Fig. 6: PCNA constrains 1D sliding and junction binding of Smc5/6.

a Representative kymograph of A555-Smc5/6 (green) and LD655-PCNA (red) on a gapped substrate in the presence of RFC, RPA, and ATP. b Examples of categories of Smc5/6 and PCNA behaviors upon their encounter on dsDNA. (Top) The two complexes exhibited transient colocalization (arrowheads) followed by a recoil upon encounter. Arrow indicates the position of DNA junction. (Middle) The two stably colocalized and co-diffused on dsDNA. (Bottom) They bypassed each other. Arrowhead indicates a bypass event. c Plot showing the fraction of events (N = 1640) for each of outcomes of Smc5/6 and PCNA encounter on dsDNA as described in panel b. Bar heights indicate means; error bars represent SEM (N = 12 kymographs). Statistical analyses are presented as in Fig. 1d. d Plot showing the numbers of Smc5/6 binding events at the 3’ junctions per 100 s. The gapped substrate was incubated with LD655-PCNA, RFC, RPA, and ATP before transitioning to channel containing A555-Smc5/6 8-mer, RPA, and ATP. For “PCNA-” condition, the first incubation was omitted. Sample sizes are: PCNA- (N = 37), PCNA+ (N = 9). e Plot showing dwell time distributions for Smc5/6 at 3’ junctions under the condition where PCNA was omitted or included. Sample sizes are: PCNA- (N = 301), PCNA+ (N = 173). d, e Plots and statistical analyses are presented as in Fig. 1d. f A working model for Smc5/6 binding to junction DNA and its regulation by RPA and PCNA. Our findings suggest that Smc5/6 can arrive at junction sites via 1D diffusion or direct binding. Smc5/6 can then engage with 3’ or 5’ junction sites, with a preference for 3’ junctions when RPA coats ssDNA. RPA’s DNA binding domain (DBD)-A/B and DBD-C/D/E are shown as small and large ovals and bind to 3’ and 5’ of ssDNA, respectively. PCNA (ring) reduces Smc5/6 junction encounter frequency and dwell times. The Nse5-6 subcomplex reduces Smc5/6 junction encounter frequency but increases junction dwell times. Frequent junction encounter requires Smc6 DNA binding residues (star) and Nse1-3-4. Additionally, Nse1-3-4 increases junction dwell times.