Investigation by two-photon fluorescence correlation spectroscopy of the interaction of the nucleocapsid protein of HIV-1 with hairpin loop DNA sequences

Yves Mely; Joel Azoulay; Herve Beltz; Jean-Pierre Clamme; Serena Bernacchi; Damien Ficheux; Bernard Pierre Roques; Jean-Luc Darlix

doi:10.1117/12.545727

8 September 2004 Investigation by two-photon fluorescence correlation spectroscopy of the interaction of the nucleocapsid protein of HIV-1 with hairpin loop DNA sequences

Yves Mely, Joel Azoulay, Herve Beltz, Jean-Pierre Clamme, Serena Bernacchi, Damien Ficheux, Bernard Pierre Roques, Jean-Luc Darlix

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Proceedings Volume 5463, Femtosecond Laser Applications in Biology; (2004) https://doi.org/10.1117/12.545727
Event: Photonics Europe, 2004, Strasbourg, France

Abstract

The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two strand transfer reactions required during reverse transcription. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response element, TAR sequence, at the 3' end of the genomic RNA and the complementary sequence cTAR at the 3’ terminus of the early product of reverse transcription. To characterize NCp7-mediated nucleic acid destabilization, we investigated by steady-state and time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly-labelled cTAR sequence with NCp7. The conformational fluctuations observed in the absence of NCp7 were associated with the rapid opening and closing (fraying) of the double stranded terminal segment of cTAR. NCp7 destabilizes cTAR mainly through a large increase of the opening rate constant. Additionally, the various destabilizing structures (bulges, internal loop, mismatches) spread all over cTAR secondary structure were found to be critical for NCp7 chaperone activity. Taken together, our data enabled us to propose a molecular mechanism for the destabilizing activity of NCp7 on cTAR which is crucial for the formation of the cTAR-TAR complex during the first strand transfer reaction.

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Yves Mely, Joel Azoulay, Herve Beltz, Jean-Pierre Clamme, Serena Bernacchi, Damien Ficheux, Bernard Pierre Roques, and Jean-Luc Darlix "Investigation by two-photon fluorescence correlation spectroscopy of the interaction of the nucleocapsid protein of HIV-1 with hairpin loop DNA sequences", Proc. SPIE 5463, Femtosecond Laser Applications in Biology, (8 September 2004); https://doi.org/10.1117/12.545727

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KEYWORDS

Proteins

Luminescence

Fluorescence correlation spectroscopy

Molecules

Diffusion

Molecular spectroscopy

Fluorescence resonance energy transfer

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