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. 2019 Feb 4;15(2):e1007956. doi: 10.1371/journal.pgen.1007956

Fig 7. Pol V Mut binding to p/t DNA visualized at single-molecule resolution in real-time.

Fig 7

(A) Sketch of smFRET experimental setup. An AF555 donor-labeled p/t DNA linked to streptavidin-biotin is attached to a glass slide surface. AF647 acceptor-labeled pol V Mut is then added, and DNA binding is observed as an increase in acceptor fluorophore emission that counter-correlates with a drop of a donor emission. (B) A representative smFRET trajectory showing multiple binding and unbinding events of ATPγS-activated pol V Mut E38K/ΔC17 (green = donor, red = acceptor, blue = FRET efficiency). ATPγS-activated pol V Mut was added at t = 30 s after the start of image acquisition. Data were collected for up to 3 min, prior to the onset of photobleaching. (C) Histogram representing smFRET efficiencies corresponding to the binding of ATPγS-activated pol V Mut E38K/ΔC17 to AF555-labeled p/t DNA. FRET efficiency is calculated as E = IA / (ID+IA), where IA and ID represent acceptor and donor emission respectively. (D-F) Representative smFRET images are shown along with representative individual FRET trajectories of ATPγS-dependent binding of pol V Mut E38K/ΔC17 to p/t DNA. AF555-labeled p/t DNA is shown as green spots, and unbound AF647-labeled pol V Mut E38K/ΔC17 is shown as red spots. The pol V Mut E38K/ΔC17-p/t DNA binding events are shown as colocalized pol V Mut E38K/ΔC17 and p/t DNA signals (yellow/orange spots). Pol V Mut E38K/ΔC17 (D-E) or ATPγS activated pol V Mut E38K/ΔC17 (F) is added at t = 30 s after the start of image acquisition, followed by addition of ATPγS (t = 60 s, middle panel). Pol V Mut does not bind p/t DNA in the absence of ATPγS (D and S1 Movie). The addition of ATPγS activates pol V Mut E38K/ΔC17, resulting in binding to p/t DNA (E and S2 Movie) and pol V Mut E38K/ΔC17-p/t DNA binding events are indicated by the arrows. If pol V Mut is activated by ATPγS prior to addition to p/t DNA (F and S3 Movie), multiple and rapid p/t DNA binding events occur, indicated by arrows. The images shown in (D-F) are smFRET data integrated over 1 min following pol V Mut addition (left panel) or first binding events (middle and right panels). Scale bar is 150 mm.