prototype foamy virus (PFV-1) integrase compared to HIV-1 integrase was compatible with protein labeling using an extrinsic fluorophore, allowing the study of the integrase/DNA interaction specificity by FRET between the fluorescently-labeled PFV-1 integrase and viral DNA ends. It has also been possible to investigate, by a combined approach of FCS and time-resolved fluorescence anisotropy, the oligomeric state of PFV-1 integrase. The results show a monomer-dimer equilibrium of PFV-1 integrase in solution and that DNA promotes protein dimerization although high protein:DNA ratios led to aggregated complexes, confirming results obtained with HIV-1 integrase. The use of fluorescence-based assays for the study of the mechanism of action of anti-integrase compounds is also discussed.

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  1. LBPA (Laboratoire de Biologie et Pharmacologie Appliquée), CNRS UMR8113, IDA FR3242, ENS Cachan, Université Paris-Saclay, 61 avenue President Wilson, F-94235, Cachan, France

    Olivier Delelis & Eric Deprez

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  1. Olivier Delelis
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  2. Eric Deprez
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Correspondence to Eric Deprez .

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  1. Institute of Fluorescence, University of Maryland Baltimore County Institute of Fluorescence, BALTIMORE, Maryland, USA

    Chris D. Geddes

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Delelis, O., Deprez, E. (2016). Interplay Between DNA-Binding/Catalytic Functions and Oligomerization of Retroviral Integrases Studied by a Combination of Time-Resolved Fluorescence Anisotropy, Fluorescence Correlation Spectroscopy and Resonance Energy Transfer. In: Geddes, C. (eds) Reviews in Fluorescence 2015. Reviews in Fluorescence, vol 8. Springer, Cham. https://doi.org/10.1007/978-3-319-24609-3_12

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