- Email: [email protected]
Scc2 Couples Replication Licensing to Sister Chromatid Cohesion in Xenopus Egg Extracts
Current Biology, Vol. 14, 1598–1603, September 7, 2004, 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j .c ub . 20 04 . 07 .0 5 3
Scc2 Couples Replication Licensing to Sister Chromatid Cohesion in Xenopus Egg Extracts Peter J. Gillespie and Tatsuya Hirano* Cold Spring Harbor Laboratory 1 Bungtown Road Cold Spring Harbor, New York 11724
Summary The cohesin complex is a central player in sister chromatid cohesion, a process that ensures the faithful segregation of chromosomes in mitosis and meiosis [1, 2]. Previous genetic studies in yeast show that Scc2/Mis4, a HEAT-repeat-containing protein, is required for the loading of cohesin onto chromatin [3, 4]. In this study, we have identified two isoforms of Scc2 in humans and Xenopus (termed Scc2A and Scc2B), which are encoded by a single gene but have different carboxyl termini created by alternative splicing. Both Scc2A and Scc2B bind to chromatin concomitant with cohesin during DNA replication in Xenopus egg extracts. Simultaneous immunodepletion of Scc2A and Scc2B from the extracts impairs the association of cohesin with chromatin, leading to severe defects in sister chromatid pairing in the subsequent mitosis. The loading of Scc2 onto chromatin is inhibited in extracts treated with geminin but not with p21CIP1, suggesting that this step depends on replication licensing but not on the initiation of DNA replication. Upon mitotic entry, Scc2 is removed from chromatin through a mechanism that requires cdc2 but not aurora B or polo-like kinase. Our results suggest that vertebrate Scc2 couples replication licensing to sister chromatid cohesion by facilitating the loading of cohesin onto chromatin. Results Identification and Characterization of Scc2 in Xenopus Egg Extracts We originally identified two polypeptides of unknown function in the human genome database that share homology with Scc2 in Saccharomyces cerevisiae [5] and Mis4 in Schizosaccharomyces pombe [6]. Most recent studies have shown that the human sequences arise by alternate splicing of a single gene termed NIPBL, which is mutated in Cornelia de Lange syndrome [7]. The two isoforms are 2697 and 2804 amino acids long, being distinguished by variant carboxyl (C) termini (Figure S1). A further BLAST search using the human sequences as query allowed us to identify two overlapping Xenopus EST sequences homologous to the C-terminal region of the longer isoform. This sequence information was then used to isolate two cDNA sequences corresponding to the Xenopus Scc2/NIPBL gene products by reverse transcription-polymerase chain reaction (RT-PCR) using Xenopus egg poly(A)⫹ RNA as template (Figure S1A). In *Correspondence: [email protected]
the current paper, we call the shorter variant XScc2A and the longer XScc2B. The XScc2A cDNA contains an insertion of 61 bp, which results in a frame shift and the creation of a termination site different from that of XScc2B (Figures S1B and S1C). We prepared isoform-specific antibodies against synthetic peptides corresponding to the C-terminal sequences of XScc2A and XScc2B. Each antibody immunoprecipitated a polypeptide of ⵑ300 kDa from a Xenopus egg extract (Figure 1A). The specificity of each immunoprecipitation was demonstrated by competition with antigen peptides. Neither of the anti-XScc2 peptide antibodies coprecipitated the alternative isoform, suggesting that XScc2A and XScc2B do not associate with each other in the extract. We also raised XScc2B-specific antibodies against a recombinant fragment unique to the C terminus of this isoform. As judged by sucrose gradient centrifugation of a whole egg extract, XScc2A and XScc2B were both fractionated into a single peak with a sedimentation coefficient of ⵑ13S (Figure 1B; data not shown). By size exclusion column chromatography, XScc2A eluted in a peak corresponding to a Stokes radius of ⵑ130 A˚ (Figure 1C). These values allowed us to estimate the native molecular mass of XScc2A to be ⵑ720 kDa [8]. The cohesin subunits and Scc2 were fractionated into different peaks by size exclusion column chromatography (Figure 1C), and they did not coimmunoprecipitate with each other (data not shown). These data suggest that XScc2 is present in a large protein complex that is distinct from cohesin in Xenopus egg extracts. While Scc2 was shown to form a complex with Scc4 in S. cerevisiae [9], no vertebrate orthologs of Scc4 have so far been detected in the database. It remains to be determined whether XScc2 forms a homo-oligomer or associates with other proteins in Xenopus egg extracts. Next, we investigated the chromatin binding properties of XScc2 and cohesin in Xenopus egg extracts [10]. When sperm chromatin was incubated with an interphase low-speed supernatant (LSS), nuclear envelope formation began ⵑ30 min after sperm addition, and full nuclear maturation was achieved by 60 min (Figure 1Da, upper panel). DNA replication began after 30 min and was completed by 90 min (Figure 1Da, lower panel). After 2 hr incubation, the extract was driven into mitosis by adding two volumes of cytostatic factor (CSF)arrested LSS. Aliquots were taken from the reaction mixture at various times, and chromatin was isolated and analyzed by immunoblotting (Figure 1Db). The recovery of chromatin was assessed by visualizing histones with Coomassie blue [11]. We found that XScc2A and XScc2B progressively bound to chromatin during interphase, reaching a plateau at 60 min (Figure 1D; data not shown). The kinetics of XScc2 chromatin binding was very similar to that of the cohesin subunits XSMC1 and XRAD21. In contrast, XMcm7, a component of the Mcm2-7 complex, reached a maximum level at 45 min and gradually dissociated from chromatin after 60 min [12]. Upon mitotic entry, the majority of XScc2, like
Coupling of Replication and Cohesion by Scc2 1599
Figure 1. Characterization of Xenopus Scc2 (A) Aliquots of interphase HSS were subject to immunoprecipitation using antibodies raised against peptides specific to XScc2A (lanes 1, 2, 5, and 6) or XScc2B (lanes 3, 4, 7, and 8) in the presence or absence of competing peptides, as indicated. The precipitated fractions were analyzed by immunoblotting using anti-XScc2A (lanes 1–4) and anti-XScc2B (lanes 5–8). (B) Interphase HSS was subject to sucrose gradient centrifugation. Individual fractions were immunoblotted for XScc2A and cohesin subunits (XSMC1, XSMC3, and XRAD21). The peaks of XScc2A (open circle), the XSMC1XSMC3 heterodimer (square) and the cohesin holocomplex (filled circle) are indicated. (C) Interphase HSS was subject to size exclusion chromatography and analyzed as in (B). (D) Sperm chromatin was incubated with interphase LSS to allow DNA replication. After 2 hr, 2 vol of CSF LSS were added to promote entry into mitosis, and the extract was incubated for another 2 hr. a, Nuclear envelope formation and DNA replication were monitored; b, chromatin fractions were isolated at the times indicated and immunoblotted for XScc2A, XSMC1, XRAD21 and XMcm7 (lanes 1–7). The lower portion of the gel was stained with Coomassie Brilliant Blue G to determine the level of histones as a control for chromatin recovery. To check the background level, an aliquot of interphase LSS was incubated with no sperm chromatin for 2 hr and processed in parallel (lane 8).
cohesin, was removed from chromatin (Figure 1Db, lane 7). Thus, XScc2 and cohesin both associate with chromatin immediately after the licensing of replication origins by the Mcm2-7 complex and almost coincident with DNA replication. Scc2 Is Required for Cohesin Loading and Sister Chromatid Cohesion in Xenopus Egg Extracts To determine the role of XScc2A and XScc2B in the establishment of sister chromatid cohesion, the two proteins were immunodepleted from interphase LSS, either individually or simultaneously. The efficiency of immunodepletion was ⵑ95% for each, as judged by quantitative immunoblotting (Figure 2Aa). As expected, cohesin was not codepleted from the extracts upon depletion of XScc2A and/or XScc2B. Nuclear envelope assembly and DNA replication occurred with apparently normal kinetics in the depleted extracts (data not shown). Chromatin fractions were isolated and analyzed by immunoblotting using antibodies against cohesin subunits and XMcm7. The depletion of either XScc2A or XScc2B alone had little, if any, effect on the recovery of cohesin in the chromatin fraction (Figure 2Ab, lanes 2 and 3). However, the association of cohesin with chromatin was severely compromised in the extract depleted of both XScc2A and XScc2B (Figure 2Ab, lane 4). Importantly,
this effect was highly specific to cohesin because the association of other factors with chromatin (such as topoisomerase II␣, ISWI, Plx1, and aurora B) was not affected in the absence of XScc2 (Figure S2). We also found that XScc2A and XScc2B were efficiently recovered on interphase chromatin assembled in an extract depleted of cohesin (Figure 2B; data not shown), suggesting that the association of XScc2 with chromatin is independent of cohesin. The inability of cohesin to associate with chromatin in the absence of XScc2 would be expected to cause defects in sister chromatid cohesion in the subsequent mitosis. To test this possibility, sperm chromatin was incubated with an interphase LSS depleted of both XScc2A and XScc2B, and after replication the mixture was driven into mitosis by the addition of two volumes of CSF LSS. An extract depleted of cohesin was also prepared as a control. The resulting metaphase chromosomes were fixed, isolated, and labeled with an antibody against an SMC core subunit of condensin (XCAP-E/ SMC2). The replication of each chromosome was confirmed by incorporation of biotin-dUTP (data not shown) [13]. No apparent defect in sister chromatid cohesion was observed in chromosomes prepared in the mockdepleted extract or in the extracts depleted of either XScc2A or XScc2B (Figure 2C, ⌬NI, ⌬Scc2A, and
Current Biology 1600
Figure 2. Scc2 Is Required for the Establishment of Sister Chromatid Cohesion in Xenopus Egg Extracts (A) a, Interphase LSS was immunodepleted with nonimmune IgG (lane 6), anti-XScc2A (lane 7), anti-XScc2B (lane 8), or both antiXScc2A and anti-XScc2B antibodies (lane 9). To estimate the efficiency of depletion, 2 l each of the depleted extracts were immunoblotted for XScc2A, XScc2B, XSMC1, and XRAD21 (lanes 6–9) next to known amounts of the mock-depleted extract (lanes 1–5). b, Sperm chromatin was incubated with the depleted LSS for 2 hr, and chromatin fractions were isolated and immunoblotted for XSMC1, XRAD21, and XMcm7. The lower portion of the gel was stained with Coomassie Brilliant Blue G to determine the level of histones as a control for chromatin recovery. (B) Interphase LSS was immunodepleted with nonimmune IgG (lane 1) or anti-cohesin antibodies (lane 2). Sperm chromatin was incubated with the depleted LSS for 2 hr, and chromatin fractions were isolated and analyzed as in (A). (C) Interphase LSS was immunodepleted with nonimmune IgG (⌬NI), anti-XScc2A (⌬Scc2A), anti-XScc2B (⌬Scc2B), both anti-XScc2A and anti-XScc2B (⌬Scc2AB), or anti-cohesin antibodies (⌬Cohesin). Sperm chromatin was incubated with the depleted LSS for 2 hr to allow DNA replication, and 2 vol of CSF LSS were added to promote entry into mitosis. After another 2 hr of incubation, samples were fixed, isolated, and stained with anticondensin (XCAP-E) antibody. Insets show close-ups of selected regions of the chromosomes (indicated by boxes). Scale bar, 10 m (5 m for the insets). (D) Sperm chromatin was incubated in interphase LSS for 2 hr, and individual chromatin samples were exposed to increasing concentrations of KCl as indicated. The chromatin fractions were isolated and analyzed as in (A).
⌬Scc2B). In contrast, chromatids prepared in the extract depleted of both XScc2A and XScc2B were unable to form discernable chromatid pairs and showed gross defects in sister chromatid cohesion (Figure 2C, ⌬Scc2AB). The morphology of these chromatids was indistinguishable from those prepared in the extract depleted of cohesin (Figure 2C, ⌬Cohesin). We next determined whether XScc2 is required to maintain the association of cohesin with chromatin. Sperm chromatin was incubated in an interphase LSS for 2 hr to allow the loading of XScc2 and cohesin, and was then exposed to increasing concentrations of KCl before being isolated. We found that whereas cohesin and XMcm7 remained stably bound to chromatin at 200 mM KCl, XScc2A and XScc2B were easily extracted from the chromatin fraction under the same condition (Figure 2D; data not shown). It is therefore most likely that XScc2 is required for the establishment but not for the maintenance of sister chromatid cohesion, as has been shown in S. cerevisiae [9]. The mechanism by which Scc2 promotes the stable association of cohesin with chromatin remains to be
determined. Scc2 may open the postulated cohesin ring by stimulating the hydrolysis of ATP bound to the SMC catalytic domains, thereby allowing its loading [14, 15]. Alternatively, Scc2 may directly attract cohesin to its chromatin binding sites through its multivalent, proteinprotein interaction surface composed of multiple HEAT repeats [16]. In any case, it is interesting to note that the distribution and activity of the condensins, a different class of SMC protein complexes, are also regulated by their distinct HEAT-repeat-containing subunits [17]. Association of Scc2 with Chromatin Is Dependent on Replication Licensing Scc2 and cohesin associate with chromatin coincident with the initiation of DNA replication (Figure 1D). To test how the establishment of sister chromatid cohesion might be functionally coupled to DNA replication, we examined the association of Scc2 and cohesin with chromatin under conditions where DNA replication is inhibited at different stages. The addition of geminin to egg extracts inhibits replication licensing by blocking the function of Cdt1, an essential component of the
Coupling of Replication and Cohesion by Scc2 1601
Figure 3. Scc2 Couples Replication Licensing to the Establishment of Sister Chromatid Cohesion in Xenopus Egg Extracts (A) Interphase LSS was treated with buffer alone, 40 nM gemininDEL, or 25 nM p21CIP1. a, Sperm chromatin was incubated in the treated extracts for 2 hr, and the extent of DNA replication was measured; b, chromatin fractions were isolated from the control (lanes 1–4), geminin-treated (lanes 5–8), and p21treated (lanes 9–12) extracts at the times indicated and immunoblotted for XScc2A, XSMC1, XRAD21, and XMcm7. The lower portion of the gel was stained with Coomassie Brilliant Blue G to determine the level of histones as a control for chromatin recovery. (B) Interphase LSS was treated with buffer alone (Buffer) or 40 nM gemininDEL prior to sperm addition (Gem. at 0 min) or 40 nM gemininDEL 20 min after sperm addition (Gem. at 20 min). a, Sperm chromatin was incubated in the treated extracts for 2 hr, and the extent of DNA replication was measured; b, chromatin fractions were isolated from the extract treated with no geminin (lanes 1–4), with geminin at 0 min (lanes 5–8), or with geminin at 20 min (lanes 9–12) and analyzed as in (A).
prereplication complex [18, 19]. On the other hand, p21CIP1 blocks the initiation of replication at a later stage by inhibiting cyclin-dependent kinase activity required for origin firing [20]. We first confirmed that the addition of either geminin or p21CIP1 to interphase LSS efficiently inhibited DNA replication (Figure 3Aa) but did not affect the kinetics of nuclear envelope assembly (data not shown). We found that geminin inhibited not only the association of XMcm7 with chromatin but also that of XScc2A and cohesin (Figure 3Ab). In contrast, however, a normal level of XScc2A, cohesin, and XMcm7 was recovered on chromatin assembled in the p21CIP1-treated extract (Figure 3Ab). When geminin was added to an interphase LSS 20 min after sperm addition, by which time replication licensing is complete but nuclear envelope has not yet formed [12], neither DNA replication nor cohesin loading were affected (Figure 3B). This result provides additional evidence for the specific inhibition of replication licensing by geminin and rules out the possibility that geminin blocks cohesin loading in a nonspecific manner. Finally, we prepared an interphase LSS depleted of XORC1, a subunit of the origin recognition complex (ORC), to block XMCM loading and replication, and found that the association of XScc2A and cohesin with chromatin was severely compromised under this condition (Figure S3). Although a number of genetic studies have implicated a functional link between DNA replication and the establishment of sister chromatid cohesion [21–23], its molecular mechanism remains elusive. The current study provides direct biochemical evidence to suggest that the Scc2-mediated association of cohesin with chromatin depends on functional replication licensing but not on the initiation of DNA replication. This finding is apparently contradictory to the previous report that cohesin can be loaded onto chromatin in G1 cells depleted of
Cdc6 in S. cerevisiae [24]. One possibility is that early embryonic cells utilize a unique regulatory mechanism that is advantageous to rapid progression of the cell cycle. It is also important to note that the egg cytoplasm contains a large stockpile of preassembled cohesin complex, whereas yeast cells newly synthesize the Scc1 subunit during the G1 phase of every cell cycle. Conceivably, multiple loading mechanisms exist to support the diverse functions of cohesin, some examples of which have been reported previously [25–27]. Mitotic Dissociation of Scc2 from Chromatin Depends on Cdc2 but Not Polo or Aurora B We have previously shown that the dissociation of bulk cohesin in prophase is regulated by two mitotic kinases, Plx1 and aurora B [13]. Our current results show that the majority of XScc2 also dissociates from chromatin upon entry into mitosis. To determine how the behavior of XScc2 is regulated during mitosis, both interphase and CSF LSS were either mock depleted, depleted of cdc2, or depleted of both Plx1 and aurora B. The efficiency of immunodepletion was ⬎99% for each protein, as judged by quantitative immunoblotting (Figure 4A). Sperm chromatin was incubated in the depleted interphase LSS for 2 hr, and each extract was then driven into mitosis by the addition of the correspondingly depleted CSF LSS. During interphase, neither depletion affected the association of XScc2A and cohesin with chromatin (Figure 4B, lanes 1–3). Consistent with our previous results [13], the mitotic dissociation of cohesin from chromatin was severely inhibited in the cdc2depleted or Plx1/aurora B-depleted LSS (Figure 4B, lanes 5 and 6, XSMC1 and XRAD21). Interestingly, we found that the dissociation of XScc2A from chromatin was inhibited only in the extract depleted of cdc2 but not in that depleted of Plx1 and aurora B (Figure 4B,
Current Biology 1602
from chromatin, whereas polo and aurora B contribute to the dissociation of cohesin itself [13]. The current study shows that Scc2 facilitates the stable association of cohesin with chromatin in Xenopus egg extracts, and that it does so in a manner dependent on functional replication licensing. Intriguingly, recent studies start to extend the role of Scc2 beyond sister chromatid cohesion. Reduced dosage of the Drosophila Scc2 ortholog called Nipped-B causes a failure in longrange gene activation of homeotic genes [28], and haploinsufficiency of its human counterpart may cause Cornelia de Lange syndrome, a disease characterized by multiple developmental defects [7, 29]. Although it remains to be determined whether these developmental defects are associated with the deregulation of cohesin loading, there is no doubt that future studies of this class of proteins will enrich our understanding of not only chromosome segregation but also the body plan of an organism. Supplemental Data Supplemental Experimental Procedures and three additional figures are available at http://www.current-biology.com/cgi/content/full/ 14/17/1598/DC1. Acknowledgments We are indebted to Anatoliy Li for recombinant geminindel and Julian Blow for XMcm7 and XORC1 antibodies and to Michiko Hirano, David MacCallum, and Ana Losada for technical advice and assistance. P.J.G. was the recipient of an Andrew Seligson Memorial Fellowship and is a Fellow of The Jane Coffin Childs Memorial Fund for Medical Research. This investigation has been aided by grants from the Jane Coffin Childs Memorial Fund for Medical Research to P.J.G. and from the National Institute of Health to T.H. Figure 4. Mitotic Regulation of XScc2 (A) Both interphase and CSF LSS were immunodepleted with nonimmune IgG (lanes 6 and 9), anti-cdc2 (lanes 7 and 10), or both antiPlx1 and anti-aurora B antibodies (lanes 8 and 11). To estimate the efficiency of extract depletion, 2 l of the depleted extracts were immunoblotted for cdc2, Plx1, or aurora B (lanes 6–11) next to known amounts of the mock-depleted extract (lanes 1–5). The relative mobility of XScc2A in the depleted extracts was determined by immunoblotting. The mitosis-specific, cdc2-dependent shift is indicated by an asterisk. (B) Sperm chromatin was added to the depleted interphase LSS to allow DNA replication. After 2 hr, 2 vol of the depleted CSF LSS extracts were added to promote entry into mitosis. Interphase (lanes 1–3) and CSF (lanes 4–6) chromatin was isolated and immunoblotted for XScc2A, XSMC1, XRAD21, and XMcm7. The lower portion of the gel was stained with Coomassie Brilliant Blue G to determine the level of histones as a control for chromatin recovery. Nuclear assembly and chromosome condensation in each extract are indicated as ⫹ or ⫺.
lanes 4–6, XScc2A). XScc2A showed a significant mobility shift upon mitotic entry in both mock-depleted and Plx1/aurora B-depleted extracts but not in the cdc2depleted extract (Figure 4A, lanes 9–11). The Scc2 orthologs from human and Drosophila contain a well-conserved cluster of consensus cdc2 phosphorylation sites in their amino-terminal domain. Thus, the dissociation of XScc2 from chromatin may be regulated directly by cdc2. We suggest that two independent mechanisms operate to tightly regulate and ensure the release of cohesin from chromatin during mitotic prophase: Cdc2 inactivates the loading pathway by dissociating Scc2
Received: July 8, 2004 Revised: July 20, 2004 Accepted: July 21, 2004 Published online: July 30, 2004 References 1. Nasmyth, K. (2001). Disseminating the genome: joining, resolving, and separating sister chromatids during mitosis and meiosis. Annu. Rev. Genet. 35, 673–745. 2. Lee, J.Y., and Orr-Weaver, T.L. (2001). The molecular basis of sister-chromatid cohesion. Annu. Rev. Cell Dev. Biol. 17, 753–777. 3. Toth, A., Ciosk, R., Uhlmann, F., Galova, M., Schleiffer, A., and Nasmyth, K. (1999). Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication. Genes Dev. 13, 320–333. 4. Tomonaga, T., Nagao, K., Kawasaki, Y., Furuya, K., Murakami, A., Morishita, J., Yuasa, T., Sutani, T., Kearsey, S.E., Uhlmann, F., et al. (2000). Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase. Genes Dev. 14, 2757–2770. 5. Michaelis, C., Ciosk, R., and Nasmyth, K. (1997). Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91, 35–45. 6. Furuya, K., Takahashi, K., and Yanagida, M. (1998). Faithful anaphase is ensured by Mis4, a sister chromatid cohesion molecule required in S phase and not destroyed in G1 phase. Genes Dev. 12, 3408–3418. 7. Tonkin, E.T., Wang, T.J., Lisgo, S., Bamshad, M.J., and Strachan, T. (2004). NIPBL, encoding a homolog of fungal Scc2type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome. Nat. Genet. 36, 636–641.
Coupling of Replication and Cohesion by Scc2 1603
8. Siegel, L.M., and Monty, K.J. (1966). Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim. Biophys. Acta 112, 346–362. 9. Ciosk, R., Shirayama, M., Shevchenko, A., Tanaka, T., Toth, A., and Nasmyth, K. (2000). Cohesin’s binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol. Cell 5, 243–254. 10. Losada, A., Hirano, M., and Hirano, T. (1998). Identification of Xenopus SMC protein complexes required for sister chromatid cohesion. Genes Dev. 12, 1986–1997. 11. Gillespie, P.J., Li, A., and Blow, J.J. (2001). Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins. BMC Biochem. 2, 15. 12. Chong, J.P., Mahbubani, H.M., Khoo, C.Y., and Blow, J.J. (1995). Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature 375, 418–421. 13. Losada, A., Hirano, M., and Hirano, T. (2002). Cohesin release is required for sister chromatid resolution, but not for condensinmediated compaction, at the onset of mitosis. Genes Dev. 16, 3004–3016. 14. Arumugam, P., Gruber, S., Tanaka, K., Haering, C.H., Mechtler, K., and Nasmyth, K. (2003). ATP hydrolysis is required for cohesin’s association with chromosomes. Curr. Biol. 13, 1941–1953. 15. Weitzer, S., Lehane, C., and Uhlmann, F. (2003). A model for ATP hydrolysis-dependent binding of cohesin to DNA. Curr. Biol. 13, 1930–1940. 16. Neuwald, A.F., and Hirano, T. (2000). HEAT repeats associated with condensins, cohesins, and other complexes involved in chromosome-related functions. Genome Res. 10, 1445–1452. 17. Ono, T., Losada, A., Hirano, M., Myers, M.P., Neuwald, A.F., and Hirano, T. (2003). Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell 115, 109–121. 18. McGarry, T.J., and Kirschner, M.W. (1998). Geminin, an inhibitor of DNA replication, is degraded during mitosis. Cell 93, 1043– 1053. 19. Tada, S., Li, A., Maiorano, D., Mechali, M., and Blow, J.J. (2001). Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. Nat. Cell Biol. 3, 107–113. 20. Strausfeld, U.P., Howell, M., Rempel, R., Maller, J.L., Hunt, T., and Blow, J.J. (1994). Cip1 blocks the initiation of DNA replication in Xenopus extracts by inhibition of cyclin-dependent kinases. Curr. Biol. 4, 876–883. 21. Hanna, J.S., Kroll, E.S., Lundblad, V., and Spencer, F.A. (2001). Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion. Mol. Cell. Biol. 21, 3144–3158. 22. Mayer, M.L., Gygi, S.P., Aebersold, R., and Hieter, P. (2001). Identification of RFC(Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S. cerevisiae. Mol. Cell 7, 959–970. 23. Edwards, S., Li, C.M., Levy, D.L., Brown, J., Snow, P.M., and Campbell, J.L. (2003). Saccharomyces cerevisiae DNA polymerase epsilon and polymerase sigma interact physically and functionally, suggesting a role for polymerase epsilon in sister chromatid cohesion. Mol. Cell. Biol. 23, 2733–2748. 24. Uhlmann, F., and Nasmyth, K. (1998). Cohesion between sister chromatids must be established during DNA replication. Curr. Biol. 8, 1095–1101. 25. Nonaka, N., Kitajima, T., Yokobayashi, S., Xiao, G., Yamamoto, M., Grewal, S.I., and Watanabe, Y. (2002). Recruitment of cohesin to heterochromatic regions by Swi6/HP1 in fission yeast. Nat. Cell Biol. 4, 89–93. 26. Kim, J.S., Krasieva, T.B., LaMorte, V., Taylor, A.M., and Yokomori, K. (2002). Specific recruitment of human cohesin to laserinduced DNA damage. J. Biol. Chem. 277, 45149–45153. 27. Hakimi, M.A., Bochar, D.A., Schmiesing, J.A., Dong, Y., Barak, O.G., Speicher, D.W., Yokomori, K., and Shiekhattar, R. (2002). A chromatin remodelling complex that loads cohesin onto human chromosomes. Nature 418, 994–998. 28. Rollins, R.A., Korom, M., Aulner, N., Martens, A., and Dorsett, D. (2004). Drosophila nipped-B protein supports sister chromatid cohesion and opposes the stromalin/Scc3 cohesion factor to
facilitate long-range activation of the cut gene. Mol. Cell. Biol. 24, 3100–3111. 29. Krantz, I.D., McCallum, J., DeScipio, C., Kaur, M., Gillis, L.A., Yaeger, D., Jukofsky, L., Wasserman, N., Bottani, A., Morris, C.A., et al. (2004). Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B. Nat. Genet. 36, 631–635. Accession Numbers The GenBank accession numbers for the sequences of XScc2A and XScc2B reported in this paper are AY661732 and AY661733, respectively.
DOI 10.1016/j .c ub . 20 04 . 07 .0 5 3
Scc2 Couples Replication Licensing to Sister Chromatid Cohesion in Xenopus Egg Extracts Peter J. Gillespie and Tatsuya Hirano* Cold Spring Harbor Laboratory 1 Bungtown Road Cold Spring Harbor, New York 11724
Summary The cohesin complex is a central player in sister chromatid cohesion, a process that ensures the faithful segregation of chromosomes in mitosis and meiosis [1, 2]. Previous genetic studies in yeast show that Scc2/Mis4, a HEAT-repeat-containing protein, is required for the loading of cohesin onto chromatin [3, 4]. In this study, we have identified two isoforms of Scc2 in humans and Xenopus (termed Scc2A and Scc2B), which are encoded by a single gene but have different carboxyl termini created by alternative splicing. Both Scc2A and Scc2B bind to chromatin concomitant with cohesin during DNA replication in Xenopus egg extracts. Simultaneous immunodepletion of Scc2A and Scc2B from the extracts impairs the association of cohesin with chromatin, leading to severe defects in sister chromatid pairing in the subsequent mitosis. The loading of Scc2 onto chromatin is inhibited in extracts treated with geminin but not with p21CIP1, suggesting that this step depends on replication licensing but not on the initiation of DNA replication. Upon mitotic entry, Scc2 is removed from chromatin through a mechanism that requires cdc2 but not aurora B or polo-like kinase. Our results suggest that vertebrate Scc2 couples replication licensing to sister chromatid cohesion by facilitating the loading of cohesin onto chromatin. Results Identification and Characterization of Scc2 in Xenopus Egg Extracts We originally identified two polypeptides of unknown function in the human genome database that share homology with Scc2 in Saccharomyces cerevisiae [5] and Mis4 in Schizosaccharomyces pombe [6]. Most recent studies have shown that the human sequences arise by alternate splicing of a single gene termed NIPBL, which is mutated in Cornelia de Lange syndrome [7]. The two isoforms are 2697 and 2804 amino acids long, being distinguished by variant carboxyl (C) termini (Figure S1). A further BLAST search using the human sequences as query allowed us to identify two overlapping Xenopus EST sequences homologous to the C-terminal region of the longer isoform. This sequence information was then used to isolate two cDNA sequences corresponding to the Xenopus Scc2/NIPBL gene products by reverse transcription-polymerase chain reaction (RT-PCR) using Xenopus egg poly(A)⫹ RNA as template (Figure S1A). In *Correspondence: [email protected]
the current paper, we call the shorter variant XScc2A and the longer XScc2B. The XScc2A cDNA contains an insertion of 61 bp, which results in a frame shift and the creation of a termination site different from that of XScc2B (Figures S1B and S1C). We prepared isoform-specific antibodies against synthetic peptides corresponding to the C-terminal sequences of XScc2A and XScc2B. Each antibody immunoprecipitated a polypeptide of ⵑ300 kDa from a Xenopus egg extract (Figure 1A). The specificity of each immunoprecipitation was demonstrated by competition with antigen peptides. Neither of the anti-XScc2 peptide antibodies coprecipitated the alternative isoform, suggesting that XScc2A and XScc2B do not associate with each other in the extract. We also raised XScc2B-specific antibodies against a recombinant fragment unique to the C terminus of this isoform. As judged by sucrose gradient centrifugation of a whole egg extract, XScc2A and XScc2B were both fractionated into a single peak with a sedimentation coefficient of ⵑ13S (Figure 1B; data not shown). By size exclusion column chromatography, XScc2A eluted in a peak corresponding to a Stokes radius of ⵑ130 A˚ (Figure 1C). These values allowed us to estimate the native molecular mass of XScc2A to be ⵑ720 kDa [8]. The cohesin subunits and Scc2 were fractionated into different peaks by size exclusion column chromatography (Figure 1C), and they did not coimmunoprecipitate with each other (data not shown). These data suggest that XScc2 is present in a large protein complex that is distinct from cohesin in Xenopus egg extracts. While Scc2 was shown to form a complex with Scc4 in S. cerevisiae [9], no vertebrate orthologs of Scc4 have so far been detected in the database. It remains to be determined whether XScc2 forms a homo-oligomer or associates with other proteins in Xenopus egg extracts. Next, we investigated the chromatin binding properties of XScc2 and cohesin in Xenopus egg extracts [10]. When sperm chromatin was incubated with an interphase low-speed supernatant (LSS), nuclear envelope formation began ⵑ30 min after sperm addition, and full nuclear maturation was achieved by 60 min (Figure 1Da, upper panel). DNA replication began after 30 min and was completed by 90 min (Figure 1Da, lower panel). After 2 hr incubation, the extract was driven into mitosis by adding two volumes of cytostatic factor (CSF)arrested LSS. Aliquots were taken from the reaction mixture at various times, and chromatin was isolated and analyzed by immunoblotting (Figure 1Db). The recovery of chromatin was assessed by visualizing histones with Coomassie blue [11]. We found that XScc2A and XScc2B progressively bound to chromatin during interphase, reaching a plateau at 60 min (Figure 1D; data not shown). The kinetics of XScc2 chromatin binding was very similar to that of the cohesin subunits XSMC1 and XRAD21. In contrast, XMcm7, a component of the Mcm2-7 complex, reached a maximum level at 45 min and gradually dissociated from chromatin after 60 min [12]. Upon mitotic entry, the majority of XScc2, like
Coupling of Replication and Cohesion by Scc2 1599
Figure 1. Characterization of Xenopus Scc2 (A) Aliquots of interphase HSS were subject to immunoprecipitation using antibodies raised against peptides specific to XScc2A (lanes 1, 2, 5, and 6) or XScc2B (lanes 3, 4, 7, and 8) in the presence or absence of competing peptides, as indicated. The precipitated fractions were analyzed by immunoblotting using anti-XScc2A (lanes 1–4) and anti-XScc2B (lanes 5–8). (B) Interphase HSS was subject to sucrose gradient centrifugation. Individual fractions were immunoblotted for XScc2A and cohesin subunits (XSMC1, XSMC3, and XRAD21). The peaks of XScc2A (open circle), the XSMC1XSMC3 heterodimer (square) and the cohesin holocomplex (filled circle) are indicated. (C) Interphase HSS was subject to size exclusion chromatography and analyzed as in (B). (D) Sperm chromatin was incubated with interphase LSS to allow DNA replication. After 2 hr, 2 vol of CSF LSS were added to promote entry into mitosis, and the extract was incubated for another 2 hr. a, Nuclear envelope formation and DNA replication were monitored; b, chromatin fractions were isolated at the times indicated and immunoblotted for XScc2A, XSMC1, XRAD21 and XMcm7 (lanes 1–7). The lower portion of the gel was stained with Coomassie Brilliant Blue G to determine the level of histones as a control for chromatin recovery. To check the background level, an aliquot of interphase LSS was incubated with no sperm chromatin for 2 hr and processed in parallel (lane 8).
cohesin, was removed from chromatin (Figure 1Db, lane 7). Thus, XScc2 and cohesin both associate with chromatin immediately after the licensing of replication origins by the Mcm2-7 complex and almost coincident with DNA replication. Scc2 Is Required for Cohesin Loading and Sister Chromatid Cohesion in Xenopus Egg Extracts To determine the role of XScc2A and XScc2B in the establishment of sister chromatid cohesion, the two proteins were immunodepleted from interphase LSS, either individually or simultaneously. The efficiency of immunodepletion was ⵑ95% for each, as judged by quantitative immunoblotting (Figure 2Aa). As expected, cohesin was not codepleted from the extracts upon depletion of XScc2A and/or XScc2B. Nuclear envelope assembly and DNA replication occurred with apparently normal kinetics in the depleted extracts (data not shown). Chromatin fractions were isolated and analyzed by immunoblotting using antibodies against cohesin subunits and XMcm7. The depletion of either XScc2A or XScc2B alone had little, if any, effect on the recovery of cohesin in the chromatin fraction (Figure 2Ab, lanes 2 and 3). However, the association of cohesin with chromatin was severely compromised in the extract depleted of both XScc2A and XScc2B (Figure 2Ab, lane 4). Importantly,
this effect was highly specific to cohesin because the association of other factors with chromatin (such as topoisomerase II␣, ISWI, Plx1, and aurora B) was not affected in the absence of XScc2 (Figure S2). We also found that XScc2A and XScc2B were efficiently recovered on interphase chromatin assembled in an extract depleted of cohesin (Figure 2B; data not shown), suggesting that the association of XScc2 with chromatin is independent of cohesin. The inability of cohesin to associate with chromatin in the absence of XScc2 would be expected to cause defects in sister chromatid cohesion in the subsequent mitosis. To test this possibility, sperm chromatin was incubated with an interphase LSS depleted of both XScc2A and XScc2B, and after replication the mixture was driven into mitosis by the addition of two volumes of CSF LSS. An extract depleted of cohesin was also prepared as a control. The resulting metaphase chromosomes were fixed, isolated, and labeled with an antibody against an SMC core subunit of condensin (XCAP-E/ SMC2). The replication of each chromosome was confirmed by incorporation of biotin-dUTP (data not shown) [13]. No apparent defect in sister chromatid cohesion was observed in chromosomes prepared in the mockdepleted extract or in the extracts depleted of either XScc2A or XScc2B (Figure 2C, ⌬NI, ⌬Scc2A, and
Current Biology 1600
Figure 2. Scc2 Is Required for the Establishment of Sister Chromatid Cohesion in Xenopus Egg Extracts (A) a, Interphase LSS was immunodepleted with nonimmune IgG (lane 6), anti-XScc2A (lane 7), anti-XScc2B (lane 8), or both antiXScc2A and anti-XScc2B antibodies (lane 9). To estimate the efficiency of depletion, 2 l each of the depleted extracts were immunoblotted for XScc2A, XScc2B, XSMC1, and XRAD21 (lanes 6–9) next to known amounts of the mock-depleted extract (lanes 1–5). b, Sperm chromatin was incubated with the depleted LSS for 2 hr, and chromatin fractions were isolated and immunoblotted for XSMC1, XRAD21, and XMcm7. The lower portion of the gel was stained with Coomassie Brilliant Blue G to determine the level of histones as a control for chromatin recovery. (B) Interphase LSS was immunodepleted with nonimmune IgG (lane 1) or anti-cohesin antibodies (lane 2). Sperm chromatin was incubated with the depleted LSS for 2 hr, and chromatin fractions were isolated and analyzed as in (A). (C) Interphase LSS was immunodepleted with nonimmune IgG (⌬NI), anti-XScc2A (⌬Scc2A), anti-XScc2B (⌬Scc2B), both anti-XScc2A and anti-XScc2B (⌬Scc2AB), or anti-cohesin antibodies (⌬Cohesin). Sperm chromatin was incubated with the depleted LSS for 2 hr to allow DNA replication, and 2 vol of CSF LSS were added to promote entry into mitosis. After another 2 hr of incubation, samples were fixed, isolated, and stained with anticondensin (XCAP-E) antibody. Insets show close-ups of selected regions of the chromosomes (indicated by boxes). Scale bar, 10 m (5 m for the insets). (D) Sperm chromatin was incubated in interphase LSS for 2 hr, and individual chromatin samples were exposed to increasing concentrations of KCl as indicated. The chromatin fractions were isolated and analyzed as in (A).
⌬Scc2B). In contrast, chromatids prepared in the extract depleted of both XScc2A and XScc2B were unable to form discernable chromatid pairs and showed gross defects in sister chromatid cohesion (Figure 2C, ⌬Scc2AB). The morphology of these chromatids was indistinguishable from those prepared in the extract depleted of cohesin (Figure 2C, ⌬Cohesin). We next determined whether XScc2 is required to maintain the association of cohesin with chromatin. Sperm chromatin was incubated in an interphase LSS for 2 hr to allow the loading of XScc2 and cohesin, and was then exposed to increasing concentrations of KCl before being isolated. We found that whereas cohesin and XMcm7 remained stably bound to chromatin at 200 mM KCl, XScc2A and XScc2B were easily extracted from the chromatin fraction under the same condition (Figure 2D; data not shown). It is therefore most likely that XScc2 is required for the establishment but not for the maintenance of sister chromatid cohesion, as has been shown in S. cerevisiae [9]. The mechanism by which Scc2 promotes the stable association of cohesin with chromatin remains to be
determined. Scc2 may open the postulated cohesin ring by stimulating the hydrolysis of ATP bound to the SMC catalytic domains, thereby allowing its loading [14, 15]. Alternatively, Scc2 may directly attract cohesin to its chromatin binding sites through its multivalent, proteinprotein interaction surface composed of multiple HEAT repeats [16]. In any case, it is interesting to note that the distribution and activity of the condensins, a different class of SMC protein complexes, are also regulated by their distinct HEAT-repeat-containing subunits [17]. Association of Scc2 with Chromatin Is Dependent on Replication Licensing Scc2 and cohesin associate with chromatin coincident with the initiation of DNA replication (Figure 1D). To test how the establishment of sister chromatid cohesion might be functionally coupled to DNA replication, we examined the association of Scc2 and cohesin with chromatin under conditions where DNA replication is inhibited at different stages. The addition of geminin to egg extracts inhibits replication licensing by blocking the function of Cdt1, an essential component of the
Coupling of Replication and Cohesion by Scc2 1601
Figure 3. Scc2 Couples Replication Licensing to the Establishment of Sister Chromatid Cohesion in Xenopus Egg Extracts (A) Interphase LSS was treated with buffer alone, 40 nM gemininDEL, or 25 nM p21CIP1. a, Sperm chromatin was incubated in the treated extracts for 2 hr, and the extent of DNA replication was measured; b, chromatin fractions were isolated from the control (lanes 1–4), geminin-treated (lanes 5–8), and p21treated (lanes 9–12) extracts at the times indicated and immunoblotted for XScc2A, XSMC1, XRAD21, and XMcm7. The lower portion of the gel was stained with Coomassie Brilliant Blue G to determine the level of histones as a control for chromatin recovery. (B) Interphase LSS was treated with buffer alone (Buffer) or 40 nM gemininDEL prior to sperm addition (Gem. at 0 min) or 40 nM gemininDEL 20 min after sperm addition (Gem. at 20 min). a, Sperm chromatin was incubated in the treated extracts for 2 hr, and the extent of DNA replication was measured; b, chromatin fractions were isolated from the extract treated with no geminin (lanes 1–4), with geminin at 0 min (lanes 5–8), or with geminin at 20 min (lanes 9–12) and analyzed as in (A).
prereplication complex [18, 19]. On the other hand, p21CIP1 blocks the initiation of replication at a later stage by inhibiting cyclin-dependent kinase activity required for origin firing [20]. We first confirmed that the addition of either geminin or p21CIP1 to interphase LSS efficiently inhibited DNA replication (Figure 3Aa) but did not affect the kinetics of nuclear envelope assembly (data not shown). We found that geminin inhibited not only the association of XMcm7 with chromatin but also that of XScc2A and cohesin (Figure 3Ab). In contrast, however, a normal level of XScc2A, cohesin, and XMcm7 was recovered on chromatin assembled in the p21CIP1-treated extract (Figure 3Ab). When geminin was added to an interphase LSS 20 min after sperm addition, by which time replication licensing is complete but nuclear envelope has not yet formed [12], neither DNA replication nor cohesin loading were affected (Figure 3B). This result provides additional evidence for the specific inhibition of replication licensing by geminin and rules out the possibility that geminin blocks cohesin loading in a nonspecific manner. Finally, we prepared an interphase LSS depleted of XORC1, a subunit of the origin recognition complex (ORC), to block XMCM loading and replication, and found that the association of XScc2A and cohesin with chromatin was severely compromised under this condition (Figure S3). Although a number of genetic studies have implicated a functional link between DNA replication and the establishment of sister chromatid cohesion [21–23], its molecular mechanism remains elusive. The current study provides direct biochemical evidence to suggest that the Scc2-mediated association of cohesin with chromatin depends on functional replication licensing but not on the initiation of DNA replication. This finding is apparently contradictory to the previous report that cohesin can be loaded onto chromatin in G1 cells depleted of
Cdc6 in S. cerevisiae [24]. One possibility is that early embryonic cells utilize a unique regulatory mechanism that is advantageous to rapid progression of the cell cycle. It is also important to note that the egg cytoplasm contains a large stockpile of preassembled cohesin complex, whereas yeast cells newly synthesize the Scc1 subunit during the G1 phase of every cell cycle. Conceivably, multiple loading mechanisms exist to support the diverse functions of cohesin, some examples of which have been reported previously [25–27]. Mitotic Dissociation of Scc2 from Chromatin Depends on Cdc2 but Not Polo or Aurora B We have previously shown that the dissociation of bulk cohesin in prophase is regulated by two mitotic kinases, Plx1 and aurora B [13]. Our current results show that the majority of XScc2 also dissociates from chromatin upon entry into mitosis. To determine how the behavior of XScc2 is regulated during mitosis, both interphase and CSF LSS were either mock depleted, depleted of cdc2, or depleted of both Plx1 and aurora B. The efficiency of immunodepletion was ⬎99% for each protein, as judged by quantitative immunoblotting (Figure 4A). Sperm chromatin was incubated in the depleted interphase LSS for 2 hr, and each extract was then driven into mitosis by the addition of the correspondingly depleted CSF LSS. During interphase, neither depletion affected the association of XScc2A and cohesin with chromatin (Figure 4B, lanes 1–3). Consistent with our previous results [13], the mitotic dissociation of cohesin from chromatin was severely inhibited in the cdc2depleted or Plx1/aurora B-depleted LSS (Figure 4B, lanes 5 and 6, XSMC1 and XRAD21). Interestingly, we found that the dissociation of XScc2A from chromatin was inhibited only in the extract depleted of cdc2 but not in that depleted of Plx1 and aurora B (Figure 4B,
Current Biology 1602
from chromatin, whereas polo and aurora B contribute to the dissociation of cohesin itself [13]. The current study shows that Scc2 facilitates the stable association of cohesin with chromatin in Xenopus egg extracts, and that it does so in a manner dependent on functional replication licensing. Intriguingly, recent studies start to extend the role of Scc2 beyond sister chromatid cohesion. Reduced dosage of the Drosophila Scc2 ortholog called Nipped-B causes a failure in longrange gene activation of homeotic genes [28], and haploinsufficiency of its human counterpart may cause Cornelia de Lange syndrome, a disease characterized by multiple developmental defects [7, 29]. Although it remains to be determined whether these developmental defects are associated with the deregulation of cohesin loading, there is no doubt that future studies of this class of proteins will enrich our understanding of not only chromosome segregation but also the body plan of an organism. Supplemental Data Supplemental Experimental Procedures and three additional figures are available at http://www.current-biology.com/cgi/content/full/ 14/17/1598/DC1. Acknowledgments We are indebted to Anatoliy Li for recombinant geminindel and Julian Blow for XMcm7 and XORC1 antibodies and to Michiko Hirano, David MacCallum, and Ana Losada for technical advice and assistance. P.J.G. was the recipient of an Andrew Seligson Memorial Fellowship and is a Fellow of The Jane Coffin Childs Memorial Fund for Medical Research. This investigation has been aided by grants from the Jane Coffin Childs Memorial Fund for Medical Research to P.J.G. and from the National Institute of Health to T.H. Figure 4. Mitotic Regulation of XScc2 (A) Both interphase and CSF LSS were immunodepleted with nonimmune IgG (lanes 6 and 9), anti-cdc2 (lanes 7 and 10), or both antiPlx1 and anti-aurora B antibodies (lanes 8 and 11). To estimate the efficiency of extract depletion, 2 l of the depleted extracts were immunoblotted for cdc2, Plx1, or aurora B (lanes 6–11) next to known amounts of the mock-depleted extract (lanes 1–5). The relative mobility of XScc2A in the depleted extracts was determined by immunoblotting. The mitosis-specific, cdc2-dependent shift is indicated by an asterisk. (B) Sperm chromatin was added to the depleted interphase LSS to allow DNA replication. After 2 hr, 2 vol of the depleted CSF LSS extracts were added to promote entry into mitosis. Interphase (lanes 1–3) and CSF (lanes 4–6) chromatin was isolated and immunoblotted for XScc2A, XSMC1, XRAD21, and XMcm7. The lower portion of the gel was stained with Coomassie Brilliant Blue G to determine the level of histones as a control for chromatin recovery. Nuclear assembly and chromosome condensation in each extract are indicated as ⫹ or ⫺.
lanes 4–6, XScc2A). XScc2A showed a significant mobility shift upon mitotic entry in both mock-depleted and Plx1/aurora B-depleted extracts but not in the cdc2depleted extract (Figure 4A, lanes 9–11). The Scc2 orthologs from human and Drosophila contain a well-conserved cluster of consensus cdc2 phosphorylation sites in their amino-terminal domain. Thus, the dissociation of XScc2 from chromatin may be regulated directly by cdc2. We suggest that two independent mechanisms operate to tightly regulate and ensure the release of cohesin from chromatin during mitotic prophase: Cdc2 inactivates the loading pathway by dissociating Scc2
Received: July 8, 2004 Revised: July 20, 2004 Accepted: July 21, 2004 Published online: July 30, 2004 References 1. Nasmyth, K. (2001). Disseminating the genome: joining, resolving, and separating sister chromatids during mitosis and meiosis. Annu. Rev. Genet. 35, 673–745. 2. Lee, J.Y., and Orr-Weaver, T.L. (2001). The molecular basis of sister-chromatid cohesion. Annu. Rev. Cell Dev. Biol. 17, 753–777. 3. Toth, A., Ciosk, R., Uhlmann, F., Galova, M., Schleiffer, A., and Nasmyth, K. (1999). Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication. Genes Dev. 13, 320–333. 4. Tomonaga, T., Nagao, K., Kawasaki, Y., Furuya, K., Murakami, A., Morishita, J., Yuasa, T., Sutani, T., Kearsey, S.E., Uhlmann, F., et al. (2000). Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase. Genes Dev. 14, 2757–2770. 5. Michaelis, C., Ciosk, R., and Nasmyth, K. (1997). Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91, 35–45. 6. Furuya, K., Takahashi, K., and Yanagida, M. (1998). Faithful anaphase is ensured by Mis4, a sister chromatid cohesion molecule required in S phase and not destroyed in G1 phase. Genes Dev. 12, 3408–3418. 7. Tonkin, E.T., Wang, T.J., Lisgo, S., Bamshad, M.J., and Strachan, T. (2004). NIPBL, encoding a homolog of fungal Scc2type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome. Nat. Genet. 36, 636–641.
Coupling of Replication and Cohesion by Scc2 1603
8. Siegel, L.M., and Monty, K.J. (1966). Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim. Biophys. Acta 112, 346–362. 9. Ciosk, R., Shirayama, M., Shevchenko, A., Tanaka, T., Toth, A., and Nasmyth, K. (2000). Cohesin’s binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol. Cell 5, 243–254. 10. Losada, A., Hirano, M., and Hirano, T. (1998). Identification of Xenopus SMC protein complexes required for sister chromatid cohesion. Genes Dev. 12, 1986–1997. 11. Gillespie, P.J., Li, A., and Blow, J.J. (2001). Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins. BMC Biochem. 2, 15. 12. Chong, J.P., Mahbubani, H.M., Khoo, C.Y., and Blow, J.J. (1995). Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature 375, 418–421. 13. Losada, A., Hirano, M., and Hirano, T. (2002). Cohesin release is required for sister chromatid resolution, but not for condensinmediated compaction, at the onset of mitosis. Genes Dev. 16, 3004–3016. 14. Arumugam, P., Gruber, S., Tanaka, K., Haering, C.H., Mechtler, K., and Nasmyth, K. (2003). ATP hydrolysis is required for cohesin’s association with chromosomes. Curr. Biol. 13, 1941–1953. 15. Weitzer, S., Lehane, C., and Uhlmann, F. (2003). A model for ATP hydrolysis-dependent binding of cohesin to DNA. Curr. Biol. 13, 1930–1940. 16. Neuwald, A.F., and Hirano, T. (2000). HEAT repeats associated with condensins, cohesins, and other complexes involved in chromosome-related functions. Genome Res. 10, 1445–1452. 17. Ono, T., Losada, A., Hirano, M., Myers, M.P., Neuwald, A.F., and Hirano, T. (2003). Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell 115, 109–121. 18. McGarry, T.J., and Kirschner, M.W. (1998). Geminin, an inhibitor of DNA replication, is degraded during mitosis. Cell 93, 1043– 1053. 19. Tada, S., Li, A., Maiorano, D., Mechali, M., and Blow, J.J. (2001). Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. Nat. Cell Biol. 3, 107–113. 20. Strausfeld, U.P., Howell, M., Rempel, R., Maller, J.L., Hunt, T., and Blow, J.J. (1994). Cip1 blocks the initiation of DNA replication in Xenopus extracts by inhibition of cyclin-dependent kinases. Curr. Biol. 4, 876–883. 21. Hanna, J.S., Kroll, E.S., Lundblad, V., and Spencer, F.A. (2001). Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion. Mol. Cell. Biol. 21, 3144–3158. 22. Mayer, M.L., Gygi, S.P., Aebersold, R., and Hieter, P. (2001). Identification of RFC(Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S. cerevisiae. Mol. Cell 7, 959–970. 23. Edwards, S., Li, C.M., Levy, D.L., Brown, J., Snow, P.M., and Campbell, J.L. (2003). Saccharomyces cerevisiae DNA polymerase epsilon and polymerase sigma interact physically and functionally, suggesting a role for polymerase epsilon in sister chromatid cohesion. Mol. Cell. Biol. 23, 2733–2748. 24. Uhlmann, F., and Nasmyth, K. (1998). Cohesion between sister chromatids must be established during DNA replication. Curr. Biol. 8, 1095–1101. 25. Nonaka, N., Kitajima, T., Yokobayashi, S., Xiao, G., Yamamoto, M., Grewal, S.I., and Watanabe, Y. (2002). Recruitment of cohesin to heterochromatic regions by Swi6/HP1 in fission yeast. Nat. Cell Biol. 4, 89–93. 26. Kim, J.S., Krasieva, T.B., LaMorte, V., Taylor, A.M., and Yokomori, K. (2002). Specific recruitment of human cohesin to laserinduced DNA damage. J. Biol. Chem. 277, 45149–45153. 27. Hakimi, M.A., Bochar, D.A., Schmiesing, J.A., Dong, Y., Barak, O.G., Speicher, D.W., Yokomori, K., and Shiekhattar, R. (2002). A chromatin remodelling complex that loads cohesin onto human chromosomes. Nature 418, 994–998. 28. Rollins, R.A., Korom, M., Aulner, N., Martens, A., and Dorsett, D. (2004). Drosophila nipped-B protein supports sister chromatid cohesion and opposes the stromalin/Scc3 cohesion factor to
facilitate long-range activation of the cut gene. Mol. Cell. Biol. 24, 3100–3111. 29. Krantz, I.D., McCallum, J., DeScipio, C., Kaur, M., Gillis, L.A., Yaeger, D., Jukofsky, L., Wasserman, N., Bottani, A., Morris, C.A., et al. (2004). Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B. Nat. Genet. 36, 631–635. Accession Numbers The GenBank accession numbers for the sequences of XScc2A and XScc2B reported in this paper are AY661732 and AY661733, respectively.