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Magic Acts with the Cohesin Ring
Molecular Cell
Preview Magic Acts with the Cohesin Ring Hongtao Yu1,* 1Department of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2016.02.003
Recent studies, including two in this issue of Molecular Cell (Elbatsh et al., 2016; Beckoue¨t et al., 2016), cast light on how cohesin regulators harness the energy of ATP hydrolysis to open the cohesin ring and enable dynamic, regulated entrapment of chromosomes. Through topologically entrapping DNA, cohesin rings regulate chromosome segregation, transcription, and DNA repair. Proper regulation of cohesin dynamics on chromosomes is essential for these diverse processes (Haarhuis et al., 2014). Scc2-Scc4 loads cohesin onto chromosomes, whereas Wapl-Pds5 releases cohesin from chromosomes. Eco1 antagonizes Wapl-Pds5 by acetylating Smc3, which stabilizes cohesin on chromosomes and establishes sisterchromatid cohesion during DNA replication and homology-directed repair. Human developmental diseases and cancer have been linked to mutations of cohesin subunits and regulators (Remeseiro et al., 2013). Two reports in this issue of Molecular Cell (Beckoue¨t et al., 2016; Elbatsh et al., 2016), along with other recent studies (C¸amdere et al., 2015; Murayama and Uhlmann, 2014, 2015), provide new insight into the mechanics and regulation of cohesin ring opening, an obligatory step during both loading and release of cohesin. The cohesin ring has three outer gates: the Smc3-Scc1N interface, the Smc1Scc1C interface, and the hinge interface (Figure 1A). Smc1 and Smc3 belong to the ATP-binding cassette (ABC) type of ATPases, which binds two ATP molecules at the dimer interface of two ATPase domains. When ATP bound, the Smc1Smc3 ATPase domains form an inner gate. ATP hydrolysis and nucleotide release drive apart the ATPase domains and open this inner gate. Using recombinant fission yeast cohesin and its loader (Mis4-Ssl3 in fission yeast), Murayama and Uhlmann show that the cohesin loader stimulates cohesin’s ATPase and promotes the topological loading of cohesin onto a circular plasmid (Murayama
and Uhlmann, 2014). Forcing the inner gate closed by a non-hydrolyzable ATP analog traps DNA inside the Smc1Smc3 closure (Murayama and Uhlmann, 2015). They further propose that cohesin loading involves opening the Smc3Scc1N interface, instead of the hinge interface suggested previously (Gruber et al., 2006). Although logically simple, this new proposal needs reconciling with the functional chromosome loading of an Smc3-Scc1 fusion protein in budding yeast. Because the cohesin loader Scc2Scc4 interacts with several cohesin subunits, one intriguing possibility is that it can open the cohesin ring at more than one interface. Strong evidence indicates that the Smc3-Scc1N interface is the conserved DNA exit gate of cohesin. Artificial tethering of Smc3 and Scc1 prevents cohesin release from chromosomes in yeast (Chan et al., 2012). Conversely, mutations that weaken the Smc3-Scc1N interface permit cohesin release in Wapl-depleted human cells (Huis in ’t Veld et al., 2014). These findings suggest involvement of this interface in cohesin release. Beckoue¨t et al. now provide direct evidence that Wapl-Pds5-dependent releasing activity actually disrupts the Smc3Scc1N interface (Beckoue¨t et al., 2016). Using chemical crosslinking, immunoprecipitation, and fluorescence microscopy, they show convincingly that, following the induced cleavage of an engineered Scc1, Wapl-Pds5 actively releases the N-terminal cleavage product of Scc1 (Scc1N) from Smc3 in budding yeast. Murayama and Uhlmann have made similar observations with an in vitro reconstituted cohesin release assay (Murayama and Uhlmann, 2015). Strikingly, this release is inhibited by mutations in
Wapl, Pds5, and core cohesin subunits that stabilize cohesin on chromosomes and bypass the need for Eco1 (Beckoue¨t et al., 2016). Wapl-Pds5-dependent releasing activity can act on soluble cohesin not bound to chromosomes. The ability of Wapl-Pds5 to open the cohesin ring (without entrapped DNA) is consistent with the finding that WaplPds5 can load cohesin onto DNA under certain conditions (Murayama and Uhlmann, 2015). In a complementary study, Elbatsh et al. have discovered that the two ATPase active sites of Smc1-Smc3 are asymmetrically required for Wapl-Pds5dependent cohesin release (Elbatsh et al., 2016). Similar observations have been independently made by Koshland and coworkers (C¸amdere et al., 2015). Smc1-Smc3 contains two asymmetric ATPase active sites, which I have termed the apical and basal sites (Figures 1B and 1C). Elbatsh et al. show that, in budding yeast, mutations of the signature motif and D loop of Smc1 at the apical site restore viability of Eco1 mutant cells and partially suppress their cohesion defects (Elbatsh et al., 2016). Despite impairing the overall ATPase activity of cohesin to a similar degree, corresponding mutations in the signature motif and D loop of Smc3 at the basal site cannot rescue phenotypes caused by Eco1 deficiency. Furthermore, the same Smc1 mutations, but not the Smc3 mutations, inhibit the release of Scc1N from Smc3 (Beckoue¨t et al., 2016). These two sets of cohesin mutants, as well as mutants with deficient ATPase activities at both sites, can be loaded onto chromosomes to some degree, presumably because Scc2-Scc4 can stimulate the ATPase activity of these mutants above the threshold needed for
Molecular Cell 61, February 18, 2016 ª2016 Elsevier Inc. 489
Molecular Cell
Preview
Figure 1. Model for DNA Capture and Release by Cohesin (A) Overall cohesion ring architecture. (B) A structural model of the Smc1-Smc3-Scc1N-Scc1C complex, with bound nucleotides and acetylation acceptor lysines shown in sticks. (C) The two ATPase sites of cohesin. (D) A speculative model of cohesin loading and release by Scc2-Scc4 and Pds5-Wapl. The model mainly serves to illustrate the complicated maneuvers that cohesin must undertake to entrap and release DNA.
loading (Beckoue¨t et al., 2016; C¸amdere et al., 2015; Elbatsh et al., 2016; Murayama and Uhlmann, 2015). These findings establish a critical requirement for ATP hydrolysis at the apical site for Wapl-Pds5-mediated cohesin release from chromosomes. The slowed cohesin release with deficient apical
ATPase activity compensates for the loss of Eco1-dependent Smc3 acetylation, a Wapl-Pds5-antagonizing mechanism. This asymmetric requirement is conserved in human cells, as expression of the same signature motif mutant of human Smc1 alleviates the requirement for the cohesion-establishment factor sororin
490 Molecular Cell 61, February 18, 2016 ª2016 Elsevier Inc.
in cell viability and sister-chromatid cohesion (Elbatsh et al., 2016). Surprisingly, a mutation of the Walker B motif of Smc3 (which cripples the ATPase activity of the apical site) does not prevent Scc1N release from Smc3 (Beckoue¨t et al., 2016). The corresponding mutation does, however, inhibit the
Molecular Cell
Preview Wapl-Pds5-stimulated release of the fission yeast cohesin from DNA in vitro (Murayama and Uhlmann, 2015). The effect of this mutation on cohesin release needs further clarification. Smc3 acetylation by Eco1 has been thought to stabilize cohesin on chromosomes by inhibiting cohesin’s ATPase activity. However, recombinant cohesin harboring acetylation-mimicking mutations has normal ATPase activity (Ladurner et al., 2014). Instead, ATP hydrolysis is required for efficient Smc3 acetylation by Eco1. Murayama and Uhlmann (2015) and C¸amdere et al. (2015) have now explained this conundrum. They show that cohesin topologically bound to DNA has higher ATPase activity (C¸amdere et al., 2015; Murayama and Uhlmann, 2015). This stimulation of ATPase activity by DNA, as well as cohesin loading and release, is inhibited by acetylation-mimicking Smc3 mutations (Murayama and Uhlmann, 2015). These findings suggest that the two acetylation acceptor lysines in Smc3 might directly contact the entrapped DNA, and their acetylation neutralizes the positive charge and is expected to weaken DNA binding. Indeed, the two lysines and neighboring positively charged residues form a basic patch adjacent to the apical ATPase site (Figure 1B). DNA binding to this patch might better orien-
tate ATP for hydrolysis or promote the release of ADP. The available data collectively support the following model for cohesin loading and release (Figure 1D). Scc2-Scc4, cohesin, and DNA form a transient complex, in which Scc2-Scc4 strengthens the engagement and ATP binding of the Smc1-Smc3 ATPase domains. ATP hydrolysis disengages the ATPase domains and opens the cohesin ring to entrap DNA. ATP rebinding closes the inner gate and locks DNA in the top Smc1-Smc3 closure. The entrapped DNA promotes ATP hydrolysis or nucleotide release at the apical site. Regardless of the nucleotide state of the basal site, removal of ATP from the apical site suffices to disengage the ATPase domains and allow DNA to escape from the top closure. Wapl-Pds5 binds to the nucleotide-free cohesin and, upon ATP binding, disrupts the Smc3-Scc1N interface to release DNA. Smc3 acetylation prevents the entrapped DNA from stimulating the apical ATPase activity, blocking inner-gate opening and subsequent Wapl-Pds5-dependent outergate opening. High-resolution structural studies of cohesin in different nucleotide states, alone or bound to its regulators and DNA, are required to prove key aspects of this model and unveil the mystery behind the magic acts of DNA
entrapment and release by the cohesin ring. REFERENCES Beckoue¨t, F., Srinivasan, M., Roig, M.B., Chan, K.-L., Scheinost, J.C., Batty, P., Hu, B., Petela, N., Gligoris, T., Smith, A.C., et al. (2016). Mol. Cell 61, this issue, 563–574. C¸amdere, G., Guacci, V., Stricklin, J., and Koshland, D. (2015). eLife 4, e11315. Chan, K.L., Roig, M.B., Hu, B., Beckoue¨t, F., Metson, J., and Nasmyth, K. (2012). Cell 150, 961–974. Elbatsh, A.M.O., Haarhuis, J.H.I., Petela, N., Chapard, C., Fish, A., Celie, P.H., Stadnik, M., Ristic, D., Wyman, C., Medema, R.H., et al. (2016). Mol. Cell 61, this issue, 575–588. Gruber, S., Arumugam, P., Katou, Y., Kuglitsch, D., Helmhart, W., Shirahige, K., and Nasmyth, K. (2006). Cell 127, 523–537. Haarhuis, J.H., Elbatsh, A.M., and Rowland, B.D. (2014). Dev. Cell 31, 7–18. Huis in ’t Veld, P.J., Herzog, F., Ladurner, R., Davidson, I.F., Piric, S., Kreidl, E., Bhaskara, V., Aebersold, R., and Peters, J.M. (2014). Science 346, 968–972. Ladurner, R., Bhaskara, V., Huis in ’t Veld, P.J., Davidson, I.F., Kreidl, E., Petzold, G., and Peters, J.M. (2014). Curr. Biol. 24, 2228–2237. Murayama, Y., and Uhlmann, F. (2014). Nature 505, 367–371. Murayama, Y., and Uhlmann, F. (2015). Cell 163, 1628–1640. Remeseiro, S., Cuadrado, A., and Losada, A. (2013). Development 140, 3715–3718.
Molecular Cell 61, February 18, 2016 ª2016 Elsevier Inc. 491
Preview Magic Acts with the Cohesin Ring Hongtao Yu1,* 1Department of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2016.02.003
Recent studies, including two in this issue of Molecular Cell (Elbatsh et al., 2016; Beckoue¨t et al., 2016), cast light on how cohesin regulators harness the energy of ATP hydrolysis to open the cohesin ring and enable dynamic, regulated entrapment of chromosomes. Through topologically entrapping DNA, cohesin rings regulate chromosome segregation, transcription, and DNA repair. Proper regulation of cohesin dynamics on chromosomes is essential for these diverse processes (Haarhuis et al., 2014). Scc2-Scc4 loads cohesin onto chromosomes, whereas Wapl-Pds5 releases cohesin from chromosomes. Eco1 antagonizes Wapl-Pds5 by acetylating Smc3, which stabilizes cohesin on chromosomes and establishes sisterchromatid cohesion during DNA replication and homology-directed repair. Human developmental diseases and cancer have been linked to mutations of cohesin subunits and regulators (Remeseiro et al., 2013). Two reports in this issue of Molecular Cell (Beckoue¨t et al., 2016; Elbatsh et al., 2016), along with other recent studies (C¸amdere et al., 2015; Murayama and Uhlmann, 2014, 2015), provide new insight into the mechanics and regulation of cohesin ring opening, an obligatory step during both loading and release of cohesin. The cohesin ring has three outer gates: the Smc3-Scc1N interface, the Smc1Scc1C interface, and the hinge interface (Figure 1A). Smc1 and Smc3 belong to the ATP-binding cassette (ABC) type of ATPases, which binds two ATP molecules at the dimer interface of two ATPase domains. When ATP bound, the Smc1Smc3 ATPase domains form an inner gate. ATP hydrolysis and nucleotide release drive apart the ATPase domains and open this inner gate. Using recombinant fission yeast cohesin and its loader (Mis4-Ssl3 in fission yeast), Murayama and Uhlmann show that the cohesin loader stimulates cohesin’s ATPase and promotes the topological loading of cohesin onto a circular plasmid (Murayama
and Uhlmann, 2014). Forcing the inner gate closed by a non-hydrolyzable ATP analog traps DNA inside the Smc1Smc3 closure (Murayama and Uhlmann, 2015). They further propose that cohesin loading involves opening the Smc3Scc1N interface, instead of the hinge interface suggested previously (Gruber et al., 2006). Although logically simple, this new proposal needs reconciling with the functional chromosome loading of an Smc3-Scc1 fusion protein in budding yeast. Because the cohesin loader Scc2Scc4 interacts with several cohesin subunits, one intriguing possibility is that it can open the cohesin ring at more than one interface. Strong evidence indicates that the Smc3-Scc1N interface is the conserved DNA exit gate of cohesin. Artificial tethering of Smc3 and Scc1 prevents cohesin release from chromosomes in yeast (Chan et al., 2012). Conversely, mutations that weaken the Smc3-Scc1N interface permit cohesin release in Wapl-depleted human cells (Huis in ’t Veld et al., 2014). These findings suggest involvement of this interface in cohesin release. Beckoue¨t et al. now provide direct evidence that Wapl-Pds5-dependent releasing activity actually disrupts the Smc3Scc1N interface (Beckoue¨t et al., 2016). Using chemical crosslinking, immunoprecipitation, and fluorescence microscopy, they show convincingly that, following the induced cleavage of an engineered Scc1, Wapl-Pds5 actively releases the N-terminal cleavage product of Scc1 (Scc1N) from Smc3 in budding yeast. Murayama and Uhlmann have made similar observations with an in vitro reconstituted cohesin release assay (Murayama and Uhlmann, 2015). Strikingly, this release is inhibited by mutations in
Wapl, Pds5, and core cohesin subunits that stabilize cohesin on chromosomes and bypass the need for Eco1 (Beckoue¨t et al., 2016). Wapl-Pds5-dependent releasing activity can act on soluble cohesin not bound to chromosomes. The ability of Wapl-Pds5 to open the cohesin ring (without entrapped DNA) is consistent with the finding that WaplPds5 can load cohesin onto DNA under certain conditions (Murayama and Uhlmann, 2015). In a complementary study, Elbatsh et al. have discovered that the two ATPase active sites of Smc1-Smc3 are asymmetrically required for Wapl-Pds5dependent cohesin release (Elbatsh et al., 2016). Similar observations have been independently made by Koshland and coworkers (C¸amdere et al., 2015). Smc1-Smc3 contains two asymmetric ATPase active sites, which I have termed the apical and basal sites (Figures 1B and 1C). Elbatsh et al. show that, in budding yeast, mutations of the signature motif and D loop of Smc1 at the apical site restore viability of Eco1 mutant cells and partially suppress their cohesion defects (Elbatsh et al., 2016). Despite impairing the overall ATPase activity of cohesin to a similar degree, corresponding mutations in the signature motif and D loop of Smc3 at the basal site cannot rescue phenotypes caused by Eco1 deficiency. Furthermore, the same Smc1 mutations, but not the Smc3 mutations, inhibit the release of Scc1N from Smc3 (Beckoue¨t et al., 2016). These two sets of cohesin mutants, as well as mutants with deficient ATPase activities at both sites, can be loaded onto chromosomes to some degree, presumably because Scc2-Scc4 can stimulate the ATPase activity of these mutants above the threshold needed for
Molecular Cell 61, February 18, 2016 ª2016 Elsevier Inc. 489
Molecular Cell
Preview
Figure 1. Model for DNA Capture and Release by Cohesin (A) Overall cohesion ring architecture. (B) A structural model of the Smc1-Smc3-Scc1N-Scc1C complex, with bound nucleotides and acetylation acceptor lysines shown in sticks. (C) The two ATPase sites of cohesin. (D) A speculative model of cohesin loading and release by Scc2-Scc4 and Pds5-Wapl. The model mainly serves to illustrate the complicated maneuvers that cohesin must undertake to entrap and release DNA.
loading (Beckoue¨t et al., 2016; C¸amdere et al., 2015; Elbatsh et al., 2016; Murayama and Uhlmann, 2015). These findings establish a critical requirement for ATP hydrolysis at the apical site for Wapl-Pds5-mediated cohesin release from chromosomes. The slowed cohesin release with deficient apical
ATPase activity compensates for the loss of Eco1-dependent Smc3 acetylation, a Wapl-Pds5-antagonizing mechanism. This asymmetric requirement is conserved in human cells, as expression of the same signature motif mutant of human Smc1 alleviates the requirement for the cohesion-establishment factor sororin
490 Molecular Cell 61, February 18, 2016 ª2016 Elsevier Inc.
in cell viability and sister-chromatid cohesion (Elbatsh et al., 2016). Surprisingly, a mutation of the Walker B motif of Smc3 (which cripples the ATPase activity of the apical site) does not prevent Scc1N release from Smc3 (Beckoue¨t et al., 2016). The corresponding mutation does, however, inhibit the
Molecular Cell
Preview Wapl-Pds5-stimulated release of the fission yeast cohesin from DNA in vitro (Murayama and Uhlmann, 2015). The effect of this mutation on cohesin release needs further clarification. Smc3 acetylation by Eco1 has been thought to stabilize cohesin on chromosomes by inhibiting cohesin’s ATPase activity. However, recombinant cohesin harboring acetylation-mimicking mutations has normal ATPase activity (Ladurner et al., 2014). Instead, ATP hydrolysis is required for efficient Smc3 acetylation by Eco1. Murayama and Uhlmann (2015) and C¸amdere et al. (2015) have now explained this conundrum. They show that cohesin topologically bound to DNA has higher ATPase activity (C¸amdere et al., 2015; Murayama and Uhlmann, 2015). This stimulation of ATPase activity by DNA, as well as cohesin loading and release, is inhibited by acetylation-mimicking Smc3 mutations (Murayama and Uhlmann, 2015). These findings suggest that the two acetylation acceptor lysines in Smc3 might directly contact the entrapped DNA, and their acetylation neutralizes the positive charge and is expected to weaken DNA binding. Indeed, the two lysines and neighboring positively charged residues form a basic patch adjacent to the apical ATPase site (Figure 1B). DNA binding to this patch might better orien-
tate ATP for hydrolysis or promote the release of ADP. The available data collectively support the following model for cohesin loading and release (Figure 1D). Scc2-Scc4, cohesin, and DNA form a transient complex, in which Scc2-Scc4 strengthens the engagement and ATP binding of the Smc1-Smc3 ATPase domains. ATP hydrolysis disengages the ATPase domains and opens the cohesin ring to entrap DNA. ATP rebinding closes the inner gate and locks DNA in the top Smc1-Smc3 closure. The entrapped DNA promotes ATP hydrolysis or nucleotide release at the apical site. Regardless of the nucleotide state of the basal site, removal of ATP from the apical site suffices to disengage the ATPase domains and allow DNA to escape from the top closure. Wapl-Pds5 binds to the nucleotide-free cohesin and, upon ATP binding, disrupts the Smc3-Scc1N interface to release DNA. Smc3 acetylation prevents the entrapped DNA from stimulating the apical ATPase activity, blocking inner-gate opening and subsequent Wapl-Pds5-dependent outergate opening. High-resolution structural studies of cohesin in different nucleotide states, alone or bound to its regulators and DNA, are required to prove key aspects of this model and unveil the mystery behind the magic acts of DNA
entrapment and release by the cohesin ring. REFERENCES Beckoue¨t, F., Srinivasan, M., Roig, M.B., Chan, K.-L., Scheinost, J.C., Batty, P., Hu, B., Petela, N., Gligoris, T., Smith, A.C., et al. (2016). Mol. Cell 61, this issue, 563–574. C¸amdere, G., Guacci, V., Stricklin, J., and Koshland, D. (2015). eLife 4, e11315. Chan, K.L., Roig, M.B., Hu, B., Beckoue¨t, F., Metson, J., and Nasmyth, K. (2012). Cell 150, 961–974. Elbatsh, A.M.O., Haarhuis, J.H.I., Petela, N., Chapard, C., Fish, A., Celie, P.H., Stadnik, M., Ristic, D., Wyman, C., Medema, R.H., et al. (2016). Mol. Cell 61, this issue, 575–588. Gruber, S., Arumugam, P., Katou, Y., Kuglitsch, D., Helmhart, W., Shirahige, K., and Nasmyth, K. (2006). Cell 127, 523–537. Haarhuis, J.H., Elbatsh, A.M., and Rowland, B.D. (2014). Dev. Cell 31, 7–18. Huis in ’t Veld, P.J., Herzog, F., Ladurner, R., Davidson, I.F., Piric, S., Kreidl, E., Bhaskara, V., Aebersold, R., and Peters, J.M. (2014). Science 346, 968–972. Ladurner, R., Bhaskara, V., Huis in ’t Veld, P.J., Davidson, I.F., Kreidl, E., Petzold, G., and Peters, J.M. (2014). Curr. Biol. 24, 2228–2237. Murayama, Y., and Uhlmann, F. (2014). Nature 505, 367–371. Murayama, Y., and Uhlmann, F. (2015). Cell 163, 1628–1640. Remeseiro, S., Cuadrado, A., and Losada, A. (2013). Development 140, 3715–3718.
Molecular Cell 61, February 18, 2016 ª2016 Elsevier Inc. 491