Eukaryotic genomes are folded into loops and topologically associating domains, which contribute to chromatin structure, gene regulation, and gene recombination. These structures depend on cohesin, a ring-shaped DNA-entrapping adenosine triphosphatase (ATPase) complex that has been proposed to form loops by extrusion. Such an activity has been observed for condensin, which forms loops in mitosis, but not for cohesin. Using biochemical reconstitution, we found that single human cohesin complexes form DNA loops symmetrically at rates up to 2.1 kilo–base pairs per second. Loop formation and maintenance depend on cohesin’s ATPase activity and on NIPBL-MAU2, but not on topological entrapment of DNA by cohesin. During loop formation, cohesin and NIPBL-MAU2 reside at the base of loops, which indicates that they generate loops by extrusion. Our results show that cohesin and NIPBL-MAU2 form an active holoenzyme that interacts with DNA either pseudo-topologically or non-topologically to extrude genomic interphase DNA into loops.

Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher-order structures, but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin-DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin-DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes.

ParABS systems facilitate chromosome segregation and plasmid partitioning in bacteria and archaea. ParB protein binds centromeric parS DNA sequences and spreads to flanking DNA. We show that ParB is an enzyme that hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP). parS DNA stimulates cooperative CTP binding by ParB and CTP hydrolysis. A nucleotide cocrystal structure elucidates the catalytic center of the dimerization-dependent ParB CTPase. Single-molecule imaging and biochemical assays recapitulate features of ParB spreading from parS in the presence but not absence of CTP. These findings suggest that centromeres assemble by self-loading of ParB DNA sliding clamps at parS. ParB CTPase is not related to known nucleotide hydrolases and might be a promising target for developing new classes of antibiotics.

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