Smc5/6 uses its head-NSE module to preferentially associate with ssDNA gaps and ss-dsDNA junctions.

Structural Maintenance of Chromosomes (SMC) complexes play critical roles in genome maintenance. While canonical SMCs, such as cohesin and condensin, organize the genome into 3D structures, Smc5/6 directly promotes genome replication and repair. Insights into Smc5/6 function have emerged from recent fluorescence-based single-molecule studies showing its binding to structures resembling replication and repair intermediates. To provide a higher-resolution view of this facet of Smc5/6's function, we employed atomic force microscopy (AFM) to visualize the budding yeast Smc5/6 at nanometer-scale resolution and investigate how it interacts with short ssDNA gaps that frequently form during genome maintenance. We report that the free or DNA-bound Smc5/6 adopts a 'bead-on-stick' shape, with its head-NSE module separated from its hinge domain by a non-bending arm region. Smc5/6 predominantly uses its head-NSE module to engage with dsDNA, and mutating DNA-binding residues within this structural module shifts the complex toward a hinge-mediated DNA-binding mode. Importantly, Smc5/6 shows an intrinsic association preference for ssDNA gap regions and ss-dsDNA junction sites over the flanking dsDNA regions, predominantly through the head-NSE module. AFM visualization of Smc5/6 and its structural elements in preferential gap and junction DNA association provides a framework for our continued understanding of Smc5/6's roles in genome maintenance.