Conformational dynamics of cohesin/Scc2 loading complex are regulated by Smc3 acetylation and ATP binding.

The ring-shaped cohesin complex is a key player in sister chromatid cohesion, DNA repair, and gene transcription. The loading of cohesin to chromosomes requires the loader Scc2 and is regulated by ATP. This process is hindered by Smc3 acetylation. However, the molecular mechanism underlying this inhibition remains mysterious. Here, using Saccharomyces cerevisiae as a model system, we identify a novel configuration of Scc2 with pre-engaged cohesin and reveal dynamic conformations of the cohesin/Scc2 complex in the loading reaction. We demonstrate that Smc3 acetylation blocks the association of Scc2 with pre-engaged cohesin by impairing the interaction of Scc2 with Smc3's head. Lastly, we show that ATP binding induces the cohesin/Scc2 complex to clamp DNA by promoting the interaction between Scc2 and Smc3 coiled coil. Our results illuminate a dynamic reconfiguration of the cohesin/Scc2 complex during loading and indicate how Smc3 acetylation and ATP regulate this process.

Smc5/6's multifaceted DNA binding capacities stabilize branched DNA structures.

Smc5/6 is an evolutionarily conserved SMC complex with roles in DNA replication and repair, as well as in viral DNA restriction. Understanding its multiple 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 question by simultaneously visualizing the behavior of Smc5/6 on three types of DNA, namely double-stranded (ds) DNA, single-stranded (ss) DNA, 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, both the Nse1-3-4 subcomplex and ATP binding enhance the complex's dsDNA association. In contrast, Smc5/6's assembly onto ssDNA emanating from junction DNA, which occurs even in the presence high-affinity ssDNA binders, is aided by Nse1-3-4, but not by ATP. Moreover, we show that Smc5/6 protects junction DNA stability by preventing ssDNA annealing. The multifaceted DNA association behaviors of Smc5/6 provide a framework for understanding its diverse functions in genome maintenance and viral DNA restriction.

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.

A folded conformation of MukBEF and cohesin.

Structural maintenance of chromosomes (SMC)-kleisin complexes organize chromosomal DNAs in all domains of life, with key roles in chromosome segregation, DNA repair and regulation of gene expression. They function through the entrapment and active translocation of DNA, but the underlying conformational changes are largely unclear. Using structural biology, mass spectrometry and cross-linking, we investigated the architecture of two evolutionarily distant SMC-kleisin complexes: MukBEF from Escherichia coli, and cohesin from Saccharomyces cerevisiae. We show that both contain a dynamic coiled-coil discontinuity, the elbow, near the middle of their arms that permits a folded conformation. Bending at the elbow brings into proximity the hinge dimerization domain and the head-kleisin module, situated at opposite ends of the arms. Our findings favour SMC activity models that include a large conformational change in the arms, such as a relative movement between DNA contact sites during DNA loading and translocation.