Journal: Nucleic Acids Research

Article Title: G-quadruplex recognition and remodeling by the FANCJ helicase

doi: 10.1093/nar/gkw574

Figure Lengend Snippet: Mechanism of FANCJ-mediated G4-unfolding and refolding. ( A ) Regions of the single-molecule G4-unfolding FRET time-courses for each FANCJ construct containing FRET transitions were globally analyzed by the ebFRET Matlab suite. The resulting transition density plots (TDPs) for each FANCJ construct show four major FRET states with mean FRET values of 0.13 (ssDNA; 1), 0.32 (G2), 0.49 (G3) and 0.64 (G4). Additional, albeit infrequent state (FRET ≈ 0.73) represents a parallel G-quadruplex (P). The scale bar for transition frequency is shown on the right of each respective TDP where the brighter peaks correspond to more frequent transitions. ( B ) Schematic representation of the TDP, observed FRET states and their corresponding DNA conformations, and transitions. ( C ) FANCJ-, bioFANCJ- and FANCJ HD -mediated G4-unfolding and refolding proceeded through the same intermediates and mechanism based on the TDPs. The TDP for the bioFANCJ K141/K142A -mediated process shows a reduction in the G2⇔G4 and G3⇔G4 interconversions.

Article Snippet: The ebFRET Matlab library was used to statistically determine the number of FRET states observed during the course of G4 melting, and the mean lower bound was used to evaluate the number of states that best described each time series.

Techniques: Construct

Journal: bioRxiv

Article Title: Cooperative action of separate interaction domains promotes high-affinity DNA binding of Arabidopsis thaliana ARF transcription factors

doi: 10.1101/2022.11.16.516730

Figure Lengend Snippet: Kinetics of the interaction between AtARF-DBDs and IR7. Kinetic parameters obtained from HMM analysis using ebFRET. The datapoints are marked with Δ, □ and ∇ in order of increasing ARF concentration. The ARF concentrations were 128, 256, 512 nM for AtARF2-DBD and AtARF1-DBD, 8, 16 and 32 nM for AtARF5-DBD, 64, 128 and 256 nM for AtARF5-DBD G279N and 128 and 512 nM for AtARF5-DBD R215A. The error bars represent the standard deviations of the mean values. AtARF2-DBD and AtARF1-DBD behaved similarly while AtARF5-DBD showed increased k on and decreased k off . Consistent with a model in which part of the difference in kinetic can be explained by an increased stability of AtARF5-DBD dimer. A weakening of AtARF5-DBD dimerisation (G279N mutant) leads to kinetic parameters that resemble the ones of AtARF1-DBD and AtARF2-DBD. In addition, AtARF5-DBD R215A mutant in a key amino acid for the interaction with DNA showed a k on reduced by one order of magnitude and a k off increased by almost two orders of magnitude compared to the wild-type.

Article Snippet: To obtain the kinetics of AtARF2/IR7 interaction, we analyzed relevant datapoints of the titrations for the series of variants using ebFRET , a MATLAB suite for Hidden Markov Model (HMM) analysis of single-molecule time traces (see Materials and Methods).

Techniques: Concentration Assay, Mutagenesis

Journal: Cell

Article Title: Cohesin mediates DNA loop extrusion by a “swing and clamp” mechanism

doi: 10.1016/j.cell.2021.09.016

Figure Lengend Snippet:

Article Snippet: Plots were generated by the plotTDP MATLAB script ( http://ebfret.github.io/ ), using a variance of 0.003, and normalized for the total number of frames in each condition (‘normbyframes’).

Techniques: Virus, Recombinant, Lambda DNA Preparation, Avidin-Biotin Assay, Software