Cohesin ATPase activities regulate DNA binding and coiled-coil configuration

The cohesin complex is required for sister chromatid cohesion and genome compaction. Cohesin coiled coils (CCs) can fold at break sites near midpoints to bring head and hinge domains, located at opposite ends of coiled coils, into proximity. Whether ATPase activities in the head play a role in this conformational change is yet to be known. Here, we dissected functions of cohesin ATPase activities in cohesin dynamics in Schizosaccharomyces pombe. Isolation and characterization of cohesin ATPase temperature-sensitive (ts) mutants indicate that both ATPase domains are required for proper chromosome segregation. Unbiased screening of spontaneous suppressor mutations rescuing the temperature lethality of cohesin ATPase mutants identified several suppressor hotspots in cohesin that located outside of ATPase domains. Then, we performed comprehensive saturation mutagenesis targeted to these suppressor hotspots. Large numbers of the identified suppressor mutations indicated several different ways to compensate for the ATPase mutants: 1) Substitutions to amino acids with smaller side chains in coiled coils at break sites around midpoints may enable folding and extension of coiled coils more easily; 2) substitutions to arginine in the DNA binding region of the head may enhance DNA binding; or 3) substitutions to hydrophobic amino acids in coiled coils, connecting the head and interacting with other subunits, may alter conformation of coiled coils close to the head. These results reflect serial structural changes in cohesin driven by its ATPase activities potentially for packaging DNAs.

The cohesin complex is required for sister chromatid cohesion, DNA damage response, gene expression, and spatial organization of the genome (1, 2). Psm1/SMC1 and Psm3/SMC3 form a stable heterodimer via both hinge–hinge interaction and ATPase heads engagement upon ATP binding (3–5). Cohesin owns two ATPase domains at its globular head. Each ATPase domain contains the Walker A and Walker B consensus sequences found in most ATPases (5, 6) and several other sequence motifs, such as signature motif and D loop (7). Both ATPase domains are required for efficient loading of cohesin (8). Rad21/SCC1, the kleisin subunit with its N-terminal domain, interacts with Psm3/SMC3 coiled coils (CCs) emerging from the head, and its C-terminal domain interacts with Psm1/SMC1 head domain (9–12). Psc3/SCC3 associates with the unstructured region in the middle of Rad21/SCC1 (13–15).Mis4/SCC2/NIPBL functions as the cohesin loader (16, 17). Mis4/SCC2/NIPBL forms a harp-shaped structure (18, 19). Its N-terminal domain binds to Psm3/SMC3 coiled coils close to the head domain and its C-terminal domain binds to Psm1/SMC1 coiled coils close to the head domain (11, 15). Mis4/SCC2/NIPBL also stimulates cohesin’s ATPase activity for efficient cohesin loading (20–22).All coiled coils of SMC complexes (cohesin, condensin, and SMC5-SMC6 complex) are ∼50 nm long and are essential for their functions (23–25). SMC coiled coils contain interruptions (break sites hereafter) that disrupt the characteristic seven-residue amino acid sequence repeats, known as heptad repeats (26, 27). It has been proposed that cohesin folds around the midpoints of its coiled coils to bring the head and hinge domains into proximity (20, 28–30). However, it is still unclear how such molecular architecture of cohesin works to fulfill its function. In this study, we isolated temperature-sensitive (ts) mutants with single amino acid substitutions in the signature motif or D loop of cohesin ATPase domains, which presumably impair ATPase activity of cohesin. Then, screening of suppressor mutations that rescued the lethality caused by ATPase defects identified several hot regions in cohesin SMC subunits, which are involved in DNA binding, interaction with non-SMC subunits, or coiled-coil dynamics around midpoints. Therefore, these results coupled the dynamics of the cohesin complex with ATPase activity.