Journal: Methods in Molecular Biology

Article Title: DNA-Protein Interactions

doi: 10.1007/978-1-4939-2877-4

Figure Lengend Snippet: Fig. 3 Example of an enzymatic DNA cleavage assay monitored by time-resolved fluorescence resonance energy transfer. (a) A 3′-flap duplex DNA is labelled with donor (fluorescein) and acceptor (Cy3) groups. In the presence of magnesium divalent ions, binding of structure-specific endonucleases such as XPF (xeroderma pigmentosum, complementation group F) induces cleavage of the 3′-flap with the subsequent release of the FRET acceptor and FRET breakdown (Penedo et al., unpublished data). (b) Monitoring the nuclease-induced cleavage of the DNA construct shown in (a) by time-resolved fluorescence spectroscopy. As the cleavage progresses, the biexponential decay together with time-dependent contributions from both product (donor only, τD = 3.8 ns) and uncleaved substrate (donor + acceptor, τFRET = 0.8 ns) evolves to a monoexponential donor-only decay. Each fluorescence decay trace was taken at fixed intervals of time (0.5 s) during the pro- gression of the cleavage reaction. The excitation was performed by a 475 nm picosecond pulsed-diode laser (Edinburgh Instruments Ltd, UK) and the emission monocromator was placed at 520 nm, the donor emission maximum. Each fluorescence trace represents the time-correlated single photon-counting histogram accu- mulated during 0.5 s. The instrument-response function (IRF) used to deconvolute the experimental decays is also shown. (c) Comparison between the lifetime decays of the donor only control and the doubly labeled DNA construct after 12-min progress of the cleavage reaction. The lifetime traces have been fitted to a mono- exponential decay in the case of the donor only DNA control and a biexponential decay following the expres- sion: F(t) = Fo(t) + A1exp(−t/τ1) + A2exp(−t/τ2) for the doubly labeled substrate. The pre-exponential factors A1 and A2, representing the amplitudes of each exponential at time zero, are proportional to the amounts of product and uncleaved substrate. As an example at 12-min reaction time, the remaining uncut substrate constitutes ~33 % of the total intensity. Inset: Residuals obtained from the fitting of the experimental decays to a monoexponential function (a, donor-only control) and a biexponential function (b, doubly labeled substrate). (d) Plot showing the average lifetime calculated according to Eq. 9 as a function of cleavage time. Inset: Percentual amplitudes corresponding to product and uncut substrate as a function of cleavage time. The complete dataset of decay traces obtained every 0.5 s was fitted to biexponential decay following global analysis (Edinburgh Instruments Ltd, UK)

Article Snippet: The complete dataset of decay traces obtained every 0.5 s was fitted to biexponential decay following global analysis (Edinburgh Instruments Ltd, UK) Functional Studies of DNA-Protein Interactions Using FRET Techniques 122 More recently, FRET experiments at single-molecule level (Sm-FRET) have become possible providing information on protein- nucleic acid dynamics that was previously hidden when using bulk-solution methods [38–41].

Techniques: DNA Cleavage Assay, Fluorescence, Förster Resonance Energy Transfer, Binding Assay, Construct, Spectroscopy, Comparison, Control, Labeling