Fig. 2: Smc5/6 exhibits ATP-dependent binding to dsDNA and ATP-independent binding to junction DNA.
From: Smc5/6’s multifaceted DNA binding capacities stabilize branched DNA structures
a Representative kymograph of a λ DNA tether being stretched from LF to HF in the presence of 20 nM Cy3-Smc5/6 (green) without ATP. b Comparison of Cy3-Smc5/6 fluorescence signals on dsDNA between −ATP and +ATP conditions under LF or HF (normalized by the number of pixels for each region). Bar heights indicate the group mean and error bars represent standard deviation. P values were determined from two-tailed unpaired t-tests (****P < 0.0001). Sample sizes are: dsDNALF − ATP (n = 10); dsDNALF + ATP (n = 10); dsDNAHF −ATP (n = 9); and dsDNAHF + ATP (n = 7), where n indicates the number of regions analyzed. c Quantification of the Smc5/6 fluorescence signals at a junction DNA site (marked by the black arrow in panel a) over time in the absence of ATP. Data points indicate the averaged photon count per frame (n = 10 frames) at the junction DNA site and error bars represent standard deviation. d Representative kymograph of a λ DNA tether being stretched from LF to HF in the presence of 20 nM Cy3-Smc5/6 (green) and 10 nM LD650-RPA (red) without ATP. dsDNA (blue), ssDNA (red), and junction DNA (green) regions of the tether are indicated to the right of the kymograph. e Comparison of the Cy3-Smc5/6 fluorescence signals at junction DNA sites between −ATP and +ATP conditions in the absence or presence of RPA. Bar heights indicate the group mean and error bars represent standard deviation. P values were determined from two-tailed unpaired t-tests (ns, not significant). P = 0.1173 for −RPA condition, and P = 0.0956 for +RPA condition. Samples sizes are: Junction −RPA −ATP (n = 20); Junction −RPA + ATP (n = 15); Junction + RPA −ATP (n = 16); Junction + RPA + ATP (n = 13), where n indicates the number of regions analyzed. Source data for panels b, c and e are provided within the Source Data file.
