SummaryCohesin organizes DNA into chromatids, regulates enhancer-promoter interactions, and confers sister chromatid cohesion. Its association with chromosomes is regulated by hook-shaped HEAT repeat proteins that bind Scc1, namely Scc3, Pds5, and Scc2. Unlike Pds5, Scc2 is not a stable cohesin constituent but, as shown here, transiently replaces Pds5. Scc1 mutations that compromise its interaction with Scc2 adversely affect cohesin’s ATPase activity and loading. Moreover, Scc2 mutations that alter how the ATPase responds to DNA abolish loading despite cohesin’s initial association with loading sites. Lastly, Scc2 mutations that permit loading in the absence of Scc4 increase Scc2’s association with chromosomal cohesin and reduce that of Pds5. We suggest that cohesin switches between two states: one with Pds5 bound that is unable to hydrolyze ATP efficiently but is capable of release from chromosomes and another in which Scc2 replaces Pds5 and stimulates ATP hydrolysis necessary for loading and translocation from loading sites.

The ring-shaped cohesin complex topologically entraps chromosomes and regulates chromosome segregation, transcription, and DNA repair. The cohesin core consists of the structural maintenance of chromosomes 1 and 3 (Smc1-Smc3) heterodimeric ATPase, the kleisin subunit sister chromatid cohesion 1 (Scc1) that links the two ATPase heads, and the Scc1-bound adaptor protein Scc3. The sister chromatid cohesion 2 and 4 (Scc2-Scc4) complex loads cohesin onto chromosomes. Mutations of cohesin and its regulators, including Scc2, cause human developmental diseases termed cohesinopathy. Here, we report the crystal structure of Chaetomium thermophilum (Ct) Scc2 and examine its interaction with cohesin. Similar to Scc3 and another Scc1-interacting cohesin regulator, precocious dissociation of sisters 5 (Pds5), Scc2 consists mostly of helical repeats that fold into a hook-shaped structure. Scc2 binds to Scc1 through an N-terminal region of Scc1 that overlaps with its Pds5-binding region. Many cohesinopathy mutations target conserved residues in Scc2 and diminish Ct Scc2 binding to Ct Scc1. Pds5 binding to Scc1 weakens the Scc2-Scc1 interaction. Our study defines a functionally important interaction between the kleisin subunit of cohesin and the hook of Scc2. Through competing with Scc2 for Scc1 binding, Pds5 might contribute to the release of Scc2 from loaded cohesin, freeing Scc2 for additional rounds of loading.transcription | cohesinopathy | X-ray crystallography | cohesin loading | HEAT repeat C ohesin consists of four core subunits: structural maintenance of chromosomes 1 and 3 (Smc1 and Smc3), and sister chromatid cohesion 1 and 3 (Scc1 and Scc3). (1-5). Smc1 and Smc3 are related ATPases that heterodimerize through their hinge domains. The C-terminal winged helix domain and the N-terminal helical domain (NHD) of Scc1 bind to the Smc1 ATPase head and a coiled-coil segment adjacent to the Smc3 ATPase head, respectively, forming a tripartite ring (6, 7). Scc3 contains Huntingtin-elongation factor 3-protein phosphatase 2A-TOR1 (HEAT) repeats, binds to the middle region of Scc1, and provides a landing pad for several cohesin regulators (8, 9). Cohesin can topologically trap DNA inside its ring (10), thereby regulating many facets of chromosome biology. During interphase, cohesin mediates the formation of chromosome loops that impact transcription (11). During S phase, cohesin physically links replicated chromosomes to establish sister-chromatid cohesion (12, 13), which is a prerequisite for faithful chromosome segregation during mitosis. Finally, cohesin contributes to homologydirected repair of DNA breaks, in part, through holding the sister chromatid in proximity of the breaks and presenting it as the repair template (14).For cohesin to accomplish these diverse tasks, its association with chromosomes has to be tightly controlled both spatially and temporally. Cohesin dynamics on chromosomes are regulated by a set of accessory proteins, including the Scc2-Scc4 loading complex and the releasing complex comprising Pds5 and wing...