Journal: Nucleic Acids Research

Article Title: APE1 coordinates its disordered region and metal cofactors to drive genome surveillance

doi: 10.1093/nar/gkag479

Figure Lengend Snippet: smFRET assay to monitor APE1 binding to AP site on DNA. ( A ) Schematic representation of the smFRET assay. Cy3-labeled APE1 interacts with surface-immobilized DNA containing a Cy5 label near a single AP site. The diagram illustrates the states before binding (left) and after APE1 binding to the AP site (right). DNA molecules were immobilized on a PEG-passivated quartz surface through biotin–neutravidin linkage. ( B, D ) Representative single-molecule fluorescence trajectories showing donor (Cy3) and acceptor (Cy5) fluorescence intensities over time together with the corresponding FRET efficiency traces. Experiments were performed in buffer containing 1 mM EDTA to suppress Mg 2+ -dependent cleavage activity. In panel B, undamaged DNA shows no detectable binding events. In panel D, On AP-DNA, binding events are characterized by simultaneous increases in Cy3 and Cy5 fluorescence intensities (black arrows), indicating formation of the bound state. Periods lacking fluorescence signal correspond to the unbound state (gray arrows). ( C, E ) FRET histograms constructed from the bound-state time points of the trajectories shown in panels B and D. No FRET population is observed for undamaged DNA, whereas AP-DNA exhibits a distinct FRET peak at ~0.39. ( F ) Histograms of the bound (τ on ) and unbound (τ off ) dwell times for APE1 on AP-DNA obtained from single-molecule trajectories. The distributions were fitted with single-exponential decay functions. Error bars represent SEM from three independent experiments. ( G ) Comparison of the apparent dissociation constant ( K d ) for APE1 binding to undamaged DNA and AP-DNA. The K d for undamaged DNA was determined by EMSA ( B), whereas the K d for AP-DNA was calculated from the single-molecule dwell times using the relationship \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${{K}_{\rm d}} = {{k}_{\mathrm{off}}}/{{k}_{\rm on}}$\end{document} . Error bars represent standard error of the mean (SEM). Unless otherwise noted, data were pooled from three independent experiments.

Article Snippet: Subsequently, Qdot fluorescence signal was imaged by NIS-Element software (Nikon) under the illumination of 488-nm laser (200 mW, OBIS, Coherent Laser) with 0.1 s exposure time for 5 min.

Techniques: Smfret Assay, Binding Assay, Labeling, Fluorescence, Activity Assay, Construct, Comparison