Biochemical reconstitution of topological DNA binding by the cohesin ring

Abstract

Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for faithful chromosome segregation in mitosis. Cohesin also has vital roles in DNA repair and transcriptional regulation. The ring-shaped cohesin complex is thought to encircle sister DNA strands, but its molecular mechanism of action is poorly understood and the biochemical reconstitution of cohesin activity in vitro has remained an unattained goal. Here we reconstitute cohesin loading onto DNA using purified fission yeast cohesin and its loader complex, Mis4(Scc2)-Ssl3(Scc4) (Schizosaccharomyces pombe gene names appear throughout with their more commonly known Saccharomyces cerevisiae counterparts added in superscript). Incubation of cohesin with DNA leads to spontaneous topological loading, but this remains inefficient. The loader contacts cohesin at multiple sites around the ring circumference, including the hitherto enigmatic Psc3(Scc3) subunit, and stimulates cohesin's ATPase, resulting in efficient topological loading. The in vitro reconstitution of cohesin loading onto DNA provides mechanistic insight into the initial steps of the establishment of sister chromatid cohesion and other chromosomal processes mediated by cohesin.

Figure 1. Mis4^Scc2/Ssl3^Scc4 is a DNA binding protein

a, Purified Mis4^Scc2/Ssl3^Scc4 complex (M4S3) and its Mis4^Scc2 subunit (M4) were analyzed by SDS-PAGE and Coomassie blue staining (CBB) or Western blotting using antibodies directed against Mis4 or its C-terminal HA epitope. b, glycerol gradient centrifugation of the Mis4^Scc2/Ssl3^Scc4 complex, followed by SDS-PAGE and silver staining. Bovine serum albumin (BSA), catalase (Cata) and thyroglobulin (Tyr) were size markers. c, DNA binding of Mis4^Scc2/Ssl3^Scc4, using an electrophoretic mobility shift assay. d, Quantification of DNA binding by Mis4^Scc2/Ssl3^Scc4 (left) and Mis4^Scc2 (right). Mean and standard deviation from 3 experiments are shown.

Figure 2. In vitro reconstitution of cohesin loading onto DNA

a, Gel filtration of the cohesin complex. b, Purified cohesin, analyzed by SDS-PAGE and CBB staining or Western blotting as indicated. c, Schematic of the cohesin loading assay. d, Agarose gel electrophoresis and quantification of recovered DNA during the loading reaction. e, Time course with or without Mis4^Scc2/Ssl3^Scc4 (M4S3) and f, Titration of Mis4^Scc2/Ssl3^Scc4, or Mis4^Scc2 only (M4). g, The loading reaction was carried out using RC DNA containing fission yeast cohesin-bound (E1, E2) or -unbound (N) sequences. Mean and standard deviation from at least 3 experiments are shown.

Figure 3. Topology-mediated DNA binding of cohesin

a, Schematic of the DNA release experiment. b, Gel image of the experiment. Covalently closed circular (CCC) input DNA and linear form (L), supernatant (S) and bead (B) fractions of experiments in presence or absence of Mis4^Scc2/Ssl3^Scc4 (M4S3), with or without PstI digestion, are shown. c, Diagram of the DNA release experiment by TEV protease (TEV) cleavage of Rad21. d, Agarose gel of recovered RC DNA and its quantification, the mean and standard deviation of three experiments are shown. Rad21 cleavage was monitored by Western blotting.

Figure 4. Mis4^Scc2/Ssl3^Scc4 stimulates cohesin’s ATPase

Loading reactions, a, in the absence or presence of ATP or derivatives, or b, comparing wild type or Walker A motif mutant cohesin. c, Timecourse analysis of ATP hydrolysis by cohesin with or without Mis4^Scc2/Ssl3^Scc4 (M4S3) or RC DNA. d, ATP hydrolysis rates of wt and mutant cohesin derived from similar timecourse analyses. RC DNA (C) or linear DNA (L) and a reaction with Mis4^Scc2 only (M4) was included. The mean and standard deviation of three experiments are shown.

Figure 5. Mis4^Scc2/Ssl3^Scc4 interactions around the cohesin ring

a, Identification of a Mis4^Scc2/Ssl3^Scc4 interaction site on a Psm1 tiling peptide array. Starting positions of the first peptide in every other row are indicated. b, Summary of Mis4^Scc2/Ssl3^Scc4 interaction sites on the four cohesin subunits c, Coimmunoprecipitation of wt and interaction site mutant Psc3-Pk with Mis4^Scc2/Ssl3^Scc4. d, Rescue of sister chromatid cohesion after psc3-303 inactivation by wild type or interaction site mutant Psc3. e, Loading reactions of a cohesin trimer were supplmented with wild type or interaction site mutant Psc3. f, Wild type and Psm1/3^LM cohesin complexes were compared. g, Wild type (+), scrambled (sc) or point mutant (m) peptides corresponding to Mis4^Scc2/Ssl3^Scc4 interactions sites were included in loading reactions. All panels present the means and standard deviations from 3 independent experiments. h, Model for cohesin loading onto DNA.