Fig. 1: Smc5/6 exhibits distinct binding behaviors on three types of DNA.
From: Smc5/6’s multifaceted DNA binding capacities stabilize branched DNA structures
a Architecture of the S. cerevisiae Smc5/6 complex, which is composed of a Smc5-Smc6 heterodimer and six other subunits (Nse1-6). Orange symbols indicate regions of Smc5/6 known to bind dsDNA. The red symbol indicates a region known to bind ssDNA. b Schematic of the single-molecule experimental setup under a low-force (LF) or high-force (HF) regime. Multiple forms of DNA co-existed on the same tether in the HF regime due to force-induced DNA peeling. The DNA binding behavior of fluorescently labelled proteins was monitored by confocal microscopy. c Representative kymograph of a λ DNA tether being stretched from LF to HF in the presence of 20 nM Cy3-Smc5/6 (green) and 2 mM ATP. Red arrows indicate twin-streaks emerging from an internal DNA nick, and blue arrows indicate single streaks emerging from untethered DNA termini. d Quantification of the Smc5/6 fluorescence signals at a junction DNA site (marked by the black arrow in panel c) over time. Data points indicate the averaged photon count per frame (n = 10 frames) at the junction DNA site and error bars represent standard deviation. e Representative kymograph of a DNA tether being stretched from LF to HF in 20 nM Cy3-Smc5/6 (green), 10 nM LD650-RPA (red), and 2 mM ATP. Smc5/6 and RPA were illuminated by two excitation lasers (532 nm and 638 nm, respectively). The red laser was turned off momentarily to confirm signals from Smc5/6. dsDNA (blue), ssDNA (red), and junction DNA (green) regions of the tether are indicated to the right of the kymograph. f Smc5/6 fluorescence signals on different types of DNA under HF. Bar heights indicate the group mean and error bars represent standard deviation. P values were determined from two-tailed unpaired t-tests with Welch’s correction (****P < 0.0001). Sample sizes are: dsDNA −RPA (n = 7); ssDNA −RPA (n = 10); Junction −RPA (n = 15); dsDNA + RPA (n = 7); ssDNA + RPA (n = 8); and Junction + RPA (n = 13), where n indicates the number of regions analyzed. Source data for panels d and f are provided within the Source Data file.
