1. Volume 3, Issue 6, Pages 1785-1794 (June 2013)
Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1 
Mohamed A. Sobhy, Luay I. Joudeh, Xiaojuan Huang, Masateru Takahashi, Samir M. Hamdan 
Cell Reports 
Volume 3, Issue 6, Pages (June 2013)
DOI: /j.celrep
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2. Cell Reports 2013 3, 1785-1794DOI: (10.1016/j.celrep.2013.05.001)
Copyright © 2013 The Authors Terms and Conditions

3. Figure 1 Dynamics of FEN1 Bending of DF-6,1 by smFRET
(A) A schematic diagram of FEN1 reaction on DF-6,1.
(B) Structure of FEN1 in complex with SF-0,1; Protein Data Bank (PDB) code 3Q8L (Tsutakawa et al., 2011). The conserved and unique structural features that interact with the bent DNA conformation are depicted and labeled in the same color.
(C) smFRET efficiency histograms at different FEN1 concentrations. The fitted Gaussians are illustrated with brown and blue solid lines for bent and unbent substrate populations, respectively. An isotherm showing percentage of bent substrate versus FEN1 concentration; KD-bending is determined using nonlinear least-squares regression fit. Error bars represent SD from two or more experiments.
(D) Representative time traces showing the fluorescence intensities of a donor (green) and acceptor (red) and their FRET efficiency (black) at different FEN1 concentrations. The dwell times for FEN1 association (τbending) and dissociation (τunbending) are illustrated on the upper FRET time trace.
(E) Transition density plot (TDP) of the two FRET states at [FEN1] of 1,000 nM.
(F) Histogram of τbending.
(G) Histograms of τunbending; τbending and τunbending are calculated from fitting their histograms with a single-exponential decay function and their inverse values are the apparent first order rate constant for DNA bending (kbending) and the rate constant for DNA unbending (kunbending), respectively.
(H) The DNA bending association rate constant (kon-bending).
(I) The DNA bending dissociation rate constant (koff-unbending). kon-bending, and koff-unbending are calculated from the slope of the linear fit of mean kbending and kunbending versus concentration, respectively. Error bars correspond to the SE in fitting the dwell time distributions with single-exponential decay function.
See also Figure S1 for SPR binding study, Figure S2 for histograms of kbending and kunbending, and Figure S3 for DF-6,1 bending at low salt concentration.
Cell Reports 2013 3, DOI: ( /j.celrep )
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4. Figure 2 The Effect of 5′ Flap on DF Bending by FEN1
(A–C) The effect of 5′ flap length. Isotherms of bent substrate versus FEN1 concentration are shown for DF-2,1 (A), DF-8,1 (B), and DF-12,1 (C). Error bars represent SD from two or more experiments.
(D) kon-bending and koff-unbending of DF-2,1. Rate constants were calculated as described in Figures 1H and 1I. Error bars correspond to the SE in fitting the dwell time distributions with single-exponential decay function.
(E and F) The donor/acceptor intensities and FRET efficiency time trace at 500 nM FEN1 are shown for DF-8,1 (E) and DF-12,1 (F).
(G) FRET efficiency histograms of DNA bending on 5′ flap-blocked DF. A schematic showing substrate and 5′ flap blockage (1) and FEN1 binding (2).
(H) DNA bending of trapped FEN1 on DF. A schematic showing binding of FEN1 at 2,000 nM (1) and then trapping it by blocking the 5′ flap (2). The FRET efficiency histograms of trapped FEN1 and after extensive washing with protein-free buffer are shown.
(I) A representative donor/acceptor intensities and FRET efficiency time trace of a trapped FEN1.
(J) FRET efficiency histograms of FEN1 bending of a DF containing a gapped 5′ flap hairpin.
(K) FRET efficiency histograms of FEN1 bending of a DF containing a fully duplex 5′ flap hairpin; the bent population is calculated after excluding the contribution from the substrate side peak.
See also Figure S1 for SPR binding study and Figure S2 for histograms of FRET efficiency, kbending, and kunbending.
Cell Reports 2013 3, DOI: ( /j.celrep )
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5. Figure 3 The Effect of 3′ Flap on DF Bending by FEN1
(A) The effect of removing the 3′ flap. A schematic of SF-6,0 is shown, together with FRET efficiency histograms.
(B) The effect of removing the 5′ flap. The SF substrate with the alternative internal labeling scheme is illustrated (ISF-0,1) with FRET efficiency histograms.
(C) The effect of internal labeling of DF-6,1. A schematic of IDF-6,1 and FRET efficiency histograms are shown.
(D) The effect of increasing the 3′ flap length. A schematic of DF-6,2 and FRET efficiency histograms are depicted.
See also Figure S1 for SPR binding study and Figure S4 for SF-0,1 histograms.
Cell Reports 2013 3, DOI: ( /j.celrep )
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6. Figure 4
The Effect of FEN1 Active-Site Metal Ions and Two-Nucleotide Unpairing on Bending of the DF Substrate
(A) Effect of omission of Ca2+ and addition of EDTA. A schematic of the DF-6,1 substrate and FRET efficiency histograms are shown. The fit Gaussians are illustrated with brown, pink, and blue solid lines for bent, partially bent and unbent substrate populations, respectively.
(B) Donor/acceptor intensities and FRET efficiency time traces. The dominant transition state at 1,000 nM is unbent whereas at 2,000 nM it is fully bent.
(C) TDP depicts the three FRET states at [FEN1] of 1,000 nM. The forward and backward transition rates between these states are shown below and are obtained from the single-exponential decay function fit to the respective dwell time distributions.
(D) The effect of bypassing the two-nucleotide unpairing step. A schematic of DF with two nucleotides flanking the unpaired scissile phosphate is shown, together with FRET efficiency histograms.
See also Figure S4.
Cell Reports 2013 3, DOI: ( /j.celrep )
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7. Figure S1 SPR Binding Studies, Related to Figures 1, 2, 3 and 4
Comparison between the binding of FEN1 to ssDNA, dsDNA and DF-6,1 (without fluorophores) in a reaction buffer containing either 25 or 100 mM KCl.
(A) At 100 mM KCl, FEN1 interaction with ssDNA was suppressed, with dsDNA became transient and with DF-6,1 unaffected. Rmax could not be reached for ssDNA and dsDNA due to their weak interaction and for DF-6,1 the stoichiometry was 1:1.
(B) At 25 mM KCl, FEN1 showed less discrimination between ssDNA, dsDNA and DF-6,1. The expected Rmax values for the amount of Rligand were 325, 304 and 295 RU for ssDNA, dsDNA and DF-6,1, respectively. Consequently, we estimated the reached stoichiometry of FEN1 binding to ssDNA, dsDNA and DF-6,1 to be 1:1, 2:1 and 2.5:1, respectively. The FEN1 interactions during the 1:1 regime were much stronger than at 100 mM KCl since less FEN1 was required to saturate the substrate and, at the start of the wash with buffer, 4.5-fold more FEN1 remained bound to DNA and dissociated at a slower rate than at 100 mM.
(C) The sensogram of FEN1 binding to DF-6,1 substrate containing the donor and acceptor fluorophores in reaction buffer containing 100 mM KCl. Similar measured KD values are obtained for both labeled and unlabeled substrates.
(D) Characterization of the binding of the different labeled substrates used in the smFRET experiments. The expected Rmax for 1:1 stoichiometry for DF-hairpinbiotin/SA,1, DF-hairpin,1, DF-hairpinduplex,1, SF-6,0, DF-6,2, SF-0,1, ISF-0,1, DF-12,1, IDF-12,1 and DF-8mismatch,1 were 296, 298, 303, 309, 290, 295, 645, 345, 463 and 305 RU, respectively.
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8. Figure S2
smFRET Measurements of DF Substrates with Variable 5′ Flap Length at Different FEN1 Concentrations, Related to Figures 1 and 2
smFRET efficiency histograms of (A) DF-2,1, (B) DF-8,1 and (C) DF-12,1. The percentage of the bent conformer at the indicated FEN1 concentration is noted on each graph. Histograms for association (bending) and dissociation (unbending) dwell times of (D) DF-6,1 and (E) DF-2,1 substrates at different FEN1 concentrations. The rates were obtained by fitting a single-exponential function to the dwell time distributions. The association rates increased on increasing the concentration of FEN1 while the dissociation rate remained constant. The optimum bin size was calculated based on the available data points followed the Scott’s normal reference rule for calculation of the bin size and implemented in the TDP calculating code and applied uniformly throughout the fittings for the rates. Furthermore, we calculated the mean lifetime for the dwell time obtained at each FEN1 concentration for DF-2,1 and DF-6,1 and they match very well with those obtained from the single-exponential decay fitting for the dwell time histograms.
Cell Reports 2013 3, DOI: ( /j.celrep )
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9. Figure S3
smFRET Measurement at Low Salt Concentration and in the Presence of Mg2+, Related to Figure 1
(A) smFRET efficiency histograms for DF-6,1 at 25 mM KCl. The percentage of bent conformer first increased by increasing FEN1 concentration and was estimated to reach 100% bent population at 600 nM before it started to decrease upon further addition of FEN1. KD-bending was calculated by relying on the bending kinetics from the FEN1 concentration range where stimulation of DNA bending was observed. At 100 mM KCl the deviation from 1:1 stoichiometry has no effect on DNA bending as shown in the case of DF-12,1 (Figures S1D and 2C).
(B) smFRET efficiency histograms of nonhydrolyzable substrate (NHDF-6,1) in presence of Mg2+. The KD-bending is determined from the bent population percentage plot versus FEN1 concentration.
Cell Reports 2013 3, DOI: ( /j.celrep )
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10. Figure S4
Histograms of SF-0,1 Labeled with Standard Scheme and DF-8mismatch,1 in the Absence of Divalent Metal Ions, Related to Figures 3 and 4
(A) The smFRET efficiency histograms of SF-0,1 substrate labeled using the standard scheme, i.e. the donor is placed at the 5′ end. This substrate did not show bending at FEN1 concentrations of 1000 and 2000 nM.
(B) The smFRET efficiency histograms of DF-8mismatch,1 in the absence of divalent metal ion (Ca2+) and presence of EDTA. Unlike the case in presence of metal ions no bending was observed at both 1000 and 2000 nM FEN1 concentrations.
Cell Reports 2013 3, DOI: ( /j.celrep )
Copyright © 2013 The Authors Terms and Conditions