Cryo-EM structure of DNA-bound Smc5/6 reveals DNA clamping enabled by multi-subunit conformational changes

Structural maintenance of chromosomes (SMC) complexes are essential for chromatin organization and functions throughout the cell cycle. The cohesin and condensin SMCs fold and tether DNA, while Smc5/6 directly promotes DNA replication and repair. The functions of SMCs rely on their abilities to engage DNA, but how Smc5/6 binds and translocates on DNA remains largely unknown. Here, we present a 3.8 Å cryogenic electron microscopy (cryo-EM) structure of DNA-bound Saccharomyces cerevisiae Smc5/6 complex containing five of its core subunits, including Smc5, Smc6, and the Nse1-3-4 subcomplex. Intricate interactions among these subunits support the formation of a clamp that encircles the DNA double helix. The positively charged inner surface of the clamp contacts DNA in a nonsequence-specific manner involving numerous DNA binding residues from four subunits. The DNA duplex is held up by Smc5 and 6 head regions and positioned between their coiled-coil arm regions, reflecting an engaged-head and open-arm configuration. The Nse3 subunit secures the DNA from above, while the hook-shaped Nse4 kleisin forms a scaffold connecting DNA and all other subunits. The Smc5/6 DNA clamp shares similarities with DNA-clamps formed by other SMCs but also exhibits differences that reflect its unique functions. Mapping cross-linking mass spectrometry data derived from DNA-free Smc5/6 to the DNA-bound Smc5/6 structure identifies multi-subunit conformational changes that enable DNA capture. Finally, mutational data from cells reveal distinct DNA binding contributions from each subunit to Smc5/6 chromatin association and cell fitness. In summary, our integrative study illuminates how a unique SMC complex engages DNA in supporting genome regulation.

Structural maintenance of chromosomes (SMC) complexes are essential genome regulators in both prokaryotes and eukaryotes. In eukaryotes, the cohesin and condensin SMC complexes organize DNA, while the Smc5/6 complex (referred to as Smc5/6) directly regulates DNA replication and repair (1). At the structural level, SMC complexes share similarities while possessing unique attributes (1). Each complex contains a pair of SMC subunits and a set of non-SMC subunits. The SMC subunits define the tripartite filamentous architecture of the complex: their approximal 50-nm long coiled coil arm region connects their dimerized hinge and adenosine triphosphatase (ATPase) head regions (1). A non-SMC kleisin subunit uses its N- and C-terminal domains to link the head of one SMC to the head-proximal arm region (neck) of another SMC, forming a trimeric SMC-kleisin structure. In cohesin and condensin, two large U-shaped HEAT (Huntington, elongation factor 3, PR65/A, TOR) repeat HAWK (HEAT proteins associated with kleisins) subunits attach to the middle region of the kleisin. By contrast, the Smc5/6 kleisin (Nse4) binds to smaller WH (winged helix)-containing KITE (kleisin interacting tandem WH elements) subunits (Nse1 and Nse3) (2).SMC-mediated functions depend on interactions with DNA. Recent cryogenic electron microscopy (cryo-EM) structures of DNA-bound cohesin and condensin revealed that their HAWK subunits and the SMC head-neck regions form a clamp to enclose a single DNA double helix (3–7). DNA clamping can be critical for cohesin and condensin to extrude DNA loops for chromatin folding (5, 7–9). DNA loop extrusion additionally requires arm bending at a region called the elbow, which is found in both cohesin and condensin (5, 7–9). By contrast, a lack of arm bending in Smc5/6 was suggested by negative stain EM and cross-linking mass spectrometry (CLMS) data (10–14). Since Smc5/6 does not contain HAWK proteins nor shows arm-bending, it has remained unclear how Smc5/6 engages DNA to accomplish its multiple functions.Here we address the molecular mechanisms by which this unique SMC complex binds DNA using an integrative approach, coupling a cryo-EM-based structural characterization with CLMS analyses and functional investigation. Our atomic structure of a DNA-bound Saccharomyces cerevisiae Smc5/6 complex reveals that the head-neck Smc5-6 regions and the Nse1-3-4 subcomplex together form a clamp entrapping the DNA helix. The structure further reveals protein subunit folds and association, as well as how the subunits collaborate to entrap DNA. Comparison of CLMS analyses of DNA-free Smc5/6 with the structure of the DNA-bound Smc5/6 unveils large scale, multi-subunit conformational changes that enable Smc5/6 to encircle DNA. Finally, our mutational data suggest distinct contributions from each of the DNA binding regions to Smc5/6 chromatin association and cellular fitness. Comparison of our findings with those of other SMCs reveals that diverse SMC complexes use a similar DNA clamping strategy despite structural differences, and that Smc5/6 possesses unique features distinct from cohesin, condensin, and prokaryotic SMCs. Our work lays the foundation for an in-depth understanding of how Smc5/6 fulfills unique roles in genome protection.