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Coordination of cohesin and DNA replication observed with purified proteins

Nature volume 626, pages 653–660 (2024)Cite this article

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Abstract

Two newly duplicated copies of genomic DNA are held together by the ring-shaped cohesin complex to ensure faithful inheritance of the genome during cell division1,2,3. Cohesin mediates sister chromatid cohesion by topologically entrapping two sister DNAs during DNA replication4,5, but how cohesion is established at the replication fork is poorly understood. Here, we studied the interplay between cohesin and replication by reconstituting a functional replisome using purified proteins. Once DNA is encircled before replication, the cohesin ring accommodates replication in its entirety, from initiation to termination, leading to topological capture of newly synthesized DNA. This suggests that topological cohesin loading is a critical molecular prerequisite to cope with replication. Paradoxically, topological loading per se is highly rate limiting and hardly occurs under the replication-competent physiological salt concentration. This inconsistency is resolved by the replisome-associated cohesion establishment factors Chl1 helicase and Ctf4 (refs. 6,7), which promote cohesin loading specifically during continuing replication. Accordingly, we found that bubble DNA, which mimics the state of DNA unwinding, induces topological cohesin loading and this is further promoted by Chl1. Thus, we propose that cohesin converts the initial electrostatic DNA-binding mode to a topological embrace when it encounters unwound DNA structures driven by enzymatic activities including replication. Together, our results show how cohesin initially responds to replication, and provide a molecular model for the establishment of sister chromatid cohesion.

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Fig. 1: Topological cohesin loading, but not initial binding, is highly salt-sensitive.
Fig. 2: Cohesin embracing DNA accommodates all replication events.
Fig. 3: Intertwines between sister DNAs are maintained by topologically bound cohesin.
Fig. 4: The replisome-associated Chl1 helicase facilitates cohesin loading specifically during DNA replication.
Fig. 5: ssDNA bubble induces DNA entrapment by cohesin.

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Data availability

The data supporting the findings of this study are available within the article and Supplementary Information files. Source data are provided with this paper.

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Acknowledgements

We are grateful to B. Argunhan and Y. Kakui for discussions and critical reading of the manuscript. This study was supported by JSPS KAKENHI grants (nos. 22H02550 and 23H04295 to Y.M.), JST-PRESTO grant (no. JPMJPR19KB to Y.M.) and the Takeda Science Foundation (to Y.M.).

Author information

Authors and Affiliations

  1. Department of Chromosome Science, National Institute of Genetics, Mishima, Japan

    Yasuto Murayama, Shizuko Endo, Yumiko Kurokawa, Ayako Kurita, Sanae Iwasaki & Hiroyuki Araki

  2. Department of Genetics, Graduate University for Advanced Studies (SOUKENDAI), Mishima, Japan

    Yasuto Murayama & Yumiko Kurokawa

  3. PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Japan

    Yasuto Murayama

  4. Joint Support-Centre for Data Science Research, Research Organisation of Information and Systems, Tachikawa, Japan

    Hiroyuki Araki

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Contributions

Y.M. conceptualized, designed and supervised the study, acquired funding, generated yeast strains, constructed DNA substrates, performed experiments (cohesin loading, DNA replication, protein interaction), analysed data and wrote the original draft of the manuscript, and reviewed and edited the manuscript. S.E. purified proteins and performed in vitro DNA replication experiments. Y.K., A.K. and S.I. purified proteins and performed in vitro cohesin loading, ATPase and helicase experiments and generated yeast strains. H.A. validated the study.

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Correspondence to Yasuto Murayama.

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Extended data figures and tables

Extended Data Fig. 1 Purification and characterization of budding yeast cohesin.

a, Purified cohesin and Scc2-Scc4 analysed by SDS-PAGE, followed by Coomassie brilliant blue (CBB) staining or western blotting with the indicated antibodies. M. W., molecular weight standard. b, Time-course analyses of ATP hydrolysis by cohesin with DNA and Scc2-Scc4 as indicated. Means ± s.d. from three independent experiments are shown. c, Representative agarose gel (cohesin-bound DNA, left) and SDS-PAGE (cohesin-IP fractions, right) analyses of the experiment carried out as depicted in Fig. 2a with the indicated factors. Note that no detectable Scc2 and Scc4 were seen in the cohesin-IP fractions, indicating that Scc2-Scc4 dissociated from cohesin after high-salt wash. d, Quantification of cohesin-bound DNA in c (n = 4 independent experiments; means ± s.d., circles denote independent experiments). e, Schematic and gel image of the DNA-release experiment mediated by restriction digestion. See also Extended Data Fig. 10b, c for validation of topological cohesin loading by the XL assay. A representative result from two independent experiments is shown. f, IP assays were carried out at the indicated salt concentration (n = 4 independent experiments; means ± s.d.). Quantified data were used for graphical presentation in Fig. 1c. g, Excess of ATP over magnesium ion is required to support topological cohesin loading. The IP assays were performed at the indicated magnesium ion and salt concentrations in the presence of 5 mM ATP (n = 3 independent experiments; means ± s.d.). h, Non-hydrolysable ATP analog AMP-PNP does not support topological cohesin loading. The IP assay was carried out in the presence of AMP-PNP at the indicated salt concentration (n = 3 independent experiments; means ± s.d.).

Source Data

Extended Data Fig. 2 Cohesin binds to DNA at physiological salt concentration in the presence of the loader.

a, Gel image of the DNA beads assay performed as depicted in Fig. 1b with the indicated proteins in the presence of AMP-PNP. b, Quantification of DNA-bound cohesin (Smc3) and Scc2-Scc4 (Scc4) in a (n = 3 independent experiments; means ± s.d.). c, The DNA beads assay was carried out with the indicated proteins in the presence of ATP. d, Quantification of DNA-bound cohesin (Smc3) and Scc2-Scc4 (Scc4) in c (n = 3 independent experiments; means ± s.d.). e, Gel image and f, quantification of the DNA beads assays containing both cohesin and Scc2-Scc4 in the presence of AMP-PNP at the indicated salt concentration (n = 3 independent experiments; means ± s.d.). Quantified data were adapted for graphical presentation in Fig. 1c. g,h, Same as e and f, but the reactions were carried out with ATP using double-stranded or single-stranded DNA beads (n = 3 independent experiments; means ± s.d.). Quantified data for dsDNA were adapted for graphical presentation in Fig. 1c. i, The DNA beads were incubated with the ssDNA-binding protein RPA to confirm actual preparation of ssDNA by NaOH treatment of the dsDNA beads (see Methods for detail). A representative result from two independent experiments is shown.

Source Data

Extended Data Fig. 3 Reconstitution of complete DNA replication with purified budding yeast proteins.

a, Additional replication proteins used in this study were analysed by SDS-PAGE and CBB staining. b, Schematic of standard in vitro DNA replication using purified budding yeast proteins. The Mcm2-7 helicase core complex was initially loaded onto the circular DNA substrate by incubating with ORC complex and Cdc6. This was then activated via phosphorylation by Dbf4-dependent Cdc7 kinase (DDK). DNA replication was initiated by addition of the indicated replication proteins. Nascent leading and lagging strands were spiked via incorporation of biotin-dUTP. As two replisomes proceeded bi-directionally and encountered each other opposite to the start site (ARS1), two nascent leading strands approximately half the length (~2.5 kb) of the template (4.1 kb) were generated, as well as shorter, discontinuous lagging strands (~0.4 kb). These strands could be separated and detected by denaturing gel analysis. When ligaseCdc9 and Fen1 were included in the reaction, nascent leading and lagging strands were ligated, generating longer, full-length strands (~4.1 kb). c, A time-course experiment of standard DNA replication detected by denaturing agarose gel analysis using 4.1 kbp circular DNA (pARS1-4.1). A representative result from two independent experiments is shown. d, as c, but the reaction contained ligaseCdc9 and Fen1, as indicated. A representative result from two independent experiments is shown. e, Reconstitution of replication termination. Replication was carried out as depicted in b, but the reactions contained either the Pif1 or Rrm3 helicase. Post-replicative DNA was subjected to restriction digestion at the ARS1 site. The resultant replication products were separated and detected by native agarose gel analysis. This converted the terminated products to 4.1 kbp of linear DNA (Full-length), whereas non-terminated intermediates were converted to X-shaped larger DNA molecules (Int. >8 kbp). A representative result from two independent experiments is shown. Note that Polδ was omitted from the experiments shown in Extended Data Fig. 3e to avoid the occurrence of strand displacement synthesis by Polδ, which is pronounced by Pif1 or Rrm332.

Extended Data Fig. 4 Additional validation experiments of the “cohesin first” replication reactions.

a, Time-course of in vitro DNA replication analysed by denaturing agarose gel electrophoresis. The indicated concentrations of cohesin and Scc2-Scc4 were loaded onto circular DNA prior to replication in low-salt buffer. A representative result from two independent experiments is shown. b, Quantitative comparison of total replication products (sum of Int. and Full-length) in Fig. 2b, expressed relative to the respective +Cohesin / −Pif1 / −KOAc reactions (denoted by stars). Means ± s.d. are shown (n = 5 independent experiments). c, Quantification of Full-length products in Fig. 2b (n = 5 independent experiments; means ± s.d.). d, as Fig. 2b, but the reactions were carried out using Rrm3 instead of Pif1. A representative result from two independent experiments is shown. e, Whole and cohesin-bound replication products were digested by the indicated restriction enzyme, sites for which are shown, and DNA was subjected to native and denaturing agarose gel analyses. This suggests that cohesin-bound products were indeed completely replicated. A representative result from two independent experiments is shown. f, Scc1-TEV cohesin was treated with TEV protease in replication buffer, followed by SDS-PAGE and CBB staining. Note that a certain fraction of Scc1-TEV appeared to be cut at the TEV site during expression and/or purification before TEV protease treatment. A representative result from two independent experiments is shown. g, Purified establishment factors analysed by SDS-PAGE and CBB staining. h, The “cohesin first” replication reactions were performed in the absence of the indicated cohesion establishment factor. A representative result from two independent experiments is shown. i, DNA-bound cohesin accommodates replication termination without Scc2-Scc4. Cohesin was first loaded onto circular DNA substrates and then immobilised on anti-Pk-bound, protein A-conjugated magnetic beads via the 3xPk tag on Smc1. After extensive high-salt washing to remove non-topological DNA associations and Scc2-Scc4, the DNA-cohesin beads were subjected to in vitro replication (see also Extended Data Fig. 1c for validation of Scc2-Scc4 removal by high-salt wash). The resultant beads were again washed with high-salt buffer before analysing the replication products by agarose gel electrophoresis. j, Native agarose gel analysis of the experiment depicted in j. Replicated DNA was recovered when cohesin loading was carried out prior to replication, with increased full-length products when Pif1 was included in the replication reaction. In contrast, no detectable signal was seen in the absence of cohesin. This suggests that cohesin by itself remains associated with DNA during replication, even in the absence of Scc2-Scc4. A representative result from two independent experiments is shown. k, As Fig. 2e, but the products were subjected to denaturing agarose gel analysis. A representative result from two independent experiments is shown. Note that Polδ was omitted from the experiments shown in Extended Data Fig. 4 to avoid the occurrence of strand displacement synthesis by Polδ, which is pronounced by Pif1 or Rrm332.

Source Data

Extended Data Fig. 5 Additional control experiments of chromatin replication.

a, Purified core histones, Nap1, ISW1a complex, FACT complex and Nhp6A were analysed by SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining. b, Micrococcal nuclease (MNase) digestion of the chromatinized circular DNA template used for chromatin replication. nuc. denotes nucleosome. The digested products were separated by agarose gel electrophoresis and stained with Sybr Gold. This confirmed the evenly spaced nucleosome assembly. A representative result from three independent experiments is shown. c, Schematic of in vitro replication with the chromatinized template. d, Denaturing and native agarose analyses of the experiments depicted in c with the indicated proteins. e, Quantification of full-length products in d (n = 3 independent experiments; means ± s.d.). Omission of Csm3-Tof1 or Mrc1 reduced the full-length products even in the presence of Pif1, suggesting that these cohesion establishment factors promote DNA replication per se in a chromatin context. f, Nucleosomes limit cohesin loading. In vitro cohesin loading reactions (IP assay) were carried out with chromatinized circular DNA. Representative gel images of the experiment performed with naked (top) or chromatinized DNA (bottom) in the presence of the indicated proteins are shown. The graph represents quantification of cohesin-bound DNA (n = 3 independent experiments; means ± s.d.). g, Confirmation of chromatin assembly using cohesin-loaded circular DNA templates. Cohesin loading was first carried out in the presence of Scc2-Scc4 and ATP, and then was sequentially subjected to nucleosome assembly using Nap1 and ISW1a. MNase digestion confirmed that the nucleosomes were efficiently and evenly assembled on cohesin-loaded DNA, as on naked DNA. A representative result from two independent experiments is shown. h, Schematic of the “cohesin-first” replication reaction with the chromatinized template. i, Denaturing and native gel analyses of the experiments depicted in h. Like replication with naked DNA, replicated DNA was recovered by cohesin-IP after chromatin replication, whereas no detectable replicated DNA was seen in the absence of cohesin. A representative result from two independent experiments is shown. Note that Pol δ was included in chromatin replication because nucleosomes limit the strand displacement synthesis by Pol δ37,38.

Source Data

Extended Data Fig. 6 Dimer products are catenated sister DNAs.

a, Gel isolated dimer products and deproteinized whole reaction products were treated with the indicated enzymes. The band denoted by a red arrowhead was excised to isolate dimer products. A representative result from two independent experiments is shown. b, Cohesin does not obviously affect DNA catenation by Top2 under these reaction conditions. Cohesin was initially loaded onto nicked circular DNA in the presence of a loader at low ionic strength conditions, and then further incubated in salt-containing replication buffer for 55 min. After addition of 100 mM NaCl, the reaction was subjected to Top2 treatment. Reaction products were then separated by agarose gel electrophoresis, followed by SYBR Gold staining. A representative gel image is shown. c, Quantification of the experiments in b (n = 3 independent experiments; means ± s.d.). d, Dimer products consist of replicated DNAs. Top2 treatment could potentially generate dimer products by promoting catenation of the replicated monomer and unreplicated substrate DNA. To rule out this possibility, purified replication products obtained from the indicated conditions were subjected to DpnI restriction enzyme treatment. Circular DNA substrates used in this study were obtained from dam+ E. coli, and are thus highly sensitive to DpnI that digests the di-methylated GATC sequence (−Mcm10 samples). e, Whole DNA (Sybr Gold staining) and f, replicated DNA detection of the experiments depicted in d with the indicated proteins. g, Quantification of the experiments in e and f. Means from two independent experiments are shown. The amounts of dimer and monomer products were unchanged upon DpnI treatment (+Mcm10 samples). This further confirmed that the dimer products were catenated, replicated sister DNAs. The graphs represent means from two independent experiments. h, Schematic of the sister DNA decatenation after cohesin cleavage by TEV protease. i, Native gel analysis of the experiments depicted in h. j, Quantification of dimer products in i (n = 4 independent experiments; means ± s.d.). Note that Polδ was omitted from the experiments shown in Extended Data Fig. 6 to avoid the occurrence of strand displacement synthesis by Polδ, which is pronounced by Pif132.

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Extended Data Fig. 7 Replication-specific stimulation of cohesin loading by Chl1.

a, Schematic of the Order 1 reaction using the chromatinized circular DNA templates. b, Native agarose gel analysis of the experiments performed as depicted in a. A representative result from two independent experiments is shown. c, Schematic of the Order 1 reaction but including Pif1 and post-replicative restriction digestion. Pfi1 was added to the reaction 10 min after replication initiation. See Methods for details. d, Native agarose gel analysis and e, quantitative comparison of the experiments performed as depicted in c, expressed relative to the −Pif1 / −Chl1 reaction (denoted by stars). Means ± s.d. from three independent experiments are shown. f, Chl1 was added in the “cohesin first” reaction as denoted in the schematic. g, Native agarose gel analysis and h, quantitative comparison of the experiments performed as depicted in f, expressed relative to the −Pif1 / −Chl1 reaction (denoted by stars). Means ± s.d. from three independent experiments are shown. i, Cohesin loading reactions were carried out with the indicated cohesin and Scc2-Scc4 concentrations, and then replication was initiated in the absence or presence of Chl1. j, Native agarose gel analysis and k, quantitative comparison of the experiments performed as depicted in i. Means from two independent experiments are shown. l, Chl1 was added in the “cohesin first” reaction using the chromatinized template as denoted in the schematic. m, Native agarose gel analysis and n, quantitative comparison of the experiments performed as depicted in l. Means of two independent experiments are shown. Note that Polδ was omitted from the in vitro replication reactions using naked DNA to avoid the occurrence of strand displacement synthesis by Polδ, which is pronounced by Pif132, whereas Pol δ was included in chromatin replication because nucleosomes limit the strand displacement synthesis37,38.

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Extended Data Fig. 8 Chl1 helicase stimulates cohesin loading through interaction with Ctf4.

a, Native agarose gel image and b, quantitative comparison of the Order 1 reaction as in Fig. 4b but with omission of Scc2-Scc4, expressed relative to the +Scc2-Scc4 / −Chl1 reaction (denoted by stars, n = 3 independent experiments; means ± s.d.). c, Representative gel image of the experiments in Fig. 4e (n = 3 independent experiments). d, Input fractions of the cohesin-IP experiments of Fig. 4f were analysed by SDS-PAGE and oriole staining (n = 2 independent experiments). e, Order 1 reactions were performed in the absence of the indicated proteins. f, Quantitative comparison of e, expressed relative to the None / −Chl1 reaction (denoted by stars, n = 3 independent experiments; means ± s.d.). g, Order 1 reactions were performed in the presence of the indicated proteins, with the addition of Polδ, Fen1 and ligaseCdc9. h, Quantitative comparison of g, expressed relative to the None / −Chl1 reaction (denoted by stars, n = 3 independent experiments; means ± s.d.). i, Wild-type Chl1, but not the K48R mutant, displays ATP-dependent helicase activity. After incubation with Y-fork structured DNA, the unwound products were detected by native PAGE. The reactions were carried out in 1 mM of ATP and Mg(oAc)2 and 50 mM NaCl. A representative result from two independent experiments is shown. j, Representative gel image of the experiments in Fig. 4g (n = 3 independent experiments). Note that Polδ was omitted from the in vitro replication reactions unless otherwise indicated.

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Extended Data Fig. 9 Single-stranded DNA facilitates topological cohesin loading.

a, Cohesin and Scc2-Scc4 were incubated with ss-gapped DNA (0.2 kb) at the indicated salt and magnesium ion concentrations. Nicked circular DNA was used for comparison. Compared to dsDNA (nicked circular DNA), increased recovery of ss-gapped DNA was seen under all conditions tested. b, Quantification of cohesin-bound DNA in a (n = 4 independent experiments; means ± s.d.). c,d, Drop-out experiments using 0.2- (c) or 0.6- (d) ss-gapped DNA at the indicated salt concentrations (n = 3 independent experiments; means ± s.d.). As with dsDNA, efficient DNA recovery was only seen in the presence of both ATP and Scc2-Scc4. e, RPA inhibits cohesin loading onto ss-gapped DNA, but not onto nicked dsDNA. The indicated amount of RPA was added to the cohesin loading reaction (n = 3 independent experiments; means ± s.d.). This suggests that cohesin contacts ssDNA to convert the initial binding into a topological embrace. f, IP assays using circular dsDNA (nicked circular DNA) were carried out with the indicated amount of 0.6 kb linear ssDNA. The separated ssDNA strand showed no detectable effect on cohesin loading onto dsDNA. g, Quantification of cohesin-bound DNA in f (n = 3 independent experiments; means ± s.d.).

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Extended Data Fig. 10 Additional validiation experiments for the crosslinking-based cohesin loading assays (XL-assay).

a, Purified wild-type, 5 C, and 6 C cohesins were treated with BMOE and analysed by SDS-PAGE and CBB staining. A representative result from two independent experiments is shown. b, Drop-out experiments performed with circular nicked dsDNA. Efficient DNA shifts were seen when 6 C cohesin was incubated with both Scc2-Scc4 and ATP at a low-salt concentration, followed by BMOE crosslinking. In the “+ProK” reaction, the reaction mixture was treated with protease K after BMOE crosslinking and SDS-denaturation. This confirmed that shifted DNA species were derived from protein crosslinking. A representative result from two independent experiments is shown. c, The XL-assay was carried out with circular nicked dsDNA at the indicated salt concentrations. Like the IP assay, shifted DNA species were generated at a low-salt concentration. A representative result from two independent experiments is shown. d, Gel image of the XL assays performed with ss-gapped DNA. Consistent with the IP assay, 6 C cohesin showed more efficient DNA shifts with ss-gapped DNA than dsDNA in the presence of 100 mM KOAc. In contrast, no detectable DNA shift was seen using 5 C cohesin, confirming topological DNA loading by 6 C cohesin (lower gel). A representative result from three independent experiments is shown. e, Drop-out experiments performed with bubble DNA at 100 mM KOAc. As with dsDNA, efficient DNA shifts were observed when 6 C cohesin was incubated with Scc2-Scc4 and ATP, followed by BMOE crosslinking. Use of 5 C cohesin as well as protease K treatment confirmed topological loading of 6 C cohesin onto bubble DNA. A representative result from two independent experiments is shown.

Extended Data Fig. 11 Chl1 facilitates topological cohesin loading on bubble DNA.

a, Helicase activity of Chl1 was measured at 100 mM of the indicated salt using Y-fork structured DNA. In all conditions tested, Chl1 displayed higher helicase activity in the presence of 5 mM magnesium ion. As shown in Fig. 4g, the helicase activity of Chl1 appears to be critical for stimulation of cohesin loading during DNA replication. Thus, we carried out IP assays at 5 mM magnesium ion (and 5 mM ATP) concentration to assess the effect of Chl1 on cohesin loading. A representative result from two independent experiments is shown. b, IP assays were performed with the bubble DNA in the presence of the indicated protein at 100 mM NaCl (n = 4 independent experiments; means ± s.d.). c, Gel image of the XL-assays performed with bubble DNA in the presence of Chl1. A representative result from two independent experiments is shown. d, Schematic of the IP assay containing RPA and Chl1 using bubble DNA. e, Gel image and f, quantification of the IP assay depicted in d. The experiment was performed once. g, Schematic of the Order 1 reaction performed with the indicated concentration of RPA. h, Native agarose gel analysis and i, quantitative comparison of the experiments performed as depicted in g, expressed relative to the −Pif1 / −Chl1 reaction using 50 nM RPA (denoted by stars). Means ± s.d. from three independent experiments are shown. Note that Polδ was omitted from the experiments shown in Extended Data Fig. 11h, i.

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Murayama, Y., Endo, S., Kurokawa, Y. et al. Coordination of cohesin and DNA replication observed with purified proteins. Nature 626, 653–660 (2024). https://doi.org/10.1038/s41586-023-07003-6

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