Figure 5: Imaging PFV intasome integration events into linear target DNA using smTIRF.
From: Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration
(a) Box plots of the frame-by-frame x- and y-displacement from the mean for (left-to-right): half-site integration example (b); the concerted integration example (c); and two random particles that were stuck to the flow cell surface (surface 1 and surface 2). Box plots show the mean (indentation), median (black line), upper and lower quartile (box ends), and outliers (whiskers). (b) (top) Representative trace of a wild-type PFV intasome (yellow) half-site integration event on DNA (red) at a 500-ms frame rate. (middle) x-position and (bottom) y-position traces over time. Note reduced x-position and increased y-position indicative of a half-site integration event where the Cy3-labelled viral U5 cDNA donor is linked to a target DNA molecule attached at both ends and undergoing Brownian movement (see a). (c) (top) Representative trace of a wild type PFV intasome (yellow) concerted integration event on DNA (red) at a 500-ms frame rate. (middle) x-position and (bottom) y-position traces over time. Note large x- and y-position Brownian movement indicative of a broken DNA attached at one end following a concerted integration event (see a). (d–g) Initial frame-by-frame traces of the four observed concerted integration events. (d) The initial tracking frames for the integration event shown in c. All tracking traces (500 ms frame rate) show an initial sliding diffusion for 1–2 s that was followed by a large movement in the x-direction (97–236 nm); ultimately resulting in a particle that randomly moved in both the x- and y-direction consistent with a broken DNA containing a Cy3 label.
