The SMC5/6 complex exhibits dynamic binding on double-stranded DNA, but binds stably to branched DNA

The Smc5/6 complex is an evolutionarily conserved genome maintenance factor with key roles in stabilizing DNA replication forks and resolving DNA repair intermediates. Understanding its genome protection functions has been hampered by a lack of mechanistic studies on how the Smc5/6 complex associates with different types of DNA. Here we address this gap by simultaneously visualizing the behavior of Smc5/6 on three types of DNA, namely double-stranded (dsDNA), single-stranded (ssDNA), and junction DNA formed by juxtaposed ss- and dsDNA, using correlative single-molecule fluorescence and force microscopy. We find that Smc5/6 displays distinct behaviors toward different types of DNA, dynamically associating with dsDNA while stably binding to junction DNA. Mechanistically, the presence of ATP stimulates increased and longer-lasting binding of Smc5/6 to dsDNA through the topological entrapment of the dsDNA. Remarkably, Smc5/6 efficiently assembles onto free ssDNA emanating from junction DNA regardless of ATP presence, with the ability to co-localize with the strong ssDNA-binding complex SSB. Finally, we show that Smc5/6 protects junction DNA stability by preventing ssDNA re-annealing. These multi-faceted DNA association patterns of Smc5/6 provide a framework for understanding the complex's functions in maintaining genome stability.