Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex

Santanu K. Ghosh; Chu-Chun Huang; Sujata Hajra; Makkuni Jayaram

doi:10.1093/nar/gkp993

Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex

Ghosh, Santanu K.; Huang, Chu-Chun; Hajra, Sujata; Jayaram, Makkuni 2010-01-17 00:00:00 570–584 Nucleic Acids Research, 2010, Vol. 38, No. 2 Published online 17 November 2009 doi:10.1093/nar/gkp993 Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex 1 2 2 2, Santanu K. Ghosh , Chu-Chun Huang , Sujata Hajra and Makkuni Jayaram * 1 2 School of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai 400076, India and Section of Molecular Genetics & Microbiology, University of Texas at Austin, Austin, TX 78712, USA Received June 20, 2009; Revised October 15, 2009; Accepted October 16, 2009 ABSTRACT until they have been bioriented on the mitotic spindle. When pairing is annulled in anaphase, the sisters split as Sister chromatid cohesion, crucial for faithful segre- under, and are pulled apart by spindle forces and dynamics gation of replicated chromosomes in eukaryotes, towards opposite cell poles. Union of sister chromatids is is mediated by the multi-subunit protein complex mediated by a multi-subunit protein complex, cohesin, cohesin. The Saccharomyces cerevisiae plasmid 2 and their separation by a site-speciﬁc protease, separase, micron circle mimics chromosomes in assembling that cleaves the cohesin component Mcd1 (1–3). By suitably modulating the ‘pairing-unpairing’ strategy, cohesin at its partitioning locus. The plasmid is a cohesin also promotes equal but reductional segregation multi-copy selfish DNA element that resides in the of chromosomes during meiosis (4). nucleus and propagates itself stably, presumably In the budding yeast Saccharomyces cerevisiae, cell cycle with assistance from cohesin. In metaphase cell dependent assembly and disassembly of cohesin occurs lysates, or fractions enriched for their cohesed not only on chromosomes but also on the 2 micron state by sedimentation, plasmid molecules are plasmid (5)—a multi-copy DNA circle that exhibits trapped topologically by the protein ring formed by nearly chromosome-like stability in host populations. cohesin. They can be released from cohesin’s Several lines of circumstantial evidence are consistent embrace either by linearizing the DNA or by with a functional role for cohesin in equal partitioning cleaving a cohesin subunit. Assays using two of the plasmid (5–8). The 2 micron circle appears to be distinctly tagged cohesin molecules argue against unique among extrachromosomal elements in its ability to the hand-cuff (an associated pair of monomeric assimilate cohesin, and raises the prospect of an evolution- ary connection between plasmid and chromosome segre- cohesin rings) or the bracelet (a dimeric cohesin gation in Saccharomyces yeast. However, the mechanism ring) model as responsible for establishing plasmid by which cohesin interacts with the plasmid is poorly cohesion. Our cumulative results most easily fit a understood. The possibility that cohesin may play a role model in which a single monomeric cohesin ring, in plasmid physiology that is unrelated, or in addition, to rather than a series of such rings, conjoins a pair segregation cannot be ruled out. of sister plasmids. These features of plasmid The biological function of cohesin is not restricted to cohesion account for its sister-to-sister mode of sister chromatid segregation alone. Consistent with its segregation by cohesin disassembly during ability to tether separate chromosomal segments, cohesin anaphase. The mechanistic similarities of cohesion participates in DNA repair, chromosome morphogenesis between mini-chromosome sisters and 2 micron and transcriptional regulation by long range activation or by blocking the spread of silencing domains (2,9–13). plasmid sisters suggest a potential kinship Several accessory factors and regulatory mechanisms between the plasmid partitioning locus and specify chromatin sites for cohesin recruitment, and deter- centromeres. mine the timing of cohesin assembly, establishment of cohesion and cohesin disassembly. Mutations in cohesin components and regulatory factors have been implicated INTRODUCTION in human developmental disorders collectively termed as The central logic in the faithful segregation of cohesinopathies (14). chromosomes during mitotic division of eukaryotic cells The conserved Smc1 and Smc3 subunits, characterized is to keep duplicated sister chromatids together in pairs by a long 45–50 nm coiled coil connecting a hinge *To whom correspondence should be addressed. Tel: +1 512 471 0966; Fax: +1 512 471 5546; Email: jayaram@icmb.utexas.edu The authors wish it to be known that, in their opinion, the ﬁrst two authors should be regarded as joint First Authors. The Author(s) 2009. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2010, Vol. 38, No. 2 571 domain to a globular head domain, lay the foundation for simultaneously surrounding the duplicated silent copies, cohesin’s architecture (15). Smc1 and Smc3 can form a encircles each of them separately. Recent data indicating V-shaped heterodimer through hydrophobic interactions potential dimerization of human cohesin in an Scc3 (SA1/ between their hinge regions. The kleisin subunit of cohesin SA2)-dependent manner has raised the possibility of a Mcd1 promotes noncovalent crosslinking of the Smc head functional cohesin hand-cuﬀ (25). It is argued that the domains, which potentiates the organization of two shared greater dynamic ﬂexibility of the hand-cuﬀ over the ATPase active sites. Mcd1 also mediates recruitment of more static embrace model is better suited for cohesin’s the ﬁnal component Scc3 to the complex. The ring role in multiple DNA transactions. In vivo modiﬁcations formed by a single cohesin unit is large enough to accom- such as acetylation or phosphorylation of cohesin subunits modate a nascent pair of duplexes, fueling the notion or associated factors (26–31), and the functional relevance that sister chromatid pairing could be established of individual modiﬁcations to speciﬁc pathways of through topological embrace of DNA by cohesin rather cohesion, may bolster the structural complexity/diversity than stable physical interaction between the two. required for modulating the mechanics and dynamics of Furthermore, variations of the basic subunit interactions cohesin’s interaction with DNA. in cohesin could engender alternative modes of embrace, We address here the nature of cohesin’s association with as in the handcuﬀ (snap) model or the bracelet model the 2 micron plasmid. The motivation stems from previous (2,15) (Figure 1A). observations that timely assembly and disassembly of Elegant in vivo and ex vivo experiments by Nasmyth and cohesin are integral steps in the plasmid partitioning colleagues lend credence to the embrace model for sister pathway (5,7). Cell biological assays suggest that cohesin chromatid cohesion in S. cerevisiae (16–20). In a cohesed recruited at the plasmid partitioning locus (STB), with complex of circular minichromosome sisters, association assistance of the plasmid coded proteins Rep1 and of DNA and cohesin can be terminated by opening the Rep2, brings about cohesion between replicated plasmid DNA ring by restriction enzyme digestion or the cohesin copies, and subsequent dissolution of cohesion mediates ring by site-speciﬁc proteolysis. When the cohesin ring is plasmid segregation in a sister-to-sister fashion. The covalently sealed by engineering cysteine crosslinks and a functional similarities between chromosome and plasmid peptide linker at the protein interfaces that mediate ring segregation prompted us to examine the generality of the closure, SDS denaturation fails to release the entrapped topological mechanism proposed for centromere-mediated DNA. Based on the eﬃciency of crosslinking and the replicative cohesion. Does this mechanism also apply to number of cohesin traps per pair of cohesed sisters, the cohesion established at a nonchromosomal locus, namely, embrace model (requiring monomeric cohesin rings) is STB? We ﬁnd that cohesin-STB interaction is topological, favored over hand-cuﬀ and bracelet models (requiring and best ﬁts the embrace model, in which a pair of sister dimeric cohesin rings). Chromosome association with plasmids are entrapped within a single cohesin ring. cohesin can be blocked by artiﬁcially cross-bridging the hinge domains of Smc1 and Smc3 but not by preventing the opening of the cohesin ring at the interfaces between MATERIALS AND METHODS Mcd1 and the Smc head domains. In sum, these Yeast strains and plasmids observations suggest that transient dissociation of hinges The yeast strains used in this study are listed in lets a chromosome into the cohesin ring, and following Supplementary Table S1. replication, cohesion and spindle attachment, cleavage of The plasmid pSG4 was derived from the plasmid Mcd1 lets sister chromatids out of the ring. TetO21CEN4 (20) by the following manipulations. The Despite the logical simplicity and parsimony of the CEN4 sequence was replaced by an EcoRI–BamHI embrace mechanism, alternative non-topological modes fragment containing the 2 micron circle replication of cohesion have not been ruled out (21). Given the origin and STB locus. The ARS1 sequence associated special architectural features of its subunits and the with TRP1 was removed by BamHI plus BglII digestion multiple cellular functions that it is involved in, distinct followed by self-ligation to obtain plasmid pSG4-1. A mechanisms for cohesin’s association with chromatin in a context dependent manner would seem plausible. Whereas DNA fragment bearing six EcoRI sites was inserted into cohesion at the arm regions of S. cerevisiae chromosomes the EcoRI site of pSG4-1. The pUC19 sequences present promotes inter-sister pairing, cohesion at pericentric in pGS4-1 were removed by SalI digestion and self- regions appears to generate DNA loops that would be ligation to derive pSG4, which was recovered by transfor- consistent with intra-sister pairing (22). Transmission mation in yeast. electron microscopy of puriﬁed minichromosome sisters Construction of plasmid pSG5 included the following with associated cohesin reveals a thick rod of cohesin, steps. The TetO21 sequence was deleted from pSG4-1 by presumably containing multiple cohesin units, interacting XhoI digestion, followed sequentially by ﬁlling-in the stag- with the replicated minichromosome copies at one end gered ends by Klenow polymerase, SmaI digestion and (23). It is diﬃcult to reconcile this picture with the self-ligation. The resulting intermediate plasmid was embrace model for cohesion. Furthermore, association named pSG5-1. After removing the pUC19 DNA by of cohesin with the silent mating type locus HMR SalI digestion and self-ligation to generate pSG5, it was during Sir-mediated transcriptional inactivation appears recovered in yeast as described for pSG4. to depart from the conventional topological mecha- To obtain pSG6, the pUC19 sequences were removed nism (24). Evidence suggests that cohesin, instead of from pSG5-1 by NdeI digestion and replaced by a 572 Nucleic Acids Research, 2010, Vol. 38, No. 2 P -CEN3 fragment derived from a previously described Protein analysis by western blotting GAL plasmid pSG1 (6). For recovery of pSG6, the recipient Protein samples for western blot analysis were obtained yeast strain was directly transformed with the ligation by precipitating with 10% trichloroacetic acid and mixture. redissolving the precipitate in SDS-sample buﬀer prior The authenticity of all plasmid constructs transformed to electrophoresis. After electro-blotting onto PVDF into yeast were veriﬁed by diagnostic PCR assays and by membranes and treatment with primary antibodies, restriction enzyme digestion and Southern analysis of total protein bands were visualized using an Amersham DNA prepared from the host strains containing them. chemiluminescence-based detection system (GE Healthcare). The sources for primary antibodies were Immunoprecipitation of cohesin-associated plasmids Covance (Princeton, NJ, USA). Peroxidase-conjugated secondary antibodies were obtained from Bio-Rad (CA, The procedures described by Ivanov and Nasmyth (20) USA). The western signals were quantitated using were followed for immunoprecipitating cohesin or Quantity One software (Bio-Rad). cohesin-associated plasmids. Typically, precultures of the Proteins were eluted from IgG beads by incubation in cdc20 strains harboring the reporter plasmid were elution buﬀer containing 1% SDS for 10 min at 23 C and inoculated into 1 liter of Sc-Trp medium and grown to 20 min at 65 C, and were precipitated with trichloroacetic mid-log phase at 24 C. In order to arrest cells in acid prior to western blot analysis. metaphase, they were incubated at 37 C for an additional period of 2.5 h. In assays employing pTetO21CEN4 (20), Sucrose gradient sedimentation for separation of cohesed 10 mg/ml nocodazole was included in the medium. plasmids: EcoRI digestion and TEV protease cleavage Nocodazole treatment was omitted in assays with STB in gradient fractions reporter plasmids, except when indicated otherwise. Spheroplasting of cells, cell lysis, preparation of cleared The conditions of centrifugation were essentially accord- lysates, immunoprecipitations, restriction enzyme diges- ing to Ivanov and Nasmyth (19), except that the gradient tion and TEV protease cleavage were carried out was from 12.5% to 37.5% sucrose. Digestions with EcoRI according to published protocols (20). Immunopre- (New England Biolabs) and TEV protease (Invitrogen) cipitates were washed thrice with the lysis buﬀer in order were carried out using 30 ml gradient fractions at 4 C for to disrupt loose/non-speciﬁc associations. 5 h. Each reaction mixture contained 20 U of EcoRI or 40 U of TEV protease. Controls were incubated for the same length of time at 4 C in reaction buﬀers without Adsorption of plasmids bound by protein A-TetR to the addition of enzyme. Aliquots of the reactions were IgG beads fractionated by electrophoresis in 1% agarose gels at A subset of the assays performed in this study required the 4 C for 7 h at 1.5 V/cm. The running buﬀer employed immobilization of reporter plasmids associated with was TAE (pH 7.8) without ethidium bromide. When Protein A-TetR on IgG beads. IgG pull-down was per- protein denaturation was required, SDS was added to formed as described by Ivanov and Nasmyth (20). samples to a ﬁnal concentration of 1%, and heated at To minimize nonspeciﬁc binding, beads were washed 65 C for 4 min prior to electrophoresis. Plasmid DNA three times with lysis buﬀer. DNA was eluted from IgG was detected by Southern blotting and hybridization to beads by two successive incubations, using a rotary radiolabeled plasmid-speciﬁc probes. shaker, in buﬀer containing 100 mM anhydrotetracycline (20) for 30 min each at 4 C. RESULTS DNA analysis by Southern blotting General experimental strategies DNA samples for Southern blot analysis were obtained by The general experimental strategies are brieﬂy outlined phenol extraction and ethanol precipitation. After at the outset for a better perspective of the logic of this electrophoresis, transfer to Hybond-XL membranes (GE study and limitations of the analytical methods. Their Healthcare) and hybridization using P-labeled probes, primary objective was to test whether cohesin interacts bands were detected by autoradiography or phosphori- with the 2 micron plasmid topologically rather than by maging. Band intensities were quantitated from establishing stable physical contacts. For simplicity, the phosphorimages. In estimating relative amounts of a two types of interactions are distinguished as ‘topological’ reporter plasmid co-immnoprecipitated with cohesin, or ‘physical’. For the case of topological interaction, the DNA was digested with a restriction enzyme to comple- subsequent aim was to distinguish among three plausible tion prior to Southern blot analysis. By doing so, models: (i) embrace, (ii) hand-cuﬀ and (iii) bracelet supercoiled, nicked and linear plasmids present in the (Figure 1A). The ﬁnal intent was to address the immunoprecipitated samples were converted to a single stoichiometry of cohesin and DNA in the cohesed state. linear form. The STB plasmid and epitope-tagged cohesin reporters DNA was extracted from protein A dynabeads incorporated, as explained below, relevant designs from (Invitrogen) by incubating them twice in succession with those employed by Nasmyth and colleagues for analysis elution buﬀer (50 mM Tris, pH 8.0, 10 mM EDTA and of cohesion in minichromosomes (19,20). Thus, valid 1% SDS) for 15 min each at 65 C. comparisons could be made between results for cohesion Nucleic Acids Research, 2010, Vol. 38, No. 2 573 Figure 1. Topological models for cohesion; trapping an STB reporter plasmid in cohesin-associated form. (A) The subunits of the yeast cohesin complex, and the ring structure they are presumed to assemble, are schematically diagrammed. Shown next to it is a simpliﬁed representation of the cohesin ring used in ﬁgures to follow. The two STB reporter plasmids, pSG4 and pSG6, employed in these studies are symbolized by blue rings. One set of control assays made use of a derivative of pSG4 lacking the TetO21 sequence (pSG5). The three models for topological interaction between cohesin and sister plasmids tested in this study are shown. The hand-cuﬀ is drawn to be consistent with the recent ﬁnding that dimerization of human cohesin is dependent on the Scc3 (SA1/SA2) subunit (25). (B) Following high-speed centrifugation of a cell lysate, DNA from the supernatant (Sup) and ‘chromatin’ pellet fractions, digested with EcoRI, was run in agarose gels and hybridized to a radiolabeled TRP1 probe. Results from a similar fractionation of a CEN4 minichromosome pTetO21CEN4 (20) are shown for comparison. Pl, plasmid; Ch, chromosome. (C) Cleared lysates + 0 (equivalent to ‘Sup’ in B) from metaphase [cir ] or [cir ] cells harboring pSG4 were immunoprecipitated with the HA-antibody and collected on Protein A beads. DNA extracted from the diﬀerent fractions (In, input; U, unbound or ﬂow-through) was probed by a radiolabeled fragment speciﬁc to pSG4. The amount of bound DNA loaded in the right lane was ﬁve times that in the left one. This ratio was kept constant in assays shown in subsequent ﬁgures as well. SC, supercoiled plasmid; L, linear plasmid; N, nicked plasmid. mediated by STB and CEN4. Myc13-tagged Mcd1 and Again, assuming 15% of the plasmid molecules to be two versions of HA-tagged Mcd1 were made use of; associated with cohesin, the actual eﬃciency was engineered into one Mcd1-HA were three adjacent between 33% and 83%. For assays simultaneously copies of the TEV protease cleavage site. Cohesin- employing cohesin(Mcd1-HA6) and cohesin(Mcd1- associated plasmid molecules were immunoprecipitated Myc13), immunoprecipitation eﬃciency for a given from metaphase cell lysates by using antibodies to HA-6 antibody would be dependent on the stoichiometry of or Myc-13 epitopes. In some experiments, the reporter cohesin with respect to DNA. For example, if only a plasmid harboring the TetO21 sequence was pulled single monomeric cohesin complex is involved in pairing down by interaction between IgG and the operator plasmid sisters, the maximum eﬃciency can only be 50%. Cohesin recruitment by the 2 micron plasmid and bound Protein A-TetR fusion protein. Plasmid DNA could be released by anhydrotetracycline, and the subset cohesion of plasmid sisters are intricately linked to of cohesin-associated molecules re-trapped by a cohesin- spindle integrity (6,8). Nocodazole treatment was unsuit- directed antibody. able in our assays for instituting metaphase arrest while Sucrose gradient centrifugation experiments performed maintaining plasmid cohesion. Instead, metaphase during this study revealed the amount of cohesed plasmids cells were enriched through cdc20 arrest or by harvesting in cleared lysates from metaphase cells to be close to 10% populations at appropriate times after release from of the total plasmids, and no >20%. Interpretations of G1 arrest. Only in control assays that employed experimental data pertain to this plasmid population. a minichromosome (a CEN4-based plasmid) or aimed The fraction of plasmid DNA that could be immunopre- to disrupt cohesin assembly at STB was nocodazole cipitated by the HA- or Myc-antibody from cleared employed. lysates ranged from 2% to 7.5% in diﬀerent assays. Cohesion assays in 2 micron circle, unlike those in a Assuming an average of 15% cohesed (or stably cohesin- circular minichromosome, faces the challenge of the associated) plasmids, this corresponded to an eﬃciency multi-copy state of the plasmid and its clustered organi- of immunoprecipitation in the range of 13–50%. zation. Previous experiments showed that a ﬂuorescence In experiments in which plasmid DNA (cohesed and tagged single copy STB reporter plasmid undergoes noncohesed) was ﬁrst pulled down, released and then cohesion in metaphase in the context of the native baited with the HA- or Myc-antibody, the amount of cluster of endogenous plasmids (6). Furthermore, two immunoprecipitated DNA varied from 5% to 12.5%. such reporters, one tagged by red ﬂuorescence and the 574 Nucleic Acids Research, 2010, Vol. 38, No. 2 other by green, segregate red-to-red and green-to-green The DNA retained on the beads was almost exclusively during anaphase. Pairing, even among a population of circular, supercoiled or nicked. That is, only the fraction plasmid molecules, appears to be restricted to sisters. that escaped SnaBI digestion remained trapped by cohesin The majority of experiments reported here, though per- (‘Bound’ in Figure 2A). The Mcd1 (and by inference formed in the multi-copy context, implicitly assume that cohesin) stayed bound to the beads under the conditions of the digestion, as indicated by western blot analysis two sister plasmids constitute the basic unit of cohesion. (data not shown). This assumption was validated by a ﬁnal set of The choice of the single SnaBI site for plasmid digestion experiments utilizing a stand-alone single copy STB was based on its presence in a relatively nucleosome-free plasmid complemented with Rep1 and Rep2 proteins locale of the STB-ORI segment of the 2 micron circle, at in trans. least in the native context of the plasmid genome (32). For veriﬁcation of the SnaBI result, plasmid digestion was also Plasmid-cohesin association is broken by linearizing performed with EcoRI. Incorporation of multiple tandem DNA or cleaving Mcd1 EcoRI sites into the design of pSG4 was intended to Three plausible topological models for chromosome increase cutting eﬃcacy by this enzyme. SnaBI or EcoR1 cohesion are diagrammed in Figure 1A. The ends of the treatment prior to immunoprecipitation left behind nearly DNA are artiﬁcially shown as closed to highlight the all of the linear pSG4 DNA in the supernatant, and pulled linkage between DNA and protein. The circular DNA down almost exclusively the undigested circular form applies to the plasmids used in the studies reported DNA (Figure 2B and C). When plasmid digestion was here. The embrace, bracelet and hand-cuﬀ models are con- carried to completion in the supernatant by an even sistent with the structural features of, and physical higher excess of the enzyme than that used in standard interactions among, cohesin subunits. Additional assays, no DNA was brought down by the antibody models, based on more complex variations of the (Supplementary Figure S2). The restriction enzymes cohesin ring theme, may be envisaged but are not consid- were active not only during the digestion phase but also ered here. While published results from one series of during the immunoprecipitation phase of the assay. experiments support the embrace model (16–20), other Continued cutting of the plasmid on the beads until the lines of evidence leave open alternative possibilities point of DNA extraction could account for the slight (22–25). In experiments described below, we address increase in linear DNA in the bound fraction from whether the interaction of cohesin with the STB locus is enzyme treated samples compared to that from non- accommodated by a ring or rings of cohesin encircling the treated samples (Figure 2B and C). DNA sisters. Digestion of Mcd1 in the cleared lysate or in the The STB reporter plasmid pSG4 (Figure 1A) harboring immunoprecipitated pSG4–cohesin complex with TEV the 2 micron plasmid replication origin, the yeast TRP1 protease annulled the association between DNA and marker and the TetO21 sequence (but no other non-yeast protein (Figure 2D and E). TEV protease treatment DNA) could be maintained with relatively high stability in per se did not aﬀect the topology of the plasmid. The a [cir ] host strain whose endogenous plasmids supplied DNA in the lysate remained almost exclusively circular the partitioning proteins Rep1 and Rep2. High-speed even though it was not pulled down by the HA-antibody centrifugation of an extract from cdc20 arrested once Mcd1 was cleaved (Figure 2D). Similarly, the DNA metaphase cells expressing Mcd1-HA6 yielded released from the beads as a result of TEV protease diges- supernatant fractions (cleared lysates) containing tion was predominantly circular (Figure 2E). The cleavage roughly 40–50% of the pSG4 minichromatin with very of Mcd1 in the lysate by TEV protease was nearly quan- little contamination from chromosomal chromatin titative, as determined by western blotting of the total (Figure 1B, left). For comparison, a similar procedure protein fraction in the lysate or the protein fraction applied to nocodazole (10 mg/ml) treated metaphase bound to the HA-antibody-Protein A beads (Figure 2F). cells partitioned nearly 80% of a CEN4 containing The combined results from DNA digestion and protein minichromosome (20) into the cleared lysate with cleavage suggest that the mainstay of the interaction slightly higher chromosomal contamination (Figure 1B, between cohesin and replicated STB plasmids in right). The pSG4 minichromatin, presumably associated metaphase is topological. The possibility that cohesin with cohesin(Mcd1-HA6), could be immunoprecipitated has poor aﬃnity for linear DNA is unlikely, as it by the HA-antibody (Figure 1C, left). Consistent with normally acts on linear chromosomes as they are being the requirement of the Rep proteins and an intact replicated. It is diﬃcult to imagine how cohesin, acting spindle for cohesin assembly at STB (5,8), immunopre- locally at or near the replication fork, can sense the cipitation of pSG4 was not detected in lysates from [cir ] global topology of DNA. If cohesin can slide or track host cells (Figure 1C, right) or nocodazole treated [cir ] along DNA, it would fall oﬀ from the ends of linear cells (Supplementary Figure S1). DNA regardless of whether DNA-protein association is Next, we inquired into the nature of the DNA–protein topological or physical. However, it would seem unlikely interaction in cohesion-associated pSG4. When the that opening a peptide bond would terminate physical plasmid was digested with SnaBI on Protein A beads association of DNA with cohesin. Ivanov and Nasmyth used to pull down the HA-antibody–cohesin–plasmid (20) showed that severance of either the Mcd1 or the Smc3 complex, the linearized DNA was released nearly quan- subunits of cohesin would suﬃce to free minichro- titatively into the supernatant (S + W in Figure 2A). mosomes from cohesin’s grasp. Furthermore, for small Nucleic Acids Research, 2010, Vol. 38, No. 2 575 Figure 2. Release of an STB reporter plasmid from cohesin’s grasp by linearizing DNA or cleaving Mcd1. The consequences of cutting DNA or protein on the topological association between plasmid and cohesin are schematically indicated. (A) Cohesin associated pSG4 was adsorbed on HA- antibody and immobilized on Protein A beads as described under Figure 1. After digestion with SnaBI, DNA released into the supernatant (S) and wash fractions (W) and that retained on the beads (Bound) were analyzed. (B and C) SnaBI or EcoRI digestion was performed in the cleared lysates prior to immunoprecipitation by HA-antibody. (D) Cleared lysates were treated with TEV protease, and then subjected to pull-down by HA- antibody and Protein A beads. (E) Cohesin bound plasmid from cleared lysates was treated with HA-antibody, trapped on Protein A beads, and subjected to TEV protease treatment. (F) Cleavage of Mcd1-HA6 by TEV protease in the cleared lysates (corresponding to the DNA analysis shown in D) was monitored by western blotting using HA-antibody. The amount of protein analyzed from the bead-bound fraction was four equivalents of that from the input. The identity of the weak band above Mcd1 seen in some of the lanes is not known. Its mobility would be consistent with phosphorylation of Mcd1, which occurs during the establishment of cohesion in response to DNA damage (27). DNA molecules, it is circularity rather than supercoiling Cohesin encircles DNA in the form of solitary rings that is important for stable association with cohesin. The and not conjoined ones eﬀect of nicking a single strand on cohesin’s association with DNA is much smaller compared to that of breaking In the embrace and bracelet models for cohesion, sis- both strands (20). The more or less unbiased association ter chromosomes are enfolded by one cohesin ring (or mul- of supercoiled or nicked plasmid circles with cohesin, con- tiple units of a solitary ring) (Figure 1A). The ring sizes are trasted by the lack of association of linear molecules, is diﬀerent in the two cases, the bracelet being a cohesin dimer. In contrast, the hand-cuﬀ model propounds two also evident in our assays (Figure 2A–C). distinct, but mutually associated, monomeric cohesin Strictly, our interpretation applies only to the fraction of plasmids that is stably associated with the cohesin rings. The one ring versus two ring models could be complex, and can be recognized by anti-cohesin distinguished by expressing two types of cohesins, antibodies. While the data favor interlinked cohesin and diﬀerentially tagged by HA6 and Myc13, in the same DNA rings, they do not discriminate among the three cell and in roughly equal amounts (Figure 3). Mcd1- models in Figure 1A. Based on previous results regarding HA6 was cleavable by TEV protease; Mcd1-Myc13 was the nature of 2 micron plasmid cohesion and segregation not. The types of cohesion resulting from the embrace, (6,33), we assume tentatively that two sister plasmid hand-cuﬀ and bracelet models are schematically dia- molecules are held together by the cohesin ring (or rings). grammed in Figure 3A. 576 Nucleic Acids Research, 2010, Vol. 38, No. 2 Figure 3. Plasmid–cohesin association in metaphase cells expressing two diﬀerentially tagged cohesin moieties. (A) The types of sister plasmid cohesion expected from the embrace, hand-cuﬀ and bracelet models in presence of cohesin(Mcd1-HA6) and cohesin(Mcd1-Myc13) in equivalent amounts are indicated (see also Table 1). Whereas Mcd1-HA6 in cohesin could be cleaved by TEV protease, Mcd1-Myc13 could not. (B) Aliquots of cell lysates were probed by western analysis using HA- or Myc-antibody to reveal relative levels of cohesin(Mcd1-HA6) or cohesin(Mcd1-Myc13). Data are shown for the haploid strains expressing either cohesin(Mcd1-HA6) (lane 1) or cohesin(Mcd1-Myc13) (lane 2) and the diploid generated from them expressing both Mcd1 variants (lanes 3–7). Signals from the two antibodies were normalized using aliquots of 75% pure Cre recombinase tagged at its carboxyl-terminus with HA6 as well as Myc13. The mean Myc13 to HA6 signal intensity was 1.83 ± 0.18. The dilution factor between Cre samples stained by Coomassie blue (right) and the corresponding ones analyzed by western blotting (left) was 500 to 1. (C) Aliquots of cell lysates from the haploid and diploid strains, run as in B, were probed using an antibody to native Mcd1. The mean ratio of Mcd1-Myc13 to Mcd1-HA6 was 0.96 ± 0.11. (D) pSG4 molecules from the cleared lysate were ﬁrst immobilized on IgG beads, and then released from them by disrupting TetO–TetR interaction using anhydrotetracycline. (E and F) Following treatment with EcoRI or TEV protease, plasmid pull-down was attempted using HA- or Myc-antibody. Table 1 summarizes the expectations from the three To ensure approximately equal amounts of cohesin models for the loss or retention of cohesin–DNA linkage tagged by HA6 and Myc13 in the diploid host harboring following the opening of the Mcd1-HA6 containing ring pSG4, the Mcd1 variants were expressed from the native by TEV protease. The simplest prediction, and the easiest MCD1 promoter and the native chromosomal locale. This to verify, is that the HA-antibody would not be able to expectation was further veriﬁed by a western blot analysis trap plasmid DNA following the action of TEV protease of the steady state levels of Mcd1-HA6 and Mcd1-Myc13 according to embrace and bracelet models. All plasmid (Figure 3B and C). Diﬀerences in the HA6 and Myc13 molecules embraced by cohesin(Mcd1-HA6) rings, and signals were normalized using Cre recombinase harboring even those associated with mixed cohesin(Mcd1-HA6)– both these epitope tags at its carboxyl-terminus as a ref- cohesin(Mcd1-Myc13) bracelets, would have escaped erence protein (Figure 3B). With appropriate correction, through the opening created in the cohesin(Mcd1-HA6) we estimated the cellular ratio of cohesin(Mcd1-Myc13) ring. This prediction is independent of the number of to cohesin(Mcd1-HA6) to be 1.09 ± 0.12. This result rings surrounding a given pair of DNA sisters. However, was further conﬁrmed by quantifying the proteins using the hand-cuﬀ model predicts 25% of the DNA to be an antibody to native Mcd1, cohesin(Mcd1-Myc13): immunoprecipitated by this antibody, so long as the cohesin(Mcd1-HA6) = 0.96 ± 0.11 (Figure 3C). The ﬁssured cohesin(Mcd1-HA6) stays associated with the extents of cohesin immunoprecipitation by the HA- and intact cohesin(Mcd1-Myc13), which is non-cleavable by Myc-antibodies were more or less equal under our exper- TEV protease. If there is more than one hand-cuﬀ per imental conditions, as indicated by the Southern signals DNA sisters, the DNA fraction immunoprecipitated by from the co-precipitated DNA (Supplementary Figure S3; the HA-antibody will be >25%. Figure 4C and D). Nucleic Acids Research, 2010, Vol. 38, No. 2 577 Table 1. Predictions by the three topological models on the nature of plasmid cohesion Schematic diagrams for plasmid cohesion established by the embrace, bracelet and hand-cuﬀ models from an equal mixture of cohesin(Mcd1-HA6) (cleavable by TEV protease) and cohesin(Mcd1-Myc13) (resistant to TEV protease) are shown in Figure 3 (top). Treatment with TEV protease will cleave all cohesin(Mcd1-HA6) containing rings (see drawings above), opening gates for trapped plasmids to escape. Only those plasmid molecules surrounded by the closed cohesin(Mcd1-Myc13) ring(s) will be stopped. R is the predicted molar ratio of plasmid that can be pulled down by the Myc-antibody before and after TEV protease cleavage; R is the observed value. Agreement between ob experiment (Figure 3B and C) and prediction is indicated by the green rectangles; disagreement by red ones. The embrace model is the winner (with two green rectangles) over the hand-cuﬀ and bracelet models (each with a red rectangle). These assays do not permit a clean distinction between the embrace and the topological hand-cuﬀ models. In order to improve the sensitivity of the assay, pSG4 double bracelet (2.14) models (Table 1). Thus, for one was enriched from metaphase cells by IgG pull-down cohesin ring or a reasonably small number of such rings (Figure 3D), followed by its release in the presence of per pair of sister plasmids, these results discount the anhydrotetracycline. The released plasmid could be bracelet in favor of the embrace model (Table 1). pulled down again by either the HA- or Myc-antibody Multiple cohesin rings per sister pair would increase the with approximately equal eﬃciency (Supplementary relative amount of DNA pulled down by the Myc Figure S3). Attempts to immunoprecipitate DNA after antibody after TEV protease cleavage, due to increased EcoRI digestion of the released plasmid with the HA- or mole fraction of cohesin(Mcd1-Myc13)/cohesin(Mcd1- Myc-antibodies failed (Figure 3E), as expected from Myc13) bracelets around sisters. However, the stoichio- earlier results. Digestion with TEV protease yielded a metry of cohesin to DNA will have to be quite high in distinct, and signiﬁcant, result. While the HA-antibody order to blur the distinction between the two models. failed, the Myc-antibody succeeded in reprecipitating A variation of the hand-cuﬀ model in which the two DNA, almost entirely as intact circles (Figure 3F). This cohesin rings are topologically, not physically, linked result is consistent with the embrace and bracelet models cannot be easily ruled out by the above experiments. In but inconsistent with the hand-cuﬀ model (Table 1). a topological hand-cuﬀ, opening of cohesin(Mcd-HA6) by Quantitations suggest that the molar ratio of plasmid TEV protease would automatically end its association brought down by the Myc-antibody in the absence of, and with cohesin(Mcd1-Myc13). As a result, in the pull- following, TEV protease digestion was 1.2, or nearly down test using the HA-antibody, it would be no diﬀerent equal to 1.0—(compare the ‘Bound’ lanes in the right from the embrace model in which two cohesin rings are two panels of Figure 3C). This value is close to that pre- formed around sister plasmids (Table 1). Further dicted by the embrace model, and signiﬁcantly smaller experiments argue against a hand-cuﬀ formed by a than that anticipated from the single bracelet (3.0) or the catenated pair of cohesin rings (see below). 578 Nucleic Acids Research, 2010, Vol. 38, No. 2 Figure 4. Distinction between embrace and bracelet models for plasmid cohesion: cohesin stoichiometry tested by sequential immunoprecipitation. (A and B) The expected outcomes for plasmid immobilization via TetO aﬃnity interaction followed by DNA linearization were experimentally veriﬁed. Plasmid molecules associated with Protein A-TetR bound to TetO were pulled down by IgG beads. DNA and protein remaining associated with the beads or released into the supernatant in the absence of EcoRI treatment or following EcoRI digestion were followed by Southern and western analyses, respectively. (C) The ﬂow-chart for the two-step immunoprecipitation assays is diagrammed at the top. Plasmids were ﬁrst trapped on IgG beads as in A, and then released by treatment with anhydrotetracycline. Equal amounts of the supernatant containing the freed plasmid were immunoprecipitated with the HA- or Myc-antibody. The leftover plasmid molecules in the supernatant were subjected to a second round of immunoprecipitations. (D) The histograms denote the mean ratios of Southern blot signals for immunoprecipitated DNA from three independent experiments, with the error bars showing standard deviations. Immunoprecipitations with HA- and Myc-antibodies are represented by ‘H’ and ‘M’, respectively. Sequential immunoprecipitations by these antibodies are indicated by the two letters separated by a dash. The ratio of the input (IN) plasmid DNA to that immunoprecipitated by the HA- and Myc-antibodies combined is given as IN/[H + M]. The immunoprecipitable plasmid fractions were 17.33%, 16.50%, 16.46% in individual assays. It may be noted that, the physical and topological (bracelet) over a cohesin hand-cuﬀ as the unit entity in handcuﬀs will give identical results for DNA pull-down, cohesion. However, they do not distinguish between the before and after TEV protease digestion, by the Myc- ring and the bracelet; nor do they reveal the number of antibody (Table 1). They do diﬀer from embrace by one rings or bracelets assembled around paired sister plasmids. or two monomeric cohesin rings in causing one third The ability to immobilize pSG4 alternatively by IgG or the reduction (from 75% to 50%), following TEV protease HA- or Myc-antibody oﬀers a potential tool to resolve cleavage, in plasmid DNA associated with cohesin(Myc- these uncertainties. Once again, the assays were performed 13). However, as is evident from Table 1, the Myc- using metaphase cells of the host strain expressing Mcd1- antibody pull down oﬀers better distinction between the HA6 and Mcd1-Myc13 in approximately equivalent embrace and bracelet models than between embrace and amounts. hand-cuﬀ models. The plasmid, trapped on IgG beads through Protein A-TetR bound to TetO, is expected to bring down Stoichiometry of cohesin-plasmid association: two-step with it associated cohesin(s), cohesin(Mcd1-HA6) and immunoprecipitations support embrace by a single cohesin(Mcd1-Myc13) monotypes (according to embrace monomeric cohesin ring or bracelet) or cohesin(Mcd1-HA6)/cohesin(Mcd1- The experimental outcomes so far favor monomeric Myc13) hybrid type (according to bracelet). cohesin ring(s) (embrace) or dimeric cohesin ring(s) Linearization of the bound pSG4 should release all Nucleic Acids Research, 2010, Vol. 38, No. 2 579 associated cohesin into the supernatant, irrespective of the (IN/[H + M] =6.0 in Figure 4D). Furthermore, the stoichiometry of cohesin rings to DNA. The linear form molar DNA ratio of approximately 4 for the HA/[HA- of pSG4, however, should stay bound to the beads. HA] or Myc/[Myc-Myc] sequence (Figure 4D) These expectations were satisﬁed (Figure 4A and B). As corresponds to an immunoprecipitation eﬃciency of shown by the results in Figure 3, plasmid released from 75% for each antibody. Note also that the DNA the beads by treatment with anhydrotetracycline could amounts pulled down by the HA-antibody and the Myc- be immunoprecipitated using antibodies to associated antibody in the respective ﬁrst step immunoprecipitations cohesin(s). A two-step immunoprecipitation assay could were nearly equal (HA/Myc of 0.88 ± 0.01 in Figure 4D). then be performed to test whether or not cohesin(Mcd1- These values are in agreement with the one ring embrace HA6) and cohesin(Mcd1-Myc13) are capable of simulta- model, which proscribes pull-down by one antibody of the neous association with the plasmid. other’s cognate epitope. One would then expect 75% of Predictions concerning plasmid pull-down by the HA- the plasmid DNA bearing cohesin of one epitope or Myc-antibody are dependent on both the cohesion type, which constitutes half of all the cohesed molecules, model and cohesin stoichiometry. According to the one to come down during the ﬁrst step, and 19% ring embrace model, immunoprecipitation by the HA- [(100 75 = 25) 75%] during the second step. antibody would deplete only plasmid molecules By the two ring embrace or the single bracelet model, embraced by cohesin containing Mcd1-HA6, and leave 50% of the cohesed DNA molecules would display dual those embraced by cohesin containing Mcd1-Myc13 epitope speciﬁcity in cohesin, and be subject to unscathed. As a result, a second round of immunopreci- immunoprecipitation by either the HA- or Myc-antibody. pitations of the ‘depleted’ supernatant individually by For 75% immunoprecipitation eﬃciency, the ﬁrst antibody HA- and Myc-antibodies would skew plasmid pull-down would bring down [(25 + 50 = 75) 75%)] =56% of all in favor of the latter. If the ﬁrst immunodepletion is per- cohesed DNA molecules. Note that 25% of the DNA formed by the Myc-13 antibody, the bias in DNA pull- molecules displaying a single epitope speciﬁcity would down during the second immunoprecipitation would also be competent for immunoprecipitation at the ﬁrst be directed oppositely. According to the one bracelet step. This step would deplete (50 75%) = 37.5 % of the model, the ﬁrst immunoprecipitation with either the HA- dual speciﬁcity molecules from the subsequent round of or Myc-antibody would pull down two thirds of the immunoprecipitation. The fraction of DNA molecules cohesin bracelets bearing the opposite epitope speciﬁcity that the virgin antibody can recognize would thus be in the form of choesin(Mcd1-HA6)–cohesin(Mcd1- (50 37.5) = 12.5% with dual epitope speciﬁcity plus Myc13) hybrid bracelets. The two ring embrace model is 25% with single speciﬁcity. The expected immunopre- also subject to a similar depletion eﬀect due to cohesion cipitation by this antibody is [(12.5 + 25 = 37.5) mediated by a mixed pair of cohesin(Mcd1-HA6) and 75%] =28%. The predicted two fold reduction in cohesin(Mcd1-Myc13) rings. Hence the relative advantage DNA pull-down between the two antibodies (from 56% for the virgin antibody in the second immunoprecipitation to 28%) for their primary encounters with cohesed step is less than that anticipated from the single ring plasmids is not in agreement with the experimental result, embrace model. which showed no such reduction (Figure 4C and D). Quantitations of the Southern signals of immunopre- The two-step immunoprecipitation data are also incon- cipitated DNA from three repeats of the two-step assay sistent with a cohesin hand-cuﬀ, even one in which the (Figure 4C) are graphed in Figure 4D. The virgin antibody individual rings are linked by catenation. displayed a strong advantage in the second step (HA-Myc IP or Myc-HA IP) over the experienced antibody Isolation of STB plasmid sisters paired by cohesin (HA-HA IP or Myc-Myc IP). The [HA-Myc]/[HA-HA] and their separation by linearizing DNA or cleaving and [Myc-HA]/[Myc-Myc] ratios were 4.74 ± 1.46 and Mcd1 ex vivo 4.15 ± 0.64, respectively. In contrast, the molar ratio of Data from Figures 1–4 support the entrapment, in DNA brought down by the HA-antibody in the ﬁrst step metaphase cells, of an STB reporter plasmid as a DNA– to that in the second step following immunoprecipitation protein catenane containing a single protein ring of by the Myc-antibody was 1 (0.95 ± 0.01; HA/[Myc-HA] cohesin. The assumption that two DNA rings are in Figure 4D). The corresponding ratio for immunopre- present within such a catenane is based on the earlier cipitation by the Myc antibody was also the same, 0.98 ± 0.07 (Myc/[HA-Myc] in Figure 4D). The absence ﬁnding that cohesin assembly at STB promotes plasmid of cross-depletion by either antibody during plasmid pull- cohesion followed by sister-to-sister plasmid segregation down is consistent with only one cohesin ring (carrying a (6). In order to directly test our assumption, we have single epitope speciﬁcity; either HA or Myc) bridging a isolated the cohesed form of a single copy STB reporter pair of plasmid sisters. A similar analysis with the CEN4 plasmid from metaphase cells by sucrose gradient sedi- containing minichromosome gave concordant results mentation (19), and interrogated ex vivo the DNA status (Supplementary Figure S4). within it. The total amount of immunoprecipitated DNA, by HA- The single copy reporter plasmid pSG6, 4 kb long, was and Myc-antibodies combined, in the two step assay fashioned after the CEN-STB reporter plasmids used in added up to approximately 15-20% of the input DNA previously published work (6,33). In this pSG4 derivative, (that was released from the IgG beads), accounting for the TetO21 locus was replaced by a P -CEN3 DNA GAL nearly the entire fraction of DNA in the cohesed state fragment. The CEN sequence, while it helped maintain 580 Nucleic Acids Research, 2010, Vol. 38, No. 2 Figure 5. Enrichment of plasmids in their cohesed form from metaphase cells by velocity sedimentation: test of the topological model for cohesion. (A) The experimental regimen for enriching metaphase cells from the appropriate [cir ] host strain harboring pSG6 and going through the cell cycle in glucose or galactose is schematically indicated. At 45 min for the cell cycle in glucose and at 75 min for that in galactose, the predominant population consisted of large budded cells with a single DAPI staining mass at the bud neck. (B) The sedimentation patterns of pSG6 in 12.5–37.5% sucrose gradient were followed under conditions where the plasmid-borne CEN alone or STB alone or neither of the two was active. Samples were run in agarose gels in the cold (4 C) and probed by pSG6-speciﬁc radio-labeled DNA. C, cohesed plasmids; NC, non-cohesed plasmids. (C) Representative fast (F), intermediate (I) and slow (S) sedimenting fractions from the gradient were reanalyzed by agarose gel electrophoresis with or without EcoRI digestion, followed by SDS treatment. For reference, S fractions treated or untreated with EcoRI but without subsequent SDS addition (left panel) and DNA prepared form the lysate by phenol extraction and ethanol precipitation (rightmost lane) were included in the run. (D and E) Representative fractions from the sucrose gradient (fast, slow and intermediate) were treated with EcoRI (D) or with TEV protease (E), and subjected to agarose gel electrophoresis under native conditions. the copy number at or close to unity, could be CEN, STB and ARS incarnations (Figure 5B) shed conditionally inactivated by galactose induced transcrip- light on the DNA stoichiometry in the cohesed form of tion. The plasmid was housed in a [cir ] strain or an the plasmid. Fractions were divided into three categories isogenic [cir ] strain expressing Rep1 and Rep2 from the based on their sedimentation velocities: fast (F; cohesed?), bidirectional GAL1–GAL10 promoter. The plasmid would slow (S; non-cohesed?) and intermediate (I; cohesed plus behave as a true CEN plasmid (or a minichromosome) in non-cohesed?). When they were analyzed by electro- either host strain in the presence of glucose as the carbon phoresis in native agarose gels in the cold (4 C), there source. In galactose, though, it would behave as an ARS was a reversal (as expected) in their relative mobility: the plasmid in the [cir ] host and as an STB plasmid in the fast-sedimenting (heavy) fractions migrated more slowly [cir ]::P [REP1 REP2] host. Fractionation of cleared than the slow-sedimenting (light) fractions. In a typical GAL lysates from metaphase cells by centrifugation through a run, the S group comprised of fractions 31 (start point 12.5–37.5% sucrose gradient resolved the plasmid into of Southern signal for DNA) to 44, the I group 45 to 54 two forms: an ‘uncohesed’ monomer form and a and the F group 55 to 65. The lower mobility band of DNA (C for cohesed) was characteristic of pSG6(CEN) ‘cohesed’ 2 monomer form (Figure 5). The general scheme for enriching metaphase cells from and pSG6(STB), and was absent or nearly so for populations arrested in G1, conditioned in glucose or pSG6(ARS). The sedimentation and gel migration galactose, and then released into the cell cycle is outlined proﬁles of the plasmid containing fractions were reminis- in Figure 5A. The sedimentation proﬁles of pSG6 in its cent of those reported by Ivanov and Nasmyth (19) for Nucleic Acids Research, 2010, Vol. 38, No. 2 581 their minichromosome assays. We inferred therefore that These observations suggest a double-gate mechanism the upper band comprised presumably of cohesed sister for the establishment and annulment of chromosomal plasmids (in association with protein factors in addition cohesion. The logic is similar to that used by DNA topoisomerase II to transport a DNA segment through to cohesin) while the lower band contained noncohesed two oppositely located entrance and exit gates during (NC) plasmid molecules. This inference was subjected to DNA relaxation (34). The diﬀerence in the case further veriﬁcation (see below). The fact that the of cohesin is that the second gate opening event involves pSG6(ARS) proﬁle was almost entirely free of the upper proteolytic cleavage of a protein subunit. Notwithstanding band implies little contribution from catenated or covalent the evidence favoring cohesion by topological DNA– plasmid dimers to the ‘cohesed’ DNA fraction. protein association, the possibility of cohesion by Digestion by EcoRI at 4 C did not alter the DNA physical interaction remains a viable alternative mobility of the S (non-cohesed) fractions of pSG6(STB) (21–24,35). We have now shown that 2 micron plasmid in electrophoresis under native conditions (Figure 5C). molecules, or at least those amongst them that remain However, electrophoresis in presence of SDS without cohesed under the assay conditions, also conform to EcoRI treatment revealed monomeric plasmid DNA, pri- cohesion by the topological dictum. marily as the supercoiled form (70–80%) and the remainder as nicked circles in S, I and F fractions. Sister plasmid cohesion in the 2 micron circle EcoRI treatment resulted in conversion of 60–70% of the DNA into linear molecules which migrated below (but The stable propagation of the 2 micron plasmid is con- almost overlapping with) the nicked circle under SDS- ferred by a partitioning system consisting of the plasmid electrophoresis. Consistent with the extent of linearization coded Rep1 and Rep2 proteins and the cis-acting STB of plasmid by EcoRI, native gel electrophoresis revealed a locus (36,37). Requirement of an active partitioning similar conversion of the cohesed form of pSG6 from the I system is mandated by the organization of the multi- and F fractions to the noncohesed form (Figure 5D). copy plasmid into a tight cluster of 3–5 foci, the cluster Conversion from the cohesed to the non-cohesed being the unit of segregation (38). The REP-STB system form was also promoted by TEV protease cleavage appears to couple plasmid segregation to chromosome (Figure 5E). Breaching of cohesion either by linearization segregation either by appropriating chromosome segrega- of pSG6(STB) or by Mcd1 cleavage was as expected for tion factors or tethering the plasmid cluster to a chromo- topology mediated cohesion. The outcomes of EcoRI and some (5). Association of cohesin with STB, assisted by the TEV protease digestions were quite similar between Rep proteins and an intact mitotic spindle (5,8,39), pSG6(STB) and pSG6(CEN) (Supplementary Figure establishes cohesion between plasmid sisters that S5A–C). Furthermore, the sedimentation assay revealed subsequently segregate one-to-one from each other (6). little cohesed dimers of pSG6 in galactose grown but Our present analyses favor the embrace model for nocodazole treated cells (Supplementary Figure S5D, plasmid cohesion, and suggest a stoichiometry of one bottom). This result attests to the complete inactivation cohesin ring per two sister plasmids (or STBs). of CEN by galactose-induced transcription, and veriﬁes The fraction of cohesin-associated STB reporter the authenticity of STB-mediated cohesion when the plasmid obtained in pull-down and sedimentation mitotic spindle is intact (Supplementary Figure S5D, assays is <20%, comparable to 10–30% reported for top; Figure 5B). minichromosomes (20). In contrast, cohesion of STB The data for plasmid sedimentation followed by ex vivo reporter plasmids as assayed by cell biological methods cleavage assays support the presence of two plasmid in metaphase populations is much higher, >70% (6). monomers per cohesed unit. Results from the previous The loss of cohesion during biochemical manipulations pull-down analyses are most easily explained by the occu- could be due to a physical, and less stable, mode of pancy of this unit by one monomeric cohesin ring. cohesin–DNA interaction. However, mutually concordant Together they are consistent with the embrace model for outcomes from distinct aﬃnity interaction and velocity plasmid cohesion, in which a monomeric cohesin ring sedimentation assays are consistent with a uniform, and holds two sister rings of the STB reporter plasmid in a topological, mode of cohesion in a ﬁnite fraction of tripartite catenane. cohesin-associated plasmids. Molar ratio of DNA rings and cohesin within cohesed DISCUSSION plasmid species: a 2:1 DNA–cohesin tri-link? The topology model for sister chromatid cohesion derives Chromatin immunoprecipitation assays reveal a periodic its support from experiments that convert DNA from distribution of cohesin at 10–15 Kb intervals along chro- circular to linear form or break a polypeptide chain in mosome arms in S. cerevisiae with a higher density cohesin or non-covalently or covalently seal borders at of localization at and around centromeres (40–42). critical points where cohesin subunits interface with each Fluorescent intensity of Smc3-GFP at kinetochores other (16–20). Closure of the hinge gate in cohesin appar- normalized to one copy of the histone H3 variant Cse4 ently blocks entry of a chromosome into its interior (16); per centromere (43), suggests the presence of one cohesin and circular minichromosomes already associated with molecule for every 4 Kb of centric/pericentric DNA (22). cohesin remain trapped until an opening is intro- Our ﬁnding that a pair of sister STB reporter plasmids is duced either in the DNA or in the protein (18–20). likely held together by one cohesin ring agrees well with 582 Nucleic Acids Research, 2010, Vol. 38, No. 2 Figure 6. A single ring formed by a monomeric cohesin complex as the unit of cohesion at STB.(A) The results from Haering et al. (18) for CEN cohesion ruling out ‘double’ rings of cohesin are schematically represented. In their experimental design, covalent protein ring closure required crosslinking two neighboring pairs of cysteines at two locations (green circles) to form a pair of chemical bridges (green triangles). Cross-linking eﬃciency (or probability ‘p’ of circle to triangle conversion) was 55% in their assays. Experiments agreed with entrapment probability of DNA 2 4 sisters ‘P’ equal to p (30%) and not p (9%). Note that, upon SDS treatment during the assay, a physical hand-cuﬀ (but not a topological one) would fall apart to yield monomeric cohesin rings, each with a single trapped plasmid molecule. (B) In the pre-cohesed state of the 2 micron circle, multiple cohesin molecules may interact physically and dynamically at or near STB. Such interactions could be promoted by the cohesin loading factors Scc2 and Scc4, which are required for cohesin assembly on the plasmid (5). Transition to the stable topological association may be mediated by passage of the replisome and closure of a single cohesin ring around a pair of STB sisters. this estimate. It is also consistent with in vivo FRET surrounding the sister DNAs (Supplementary Figure results indicating lack of interaction between more than S6A). In the case of two rings, the expected P is 2 2 2 4 one cohesin complex (44) and biochemical outcomes 1[1p ] =2p p = 51%. However, double ring from induced cohesin ring closure within cohesed models in which the DNA sisters reside within one ring minichromosomes (18). cannot be ruled out (Supplementary Figure S6B). Based on the extent of cohesed minichromosomes A biased hand-cuﬀ, physical or topological, predicts entrapped by chemically crosslinking cohesin subunits, P=p =30%, since two crosslinks would suﬃce to trap Haering et al. (18) argue in favor of cohesion by both sisters. monomeric cohesin rings (embrace model) over dimeric Although multiple cohesin molecules could be involved cohesin rings (hand-cuﬀ and bracelet models). In their in precohesive interactions, likely mediated through assays, covalent protein closure in a monomeric ring DNA-bound cohesin loading factors Scc2 and Scc4 (45). required crosslinking of two pairs of cysteine neighbors Only a subset of such interactions may be consummated resident at distinct locations (green circles) to form a to stable topological cohesion by passage of the replisome. pair of chemical bridges (green triangles) [Figure 6A; In the case of the 2 micron plasmid, only one cohesin ring, adapted from (18)]. For a probability ‘p’ of forming a in most cases, may be closed to encircle the nascent STB single cross-link (p = eﬃciency of the crosslinking duplexes (Figure 6B). agent), the expectation for entrapment of DNA sisters by a monomeric ring is p , since two crosslinks have to Conserved mode of cohesion at CEN and STB: be formed to seal the ring. For a dimeric ring of the evolutionary implications bracelet or hand-cuﬀ type, this value is p . For a value of ‘p’ =55%, experimentally determined by Haering The genetically deﬁned ‘point’ centromere of S. cerevisiae, et al., the observed extent of entrapment ‘P’ matches contrasted by the epigenetically speciﬁed centromeres of 2 4 p = 30%, and not p = 9%. The Haering et al. result is fungi in general, poses a rather puzzling transitional also contrary to two or more monomeric cohesin rings oddity in centromere evolution (46). It has been suggested Nucleic Acids Research, 2010, Vol. 38, No. 2 583 2. Onn,I., Heidinger-Pauli,J.M., Guacci,V., Unal,E. and that the point centromere is a domesticated version of the Koshland,D.E. (2008) Sister chromatid cohesion: a simple partitioning locus of an ancestral 2 micron-like plasmid concept with a complex reality. Annu. Rev. Cell Dev. Biol., 24, that functionally replaced the canonical epigenetic fungal 105–129. centromere. Miniaturization of the centromere and 3. Uhlmann,F. (2004) The mechanism of sister chromatid cohesion. loss of the machinery for establishing pericentric hetero- Exp. Cell Res., 296, 80–85. 4. Watanabe,Y. (2005) Sister chromatid cohesion along arms and at chromatin and RNA interference (47) appear to have been centromeres. Trends Genet., 21, 405–412. related events. It is noteworthy in this regard that the exis- 5. Mehta,S., Yang,X.M., Chan,C.S., Dobson,M.J., Jayaram,M. and tence of 2 micron-related plasmids is limited to fungal Velmurugan,S. 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SUPPLEMENTARY DATA Annu. Rev. Genomics Hum. Genet., 9, 303–320. 15. Nasmyth,K. and Haering,C.H. (2005) The structure and function of Supplementary Data are available at NAR Online. SMC and kleisin complexes. Annu. Rev. Biochem., 74, 595–648. 16. Gruber,S., Arumugam,P., Katou,Y., Kuglitsch,D., Helmhart,W., Shirahige,K. and Nasmyth,K. (2006) Evidence that loading of ACKNOWLEDGEMENTS cohesin onto chromosomes involves opening of its SMC hinge. Cell, 127, 523–537. We thank D. Ivanov, K. Nasmyth, A. Johnson, V. Guacci 17. Gruber,S., Haering,C.H. and Nasmyth,K. (2003) Chromosomal and Doug Koshland for generously sharing yeast strains, cohesin forms a ring. Cell, 112, 765–777. 18. Haering,C.H., Farcas,A.M., Arumugam,P., Metson,J. and plasmids, reagents and equipment. We are grateful to Nasmyth,K. (2008) The cohesin ring concatenates sister DNA C. Haering and D. Ivanov for helpful comments on the molecules. Nature, 454, 297–301. article. We acknowledge Chien-Hui Ma for excellent tech- 19. Ivanov,D. and Nasmyth,K. (2007) A physical assay for sister nical assistance. We appreciate the insightful critique from chromatid cohesion in vitro. Mol. Cell, 27, 300–310. the reviewers that helped improve the article in style and 20. Ivanov,D. and Nasmyth,K. (2005) A topological interaction between cohesin rings and a circular minichromosome. Cell, 122, content. 849–860. 21. Guacci,V. (2007) Sister chromatid cohesion: the cohesin cleavage model does not ring true. Genes Cells, 12, 693–708. FUNDING 22. Yeh,E., Haase,J., Paliulis,L.V., Joglekar,A., Bond,L., Bouck,D., Salmon,E.D. and Bloom,K.S. (2008) Pericentric chromatin is National Institutes of Health (GM064363); Robert F organized into an intramolecular loop in mitosis. Curr. Biol., 18, Welch Foundation award (F-1274, partial). Funding 81–90. for open access charge: National Institutes of Health 23. Surcel,A., Koshland,D., Ma,H. and Simpson,R.T. (2008) (GM064363). 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Publisher: Oxford University Press
Copyright: © The Author(s) 2009. Published by Oxford University Press.
ISSN: 0305-1048
eISSN: 1362-4962
DOI: 10.1093/nar/gkp993
pmid: 19920123
Publisher site: See Article on Publisher Site

Abstract

570–584 Nucleic Acids Research, 2010, Vol. 38, No. 2 Published online 17 November 2009 doi:10.1093/nar/gkp993 Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex 1 2 2 2, Santanu K. Ghosh , Chu-Chun Huang , Sujata Hajra and Makkuni Jayaram * 1 2 School of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai 400076, India and Section of Molecular Genetics & Microbiology, University of Texas at Austin, Austin, TX 78712, USA Received June 20, 2009; Revised October 15, 2009; Accepted October 16, 2009 ABSTRACT until they have been bioriented on the mitotic spindle. When pairing is annulled in anaphase, the sisters split as Sister chromatid cohesion, crucial for faithful segre- under, and are pulled apart by spindle forces and dynamics gation of replicated chromosomes in eukaryotes, towards opposite cell poles. Union of sister chromatids is is mediated by the multi-subunit protein complex mediated by a multi-subunit protein complex, cohesin, cohesin. The Saccharomyces cerevisiae plasmid 2 and their separation by a site-speciﬁc protease, separase, micron circle mimics chromosomes in assembling that cleaves the cohesin component Mcd1 (1–3). By suitably modulating the ‘pairing-unpairing’ strategy, cohesin at its partitioning locus. The plasmid is a cohesin also promotes equal but reductional segregation multi-copy selfish DNA element that resides in the of chromosomes during meiosis (4). nucleus and propagates itself stably, presumably In the budding yeast Saccharomyces cerevisiae, cell cycle with assistance from cohesin. In metaphase cell dependent assembly and disassembly of cohesin occurs lysates, or fractions enriched for their cohesed not only on chromosomes but also on the 2 micron state by sedimentation, plasmid molecules are plasmid (5)—a multi-copy DNA circle that exhibits trapped topologically by the protein ring formed by nearly chromosome-like stability in host populations. cohesin. They can be released from cohesin’s Several lines of circumstantial evidence are consistent embrace either by linearizing the DNA or by with a functional role for cohesin in equal partitioning cleaving a cohesin subunit. Assays using two of the plasmid (5–8). The 2 micron circle appears to be distinctly tagged cohesin molecules argue against unique among extrachromosomal elements in its ability to the hand-cuff (an associated pair of monomeric assimilate cohesin, and raises the prospect of an evolution- ary connection between plasmid and chromosome segre- cohesin rings) or the bracelet (a dimeric cohesin gation in Saccharomyces yeast. However, the mechanism ring) model as responsible for establishing plasmid by which cohesin interacts with the plasmid is poorly cohesion. Our cumulative results most easily fit a understood. The possibility that cohesin may play a role model in which a single monomeric cohesin ring, in plasmid physiology that is unrelated, or in addition, to rather than a series of such rings, conjoins a pair segregation cannot be ruled out. of sister plasmids. These features of plasmid The biological function of cohesin is not restricted to cohesion account for its sister-to-sister mode of sister chromatid segregation alone. Consistent with its segregation by cohesin disassembly during ability to tether separate chromosomal segments, cohesin anaphase. The mechanistic similarities of cohesion participates in DNA repair, chromosome morphogenesis between mini-chromosome sisters and 2 micron and transcriptional regulation by long range activation or by blocking the spread of silencing domains (2,9–13). plasmid sisters suggest a potential kinship Several accessory factors and regulatory mechanisms between the plasmid partitioning locus and specify chromatin sites for cohesin recruitment, and deter- centromeres. mine the timing of cohesin assembly, establishment of cohesion and cohesin disassembly. Mutations in cohesin components and regulatory factors have been implicated INTRODUCTION in human developmental disorders collectively termed as The central logic in the faithful segregation of cohesinopathies (14). chromosomes during mitotic division of eukaryotic cells The conserved Smc1 and Smc3 subunits, characterized is to keep duplicated sister chromatids together in pairs by a long 45–50 nm coiled coil connecting a hinge *To whom correspondence should be addressed. Tel: +1 512 471 0966; Fax: +1 512 471 5546; Email: jayaram@icmb.utexas.edu The authors wish it to be known that, in their opinion, the ﬁrst two authors should be regarded as joint First Authors. The Author(s) 2009. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2010, Vol. 38, No. 2 571 domain to a globular head domain, lay the foundation for simultaneously surrounding the duplicated silent copies, cohesin’s architecture (15). Smc1 and Smc3 can form a encircles each of them separately. Recent data indicating V-shaped heterodimer through hydrophobic interactions potential dimerization of human cohesin in an Scc3 (SA1/ between their hinge regions. The kleisin subunit of cohesin SA2)-dependent manner has raised the possibility of a Mcd1 promotes noncovalent crosslinking of the Smc head functional cohesin hand-cuﬀ (25). It is argued that the domains, which potentiates the organization of two shared greater dynamic ﬂexibility of the hand-cuﬀ over the ATPase active sites. Mcd1 also mediates recruitment of more static embrace model is better suited for cohesin’s the ﬁnal component Scc3 to the complex. The ring role in multiple DNA transactions. In vivo modiﬁcations formed by a single cohesin unit is large enough to accom- such as acetylation or phosphorylation of cohesin subunits modate a nascent pair of duplexes, fueling the notion or associated factors (26–31), and the functional relevance that sister chromatid pairing could be established of individual modiﬁcations to speciﬁc pathways of through topological embrace of DNA by cohesin rather cohesion, may bolster the structural complexity/diversity than stable physical interaction between the two. required for modulating the mechanics and dynamics of Furthermore, variations of the basic subunit interactions cohesin’s interaction with DNA. in cohesin could engender alternative modes of embrace, We address here the nature of cohesin’s association with as in the handcuﬀ (snap) model or the bracelet model the 2 micron plasmid. The motivation stems from previous (2,15) (Figure 1A). observations that timely assembly and disassembly of Elegant in vivo and ex vivo experiments by Nasmyth and cohesin are integral steps in the plasmid partitioning colleagues lend credence to the embrace model for sister pathway (5,7). Cell biological assays suggest that cohesin chromatid cohesion in S. cerevisiae (16–20). In a cohesed recruited at the plasmid partitioning locus (STB), with complex of circular minichromosome sisters, association assistance of the plasmid coded proteins Rep1 and of DNA and cohesin can be terminated by opening the Rep2, brings about cohesion between replicated plasmid DNA ring by restriction enzyme digestion or the cohesin copies, and subsequent dissolution of cohesion mediates ring by site-speciﬁc proteolysis. When the cohesin ring is plasmid segregation in a sister-to-sister fashion. The covalently sealed by engineering cysteine crosslinks and a functional similarities between chromosome and plasmid peptide linker at the protein interfaces that mediate ring segregation prompted us to examine the generality of the closure, SDS denaturation fails to release the entrapped topological mechanism proposed for centromere-mediated DNA. Based on the eﬃciency of crosslinking and the replicative cohesion. Does this mechanism also apply to number of cohesin traps per pair of cohesed sisters, the cohesion established at a nonchromosomal locus, namely, embrace model (requiring monomeric cohesin rings) is STB? We ﬁnd that cohesin-STB interaction is topological, favored over hand-cuﬀ and bracelet models (requiring and best ﬁts the embrace model, in which a pair of sister dimeric cohesin rings). Chromosome association with plasmids are entrapped within a single cohesin ring. cohesin can be blocked by artiﬁcially cross-bridging the hinge domains of Smc1 and Smc3 but not by preventing the opening of the cohesin ring at the interfaces between MATERIALS AND METHODS Mcd1 and the Smc head domains. In sum, these Yeast strains and plasmids observations suggest that transient dissociation of hinges The yeast strains used in this study are listed in lets a chromosome into the cohesin ring, and following Supplementary Table S1. replication, cohesion and spindle attachment, cleavage of The plasmid pSG4 was derived from the plasmid Mcd1 lets sister chromatids out of the ring. TetO21CEN4 (20) by the following manipulations. The Despite the logical simplicity and parsimony of the CEN4 sequence was replaced by an EcoRI–BamHI embrace mechanism, alternative non-topological modes fragment containing the 2 micron circle replication of cohesion have not been ruled out (21). Given the origin and STB locus. The ARS1 sequence associated special architectural features of its subunits and the with TRP1 was removed by BamHI plus BglII digestion multiple cellular functions that it is involved in, distinct followed by self-ligation to obtain plasmid pSG4-1. A mechanisms for cohesin’s association with chromatin in a context dependent manner would seem plausible. Whereas DNA fragment bearing six EcoRI sites was inserted into cohesion at the arm regions of S. cerevisiae chromosomes the EcoRI site of pSG4-1. The pUC19 sequences present promotes inter-sister pairing, cohesion at pericentric in pGS4-1 were removed by SalI digestion and self- regions appears to generate DNA loops that would be ligation to derive pSG4, which was recovered by transfor- consistent with intra-sister pairing (22). Transmission mation in yeast. electron microscopy of puriﬁed minichromosome sisters Construction of plasmid pSG5 included the following with associated cohesin reveals a thick rod of cohesin, steps. The TetO21 sequence was deleted from pSG4-1 by presumably containing multiple cohesin units, interacting XhoI digestion, followed sequentially by ﬁlling-in the stag- with the replicated minichromosome copies at one end gered ends by Klenow polymerase, SmaI digestion and (23). It is diﬃcult to reconcile this picture with the self-ligation. The resulting intermediate plasmid was embrace model for cohesion. Furthermore, association named pSG5-1. After removing the pUC19 DNA by of cohesin with the silent mating type locus HMR SalI digestion and self-ligation to generate pSG5, it was during Sir-mediated transcriptional inactivation appears recovered in yeast as described for pSG4. to depart from the conventional topological mecha- To obtain pSG6, the pUC19 sequences were removed nism (24). Evidence suggests that cohesin, instead of from pSG5-1 by NdeI digestion and replaced by a 572 Nucleic Acids Research, 2010, Vol. 38, No. 2 P -CEN3 fragment derived from a previously described Protein analysis by western blotting GAL plasmid pSG1 (6). For recovery of pSG6, the recipient Protein samples for western blot analysis were obtained yeast strain was directly transformed with the ligation by precipitating with 10% trichloroacetic acid and mixture. redissolving the precipitate in SDS-sample buﬀer prior The authenticity of all plasmid constructs transformed to electrophoresis. After electro-blotting onto PVDF into yeast were veriﬁed by diagnostic PCR assays and by membranes and treatment with primary antibodies, restriction enzyme digestion and Southern analysis of total protein bands were visualized using an Amersham DNA prepared from the host strains containing them. chemiluminescence-based detection system (GE Healthcare). The sources for primary antibodies were Immunoprecipitation of cohesin-associated plasmids Covance (Princeton, NJ, USA). Peroxidase-conjugated secondary antibodies were obtained from Bio-Rad (CA, The procedures described by Ivanov and Nasmyth (20) USA). The western signals were quantitated using were followed for immunoprecipitating cohesin or Quantity One software (Bio-Rad). cohesin-associated plasmids. Typically, precultures of the Proteins were eluted from IgG beads by incubation in cdc20 strains harboring the reporter plasmid were elution buﬀer containing 1% SDS for 10 min at 23 C and inoculated into 1 liter of Sc-Trp medium and grown to 20 min at 65 C, and were precipitated with trichloroacetic mid-log phase at 24 C. In order to arrest cells in acid prior to western blot analysis. metaphase, they were incubated at 37 C for an additional period of 2.5 h. In assays employing pTetO21CEN4 (20), Sucrose gradient sedimentation for separation of cohesed 10 mg/ml nocodazole was included in the medium. plasmids: EcoRI digestion and TEV protease cleavage Nocodazole treatment was omitted in assays with STB in gradient fractions reporter plasmids, except when indicated otherwise. Spheroplasting of cells, cell lysis, preparation of cleared The conditions of centrifugation were essentially accord- lysates, immunoprecipitations, restriction enzyme diges- ing to Ivanov and Nasmyth (19), except that the gradient tion and TEV protease cleavage were carried out was from 12.5% to 37.5% sucrose. Digestions with EcoRI according to published protocols (20). Immunopre- (New England Biolabs) and TEV protease (Invitrogen) cipitates were washed thrice with the lysis buﬀer in order were carried out using 30 ml gradient fractions at 4 C for to disrupt loose/non-speciﬁc associations. 5 h. Each reaction mixture contained 20 U of EcoRI or 40 U of TEV protease. Controls were incubated for the same length of time at 4 C in reaction buﬀers without Adsorption of plasmids bound by protein A-TetR to the addition of enzyme. Aliquots of the reactions were IgG beads fractionated by electrophoresis in 1% agarose gels at A subset of the assays performed in this study required the 4 C for 7 h at 1.5 V/cm. The running buﬀer employed immobilization of reporter plasmids associated with was TAE (pH 7.8) without ethidium bromide. When Protein A-TetR on IgG beads. IgG pull-down was per- protein denaturation was required, SDS was added to formed as described by Ivanov and Nasmyth (20). samples to a ﬁnal concentration of 1%, and heated at To minimize nonspeciﬁc binding, beads were washed 65 C for 4 min prior to electrophoresis. Plasmid DNA three times with lysis buﬀer. DNA was eluted from IgG was detected by Southern blotting and hybridization to beads by two successive incubations, using a rotary radiolabeled plasmid-speciﬁc probes. shaker, in buﬀer containing 100 mM anhydrotetracycline (20) for 30 min each at 4 C. RESULTS DNA analysis by Southern blotting General experimental strategies DNA samples for Southern blot analysis were obtained by The general experimental strategies are brieﬂy outlined phenol extraction and ethanol precipitation. After at the outset for a better perspective of the logic of this electrophoresis, transfer to Hybond-XL membranes (GE study and limitations of the analytical methods. Their Healthcare) and hybridization using P-labeled probes, primary objective was to test whether cohesin interacts bands were detected by autoradiography or phosphori- with the 2 micron plasmid topologically rather than by maging. Band intensities were quantitated from establishing stable physical contacts. For simplicity, the phosphorimages. In estimating relative amounts of a two types of interactions are distinguished as ‘topological’ reporter plasmid co-immnoprecipitated with cohesin, or ‘physical’. For the case of topological interaction, the DNA was digested with a restriction enzyme to comple- subsequent aim was to distinguish among three plausible tion prior to Southern blot analysis. By doing so, models: (i) embrace, (ii) hand-cuﬀ and (iii) bracelet supercoiled, nicked and linear plasmids present in the (Figure 1A). The ﬁnal intent was to address the immunoprecipitated samples were converted to a single stoichiometry of cohesin and DNA in the cohesed state. linear form. The STB plasmid and epitope-tagged cohesin reporters DNA was extracted from protein A dynabeads incorporated, as explained below, relevant designs from (Invitrogen) by incubating them twice in succession with those employed by Nasmyth and colleagues for analysis elution buﬀer (50 mM Tris, pH 8.0, 10 mM EDTA and of cohesion in minichromosomes (19,20). Thus, valid 1% SDS) for 15 min each at 65 C. comparisons could be made between results for cohesion Nucleic Acids Research, 2010, Vol. 38, No. 2 573 Figure 1. Topological models for cohesion; trapping an STB reporter plasmid in cohesin-associated form. (A) The subunits of the yeast cohesin complex, and the ring structure they are presumed to assemble, are schematically diagrammed. Shown next to it is a simpliﬁed representation of the cohesin ring used in ﬁgures to follow. The two STB reporter plasmids, pSG4 and pSG6, employed in these studies are symbolized by blue rings. One set of control assays made use of a derivative of pSG4 lacking the TetO21 sequence (pSG5). The three models for topological interaction between cohesin and sister plasmids tested in this study are shown. The hand-cuﬀ is drawn to be consistent with the recent ﬁnding that dimerization of human cohesin is dependent on the Scc3 (SA1/SA2) subunit (25). (B) Following high-speed centrifugation of a cell lysate, DNA from the supernatant (Sup) and ‘chromatin’ pellet fractions, digested with EcoRI, was run in agarose gels and hybridized to a radiolabeled TRP1 probe. Results from a similar fractionation of a CEN4 minichromosome pTetO21CEN4 (20) are shown for comparison. Pl, plasmid; Ch, chromosome. (C) Cleared lysates + 0 (equivalent to ‘Sup’ in B) from metaphase [cir ] or [cir ] cells harboring pSG4 were immunoprecipitated with the HA-antibody and collected on Protein A beads. DNA extracted from the diﬀerent fractions (In, input; U, unbound or ﬂow-through) was probed by a radiolabeled fragment speciﬁc to pSG4. The amount of bound DNA loaded in the right lane was ﬁve times that in the left one. This ratio was kept constant in assays shown in subsequent ﬁgures as well. SC, supercoiled plasmid; L, linear plasmid; N, nicked plasmid. mediated by STB and CEN4. Myc13-tagged Mcd1 and Again, assuming 15% of the plasmid molecules to be two versions of HA-tagged Mcd1 were made use of; associated with cohesin, the actual eﬃciency was engineered into one Mcd1-HA were three adjacent between 33% and 83%. For assays simultaneously copies of the TEV protease cleavage site. Cohesin- employing cohesin(Mcd1-HA6) and cohesin(Mcd1- associated plasmid molecules were immunoprecipitated Myc13), immunoprecipitation eﬃciency for a given from metaphase cell lysates by using antibodies to HA-6 antibody would be dependent on the stoichiometry of or Myc-13 epitopes. In some experiments, the reporter cohesin with respect to DNA. For example, if only a plasmid harboring the TetO21 sequence was pulled single monomeric cohesin complex is involved in pairing down by interaction between IgG and the operator plasmid sisters, the maximum eﬃciency can only be 50%. Cohesin recruitment by the 2 micron plasmid and bound Protein A-TetR fusion protein. Plasmid DNA could be released by anhydrotetracycline, and the subset cohesion of plasmid sisters are intricately linked to of cohesin-associated molecules re-trapped by a cohesin- spindle integrity (6,8). Nocodazole treatment was unsuit- directed antibody. able in our assays for instituting metaphase arrest while Sucrose gradient centrifugation experiments performed maintaining plasmid cohesion. Instead, metaphase during this study revealed the amount of cohesed plasmids cells were enriched through cdc20 arrest or by harvesting in cleared lysates from metaphase cells to be close to 10% populations at appropriate times after release from of the total plasmids, and no >20%. Interpretations of G1 arrest. Only in control assays that employed experimental data pertain to this plasmid population. a minichromosome (a CEN4-based plasmid) or aimed The fraction of plasmid DNA that could be immunopre- to disrupt cohesin assembly at STB was nocodazole cipitated by the HA- or Myc-antibody from cleared employed. lysates ranged from 2% to 7.5% in diﬀerent assays. Cohesion assays in 2 micron circle, unlike those in a Assuming an average of 15% cohesed (or stably cohesin- circular minichromosome, faces the challenge of the associated) plasmids, this corresponded to an eﬃciency multi-copy state of the plasmid and its clustered organi- of immunoprecipitation in the range of 13–50%. zation. Previous experiments showed that a ﬂuorescence In experiments in which plasmid DNA (cohesed and tagged single copy STB reporter plasmid undergoes noncohesed) was ﬁrst pulled down, released and then cohesion in metaphase in the context of the native baited with the HA- or Myc-antibody, the amount of cluster of endogenous plasmids (6). Furthermore, two immunoprecipitated DNA varied from 5% to 12.5%. such reporters, one tagged by red ﬂuorescence and the 574 Nucleic Acids Research, 2010, Vol. 38, No. 2 other by green, segregate red-to-red and green-to-green The DNA retained on the beads was almost exclusively during anaphase. Pairing, even among a population of circular, supercoiled or nicked. That is, only the fraction plasmid molecules, appears to be restricted to sisters. that escaped SnaBI digestion remained trapped by cohesin The majority of experiments reported here, though per- (‘Bound’ in Figure 2A). The Mcd1 (and by inference formed in the multi-copy context, implicitly assume that cohesin) stayed bound to the beads under the conditions of the digestion, as indicated by western blot analysis two sister plasmids constitute the basic unit of cohesion. (data not shown). This assumption was validated by a ﬁnal set of The choice of the single SnaBI site for plasmid digestion experiments utilizing a stand-alone single copy STB was based on its presence in a relatively nucleosome-free plasmid complemented with Rep1 and Rep2 proteins locale of the STB-ORI segment of the 2 micron circle, at in trans. least in the native context of the plasmid genome (32). For veriﬁcation of the SnaBI result, plasmid digestion was also Plasmid-cohesin association is broken by linearizing performed with EcoRI. Incorporation of multiple tandem DNA or cleaving Mcd1 EcoRI sites into the design of pSG4 was intended to Three plausible topological models for chromosome increase cutting eﬃcacy by this enzyme. SnaBI or EcoR1 cohesion are diagrammed in Figure 1A. The ends of the treatment prior to immunoprecipitation left behind nearly DNA are artiﬁcially shown as closed to highlight the all of the linear pSG4 DNA in the supernatant, and pulled linkage between DNA and protein. The circular DNA down almost exclusively the undigested circular form applies to the plasmids used in the studies reported DNA (Figure 2B and C). When plasmid digestion was here. The embrace, bracelet and hand-cuﬀ models are con- carried to completion in the supernatant by an even sistent with the structural features of, and physical higher excess of the enzyme than that used in standard interactions among, cohesin subunits. Additional assays, no DNA was brought down by the antibody models, based on more complex variations of the (Supplementary Figure S2). The restriction enzymes cohesin ring theme, may be envisaged but are not consid- were active not only during the digestion phase but also ered here. While published results from one series of during the immunoprecipitation phase of the assay. experiments support the embrace model (16–20), other Continued cutting of the plasmid on the beads until the lines of evidence leave open alternative possibilities point of DNA extraction could account for the slight (22–25). In experiments described below, we address increase in linear DNA in the bound fraction from whether the interaction of cohesin with the STB locus is enzyme treated samples compared to that from non- accommodated by a ring or rings of cohesin encircling the treated samples (Figure 2B and C). DNA sisters. Digestion of Mcd1 in the cleared lysate or in the The STB reporter plasmid pSG4 (Figure 1A) harboring immunoprecipitated pSG4–cohesin complex with TEV the 2 micron plasmid replication origin, the yeast TRP1 protease annulled the association between DNA and marker and the TetO21 sequence (but no other non-yeast protein (Figure 2D and E). TEV protease treatment DNA) could be maintained with relatively high stability in per se did not aﬀect the topology of the plasmid. The a [cir ] host strain whose endogenous plasmids supplied DNA in the lysate remained almost exclusively circular the partitioning proteins Rep1 and Rep2. High-speed even though it was not pulled down by the HA-antibody centrifugation of an extract from cdc20 arrested once Mcd1 was cleaved (Figure 2D). Similarly, the DNA metaphase cells expressing Mcd1-HA6 yielded released from the beads as a result of TEV protease diges- supernatant fractions (cleared lysates) containing tion was predominantly circular (Figure 2E). The cleavage roughly 40–50% of the pSG4 minichromatin with very of Mcd1 in the lysate by TEV protease was nearly quan- little contamination from chromosomal chromatin titative, as determined by western blotting of the total (Figure 1B, left). For comparison, a similar procedure protein fraction in the lysate or the protein fraction applied to nocodazole (10 mg/ml) treated metaphase bound to the HA-antibody-Protein A beads (Figure 2F). cells partitioned nearly 80% of a CEN4 containing The combined results from DNA digestion and protein minichromosome (20) into the cleared lysate with cleavage suggest that the mainstay of the interaction slightly higher chromosomal contamination (Figure 1B, between cohesin and replicated STB plasmids in right). The pSG4 minichromatin, presumably associated metaphase is topological. The possibility that cohesin with cohesin(Mcd1-HA6), could be immunoprecipitated has poor aﬃnity for linear DNA is unlikely, as it by the HA-antibody (Figure 1C, left). Consistent with normally acts on linear chromosomes as they are being the requirement of the Rep proteins and an intact replicated. It is diﬃcult to imagine how cohesin, acting spindle for cohesin assembly at STB (5,8), immunopre- locally at or near the replication fork, can sense the cipitation of pSG4 was not detected in lysates from [cir ] global topology of DNA. If cohesin can slide or track host cells (Figure 1C, right) or nocodazole treated [cir ] along DNA, it would fall oﬀ from the ends of linear cells (Supplementary Figure S1). DNA regardless of whether DNA-protein association is Next, we inquired into the nature of the DNA–protein topological or physical. However, it would seem unlikely interaction in cohesion-associated pSG4. When the that opening a peptide bond would terminate physical plasmid was digested with SnaBI on Protein A beads association of DNA with cohesin. Ivanov and Nasmyth used to pull down the HA-antibody–cohesin–plasmid (20) showed that severance of either the Mcd1 or the Smc3 complex, the linearized DNA was released nearly quan- subunits of cohesin would suﬃce to free minichro- titatively into the supernatant (S + W in Figure 2A). mosomes from cohesin’s grasp. Furthermore, for small Nucleic Acids Research, 2010, Vol. 38, No. 2 575 Figure 2. Release of an STB reporter plasmid from cohesin’s grasp by linearizing DNA or cleaving Mcd1. The consequences of cutting DNA or protein on the topological association between plasmid and cohesin are schematically indicated. (A) Cohesin associated pSG4 was adsorbed on HA- antibody and immobilized on Protein A beads as described under Figure 1. After digestion with SnaBI, DNA released into the supernatant (S) and wash fractions (W) and that retained on the beads (Bound) were analyzed. (B and C) SnaBI or EcoRI digestion was performed in the cleared lysates prior to immunoprecipitation by HA-antibody. (D) Cleared lysates were treated with TEV protease, and then subjected to pull-down by HA- antibody and Protein A beads. (E) Cohesin bound plasmid from cleared lysates was treated with HA-antibody, trapped on Protein A beads, and subjected to TEV protease treatment. (F) Cleavage of Mcd1-HA6 by TEV protease in the cleared lysates (corresponding to the DNA analysis shown in D) was monitored by western blotting using HA-antibody. The amount of protein analyzed from the bead-bound fraction was four equivalents of that from the input. The identity of the weak band above Mcd1 seen in some of the lanes is not known. Its mobility would be consistent with phosphorylation of Mcd1, which occurs during the establishment of cohesion in response to DNA damage (27). DNA molecules, it is circularity rather than supercoiling Cohesin encircles DNA in the form of solitary rings that is important for stable association with cohesin. The and not conjoined ones eﬀect of nicking a single strand on cohesin’s association with DNA is much smaller compared to that of breaking In the embrace and bracelet models for cohesion, sis- both strands (20). The more or less unbiased association ter chromosomes are enfolded by one cohesin ring (or mul- of supercoiled or nicked plasmid circles with cohesin, con- tiple units of a solitary ring) (Figure 1A). The ring sizes are trasted by the lack of association of linear molecules, is diﬀerent in the two cases, the bracelet being a cohesin dimer. In contrast, the hand-cuﬀ model propounds two also evident in our assays (Figure 2A–C). distinct, but mutually associated, monomeric cohesin Strictly, our interpretation applies only to the fraction of plasmids that is stably associated with the cohesin rings. The one ring versus two ring models could be complex, and can be recognized by anti-cohesin distinguished by expressing two types of cohesins, antibodies. While the data favor interlinked cohesin and diﬀerentially tagged by HA6 and Myc13, in the same DNA rings, they do not discriminate among the three cell and in roughly equal amounts (Figure 3). Mcd1- models in Figure 1A. Based on previous results regarding HA6 was cleavable by TEV protease; Mcd1-Myc13 was the nature of 2 micron plasmid cohesion and segregation not. The types of cohesion resulting from the embrace, (6,33), we assume tentatively that two sister plasmid hand-cuﬀ and bracelet models are schematically dia- molecules are held together by the cohesin ring (or rings). grammed in Figure 3A. 576 Nucleic Acids Research, 2010, Vol. 38, No. 2 Figure 3. Plasmid–cohesin association in metaphase cells expressing two diﬀerentially tagged cohesin moieties. (A) The types of sister plasmid cohesion expected from the embrace, hand-cuﬀ and bracelet models in presence of cohesin(Mcd1-HA6) and cohesin(Mcd1-Myc13) in equivalent amounts are indicated (see also Table 1). Whereas Mcd1-HA6 in cohesin could be cleaved by TEV protease, Mcd1-Myc13 could not. (B) Aliquots of cell lysates were probed by western analysis using HA- or Myc-antibody to reveal relative levels of cohesin(Mcd1-HA6) or cohesin(Mcd1-Myc13). Data are shown for the haploid strains expressing either cohesin(Mcd1-HA6) (lane 1) or cohesin(Mcd1-Myc13) (lane 2) and the diploid generated from them expressing both Mcd1 variants (lanes 3–7). Signals from the two antibodies were normalized using aliquots of 75% pure Cre recombinase tagged at its carboxyl-terminus with HA6 as well as Myc13. The mean Myc13 to HA6 signal intensity was 1.83 ± 0.18. The dilution factor between Cre samples stained by Coomassie blue (right) and the corresponding ones analyzed by western blotting (left) was 500 to 1. (C) Aliquots of cell lysates from the haploid and diploid strains, run as in B, were probed using an antibody to native Mcd1. The mean ratio of Mcd1-Myc13 to Mcd1-HA6 was 0.96 ± 0.11. (D) pSG4 molecules from the cleared lysate were ﬁrst immobilized on IgG beads, and then released from them by disrupting TetO–TetR interaction using anhydrotetracycline. (E and F) Following treatment with EcoRI or TEV protease, plasmid pull-down was attempted using HA- or Myc-antibody. Table 1 summarizes the expectations from the three To ensure approximately equal amounts of cohesin models for the loss or retention of cohesin–DNA linkage tagged by HA6 and Myc13 in the diploid host harboring following the opening of the Mcd1-HA6 containing ring pSG4, the Mcd1 variants were expressed from the native by TEV protease. The simplest prediction, and the easiest MCD1 promoter and the native chromosomal locale. This to verify, is that the HA-antibody would not be able to expectation was further veriﬁed by a western blot analysis trap plasmid DNA following the action of TEV protease of the steady state levels of Mcd1-HA6 and Mcd1-Myc13 according to embrace and bracelet models. All plasmid (Figure 3B and C). Diﬀerences in the HA6 and Myc13 molecules embraced by cohesin(Mcd1-HA6) rings, and signals were normalized using Cre recombinase harboring even those associated with mixed cohesin(Mcd1-HA6)– both these epitope tags at its carboxyl-terminus as a ref- cohesin(Mcd1-Myc13) bracelets, would have escaped erence protein (Figure 3B). With appropriate correction, through the opening created in the cohesin(Mcd1-HA6) we estimated the cellular ratio of cohesin(Mcd1-Myc13) ring. This prediction is independent of the number of to cohesin(Mcd1-HA6) to be 1.09 ± 0.12. This result rings surrounding a given pair of DNA sisters. However, was further conﬁrmed by quantifying the proteins using the hand-cuﬀ model predicts 25% of the DNA to be an antibody to native Mcd1, cohesin(Mcd1-Myc13): immunoprecipitated by this antibody, so long as the cohesin(Mcd1-HA6) = 0.96 ± 0.11 (Figure 3C). The ﬁssured cohesin(Mcd1-HA6) stays associated with the extents of cohesin immunoprecipitation by the HA- and intact cohesin(Mcd1-Myc13), which is non-cleavable by Myc-antibodies were more or less equal under our exper- TEV protease. If there is more than one hand-cuﬀ per imental conditions, as indicated by the Southern signals DNA sisters, the DNA fraction immunoprecipitated by from the co-precipitated DNA (Supplementary Figure S3; the HA-antibody will be >25%. Figure 4C and D). Nucleic Acids Research, 2010, Vol. 38, No. 2 577 Table 1. Predictions by the three topological models on the nature of plasmid cohesion Schematic diagrams for plasmid cohesion established by the embrace, bracelet and hand-cuﬀ models from an equal mixture of cohesin(Mcd1-HA6) (cleavable by TEV protease) and cohesin(Mcd1-Myc13) (resistant to TEV protease) are shown in Figure 3 (top). Treatment with TEV protease will cleave all cohesin(Mcd1-HA6) containing rings (see drawings above), opening gates for trapped plasmids to escape. Only those plasmid molecules surrounded by the closed cohesin(Mcd1-Myc13) ring(s) will be stopped. R is the predicted molar ratio of plasmid that can be pulled down by the Myc-antibody before and after TEV protease cleavage; R is the observed value. Agreement between ob experiment (Figure 3B and C) and prediction is indicated by the green rectangles; disagreement by red ones. The embrace model is the winner (with two green rectangles) over the hand-cuﬀ and bracelet models (each with a red rectangle). These assays do not permit a clean distinction between the embrace and the topological hand-cuﬀ models. In order to improve the sensitivity of the assay, pSG4 double bracelet (2.14) models (Table 1). Thus, for one was enriched from metaphase cells by IgG pull-down cohesin ring or a reasonably small number of such rings (Figure 3D), followed by its release in the presence of per pair of sister plasmids, these results discount the anhydrotetracycline. The released plasmid could be bracelet in favor of the embrace model (Table 1). pulled down again by either the HA- or Myc-antibody Multiple cohesin rings per sister pair would increase the with approximately equal eﬃciency (Supplementary relative amount of DNA pulled down by the Myc Figure S3). Attempts to immunoprecipitate DNA after antibody after TEV protease cleavage, due to increased EcoRI digestion of the released plasmid with the HA- or mole fraction of cohesin(Mcd1-Myc13)/cohesin(Mcd1- Myc-antibodies failed (Figure 3E), as expected from Myc13) bracelets around sisters. However, the stoichio- earlier results. Digestion with TEV protease yielded a metry of cohesin to DNA will have to be quite high in distinct, and signiﬁcant, result. While the HA-antibody order to blur the distinction between the two models. failed, the Myc-antibody succeeded in reprecipitating A variation of the hand-cuﬀ model in which the two DNA, almost entirely as intact circles (Figure 3F). This cohesin rings are topologically, not physically, linked result is consistent with the embrace and bracelet models cannot be easily ruled out by the above experiments. In but inconsistent with the hand-cuﬀ model (Table 1). a topological hand-cuﬀ, opening of cohesin(Mcd-HA6) by Quantitations suggest that the molar ratio of plasmid TEV protease would automatically end its association brought down by the Myc-antibody in the absence of, and with cohesin(Mcd1-Myc13). As a result, in the pull- following, TEV protease digestion was 1.2, or nearly down test using the HA-antibody, it would be no diﬀerent equal to 1.0—(compare the ‘Bound’ lanes in the right from the embrace model in which two cohesin rings are two panels of Figure 3C). This value is close to that pre- formed around sister plasmids (Table 1). Further dicted by the embrace model, and signiﬁcantly smaller experiments argue against a hand-cuﬀ formed by a than that anticipated from the single bracelet (3.0) or the catenated pair of cohesin rings (see below). 578 Nucleic Acids Research, 2010, Vol. 38, No. 2 Figure 4. Distinction between embrace and bracelet models for plasmid cohesion: cohesin stoichiometry tested by sequential immunoprecipitation. (A and B) The expected outcomes for plasmid immobilization via TetO aﬃnity interaction followed by DNA linearization were experimentally veriﬁed. Plasmid molecules associated with Protein A-TetR bound to TetO were pulled down by IgG beads. DNA and protein remaining associated with the beads or released into the supernatant in the absence of EcoRI treatment or following EcoRI digestion were followed by Southern and western analyses, respectively. (C) The ﬂow-chart for the two-step immunoprecipitation assays is diagrammed at the top. Plasmids were ﬁrst trapped on IgG beads as in A, and then released by treatment with anhydrotetracycline. Equal amounts of the supernatant containing the freed plasmid were immunoprecipitated with the HA- or Myc-antibody. The leftover plasmid molecules in the supernatant were subjected to a second round of immunoprecipitations. (D) The histograms denote the mean ratios of Southern blot signals for immunoprecipitated DNA from three independent experiments, with the error bars showing standard deviations. Immunoprecipitations with HA- and Myc-antibodies are represented by ‘H’ and ‘M’, respectively. Sequential immunoprecipitations by these antibodies are indicated by the two letters separated by a dash. The ratio of the input (IN) plasmid DNA to that immunoprecipitated by the HA- and Myc-antibodies combined is given as IN/[H + M]. The immunoprecipitable plasmid fractions were 17.33%, 16.50%, 16.46% in individual assays. It may be noted that, the physical and topological (bracelet) over a cohesin hand-cuﬀ as the unit entity in handcuﬀs will give identical results for DNA pull-down, cohesion. However, they do not distinguish between the before and after TEV protease digestion, by the Myc- ring and the bracelet; nor do they reveal the number of antibody (Table 1). They do diﬀer from embrace by one rings or bracelets assembled around paired sister plasmids. or two monomeric cohesin rings in causing one third The ability to immobilize pSG4 alternatively by IgG or the reduction (from 75% to 50%), following TEV protease HA- or Myc-antibody oﬀers a potential tool to resolve cleavage, in plasmid DNA associated with cohesin(Myc- these uncertainties. Once again, the assays were performed 13). However, as is evident from Table 1, the Myc- using metaphase cells of the host strain expressing Mcd1- antibody pull down oﬀers better distinction between the HA6 and Mcd1-Myc13 in approximately equivalent embrace and bracelet models than between embrace and amounts. hand-cuﬀ models. The plasmid, trapped on IgG beads through Protein A-TetR bound to TetO, is expected to bring down Stoichiometry of cohesin-plasmid association: two-step with it associated cohesin(s), cohesin(Mcd1-HA6) and immunoprecipitations support embrace by a single cohesin(Mcd1-Myc13) monotypes (according to embrace monomeric cohesin ring or bracelet) or cohesin(Mcd1-HA6)/cohesin(Mcd1- The experimental outcomes so far favor monomeric Myc13) hybrid type (according to bracelet). cohesin ring(s) (embrace) or dimeric cohesin ring(s) Linearization of the bound pSG4 should release all Nucleic Acids Research, 2010, Vol. 38, No. 2 579 associated cohesin into the supernatant, irrespective of the (IN/[H + M] =6.0 in Figure 4D). Furthermore, the stoichiometry of cohesin rings to DNA. The linear form molar DNA ratio of approximately 4 for the HA/[HA- of pSG4, however, should stay bound to the beads. HA] or Myc/[Myc-Myc] sequence (Figure 4D) These expectations were satisﬁed (Figure 4A and B). As corresponds to an immunoprecipitation eﬃciency of shown by the results in Figure 3, plasmid released from 75% for each antibody. Note also that the DNA the beads by treatment with anhydrotetracycline could amounts pulled down by the HA-antibody and the Myc- be immunoprecipitated using antibodies to associated antibody in the respective ﬁrst step immunoprecipitations cohesin(s). A two-step immunoprecipitation assay could were nearly equal (HA/Myc of 0.88 ± 0.01 in Figure 4D). then be performed to test whether or not cohesin(Mcd1- These values are in agreement with the one ring embrace HA6) and cohesin(Mcd1-Myc13) are capable of simulta- model, which proscribes pull-down by one antibody of the neous association with the plasmid. other’s cognate epitope. One would then expect 75% of Predictions concerning plasmid pull-down by the HA- the plasmid DNA bearing cohesin of one epitope or Myc-antibody are dependent on both the cohesion type, which constitutes half of all the cohesed molecules, model and cohesin stoichiometry. According to the one to come down during the ﬁrst step, and 19% ring embrace model, immunoprecipitation by the HA- [(100 75 = 25) 75%] during the second step. antibody would deplete only plasmid molecules By the two ring embrace or the single bracelet model, embraced by cohesin containing Mcd1-HA6, and leave 50% of the cohesed DNA molecules would display dual those embraced by cohesin containing Mcd1-Myc13 epitope speciﬁcity in cohesin, and be subject to unscathed. As a result, a second round of immunopreci- immunoprecipitation by either the HA- or Myc-antibody. pitations of the ‘depleted’ supernatant individually by For 75% immunoprecipitation eﬃciency, the ﬁrst antibody HA- and Myc-antibodies would skew plasmid pull-down would bring down [(25 + 50 = 75) 75%)] =56% of all in favor of the latter. If the ﬁrst immunodepletion is per- cohesed DNA molecules. Note that 25% of the DNA formed by the Myc-13 antibody, the bias in DNA pull- molecules displaying a single epitope speciﬁcity would down during the second immunoprecipitation would also be competent for immunoprecipitation at the ﬁrst be directed oppositely. According to the one bracelet step. This step would deplete (50 75%) = 37.5 % of the model, the ﬁrst immunoprecipitation with either the HA- dual speciﬁcity molecules from the subsequent round of or Myc-antibody would pull down two thirds of the immunoprecipitation. The fraction of DNA molecules cohesin bracelets bearing the opposite epitope speciﬁcity that the virgin antibody can recognize would thus be in the form of choesin(Mcd1-HA6)–cohesin(Mcd1- (50 37.5) = 12.5% with dual epitope speciﬁcity plus Myc13) hybrid bracelets. The two ring embrace model is 25% with single speciﬁcity. The expected immunopre- also subject to a similar depletion eﬀect due to cohesion cipitation by this antibody is [(12.5 + 25 = 37.5) mediated by a mixed pair of cohesin(Mcd1-HA6) and 75%] =28%. The predicted two fold reduction in cohesin(Mcd1-Myc13) rings. Hence the relative advantage DNA pull-down between the two antibodies (from 56% for the virgin antibody in the second immunoprecipitation to 28%) for their primary encounters with cohesed step is less than that anticipated from the single ring plasmids is not in agreement with the experimental result, embrace model. which showed no such reduction (Figure 4C and D). Quantitations of the Southern signals of immunopre- The two-step immunoprecipitation data are also incon- cipitated DNA from three repeats of the two-step assay sistent with a cohesin hand-cuﬀ, even one in which the (Figure 4C) are graphed in Figure 4D. The virgin antibody individual rings are linked by catenation. displayed a strong advantage in the second step (HA-Myc IP or Myc-HA IP) over the experienced antibody Isolation of STB plasmid sisters paired by cohesin (HA-HA IP or Myc-Myc IP). The [HA-Myc]/[HA-HA] and their separation by linearizing DNA or cleaving and [Myc-HA]/[Myc-Myc] ratios were 4.74 ± 1.46 and Mcd1 ex vivo 4.15 ± 0.64, respectively. In contrast, the molar ratio of Data from Figures 1–4 support the entrapment, in DNA brought down by the HA-antibody in the ﬁrst step metaphase cells, of an STB reporter plasmid as a DNA– to that in the second step following immunoprecipitation protein catenane containing a single protein ring of by the Myc-antibody was 1 (0.95 ± 0.01; HA/[Myc-HA] cohesin. The assumption that two DNA rings are in Figure 4D). The corresponding ratio for immunopre- present within such a catenane is based on the earlier cipitation by the Myc antibody was also the same, 0.98 ± 0.07 (Myc/[HA-Myc] in Figure 4D). The absence ﬁnding that cohesin assembly at STB promotes plasmid of cross-depletion by either antibody during plasmid pull- cohesion followed by sister-to-sister plasmid segregation down is consistent with only one cohesin ring (carrying a (6). In order to directly test our assumption, we have single epitope speciﬁcity; either HA or Myc) bridging a isolated the cohesed form of a single copy STB reporter pair of plasmid sisters. A similar analysis with the CEN4 plasmid from metaphase cells by sucrose gradient sedi- containing minichromosome gave concordant results mentation (19), and interrogated ex vivo the DNA status (Supplementary Figure S4). within it. The total amount of immunoprecipitated DNA, by HA- The single copy reporter plasmid pSG6, 4 kb long, was and Myc-antibodies combined, in the two step assay fashioned after the CEN-STB reporter plasmids used in added up to approximately 15-20% of the input DNA previously published work (6,33). In this pSG4 derivative, (that was released from the IgG beads), accounting for the TetO21 locus was replaced by a P -CEN3 DNA GAL nearly the entire fraction of DNA in the cohesed state fragment. The CEN sequence, while it helped maintain 580 Nucleic Acids Research, 2010, Vol. 38, No. 2 Figure 5. Enrichment of plasmids in their cohesed form from metaphase cells by velocity sedimentation: test of the topological model for cohesion. (A) The experimental regimen for enriching metaphase cells from the appropriate [cir ] host strain harboring pSG6 and going through the cell cycle in glucose or galactose is schematically indicated. At 45 min for the cell cycle in glucose and at 75 min for that in galactose, the predominant population consisted of large budded cells with a single DAPI staining mass at the bud neck. (B) The sedimentation patterns of pSG6 in 12.5–37.5% sucrose gradient were followed under conditions where the plasmid-borne CEN alone or STB alone or neither of the two was active. Samples were run in agarose gels in the cold (4 C) and probed by pSG6-speciﬁc radio-labeled DNA. C, cohesed plasmids; NC, non-cohesed plasmids. (C) Representative fast (F), intermediate (I) and slow (S) sedimenting fractions from the gradient were reanalyzed by agarose gel electrophoresis with or without EcoRI digestion, followed by SDS treatment. For reference, S fractions treated or untreated with EcoRI but without subsequent SDS addition (left panel) and DNA prepared form the lysate by phenol extraction and ethanol precipitation (rightmost lane) were included in the run. (D and E) Representative fractions from the sucrose gradient (fast, slow and intermediate) were treated with EcoRI (D) or with TEV protease (E), and subjected to agarose gel electrophoresis under native conditions. the copy number at or close to unity, could be CEN, STB and ARS incarnations (Figure 5B) shed conditionally inactivated by galactose induced transcrip- light on the DNA stoichiometry in the cohesed form of tion. The plasmid was housed in a [cir ] strain or an the plasmid. Fractions were divided into three categories isogenic [cir ] strain expressing Rep1 and Rep2 from the based on their sedimentation velocities: fast (F; cohesed?), bidirectional GAL1–GAL10 promoter. The plasmid would slow (S; non-cohesed?) and intermediate (I; cohesed plus behave as a true CEN plasmid (or a minichromosome) in non-cohesed?). When they were analyzed by electro- either host strain in the presence of glucose as the carbon phoresis in native agarose gels in the cold (4 C), there source. In galactose, though, it would behave as an ARS was a reversal (as expected) in their relative mobility: the plasmid in the [cir ] host and as an STB plasmid in the fast-sedimenting (heavy) fractions migrated more slowly [cir ]::P [REP1 REP2] host. Fractionation of cleared than the slow-sedimenting (light) fractions. In a typical GAL lysates from metaphase cells by centrifugation through a run, the S group comprised of fractions 31 (start point 12.5–37.5% sucrose gradient resolved the plasmid into of Southern signal for DNA) to 44, the I group 45 to 54 two forms: an ‘uncohesed’ monomer form and a and the F group 55 to 65. The lower mobility band of DNA (C for cohesed) was characteristic of pSG6(CEN) ‘cohesed’ 2 monomer form (Figure 5). The general scheme for enriching metaphase cells from and pSG6(STB), and was absent or nearly so for populations arrested in G1, conditioned in glucose or pSG6(ARS). The sedimentation and gel migration galactose, and then released into the cell cycle is outlined proﬁles of the plasmid containing fractions were reminis- in Figure 5A. The sedimentation proﬁles of pSG6 in its cent of those reported by Ivanov and Nasmyth (19) for Nucleic Acids Research, 2010, Vol. 38, No. 2 581 their minichromosome assays. We inferred therefore that These observations suggest a double-gate mechanism the upper band comprised presumably of cohesed sister for the establishment and annulment of chromosomal plasmids (in association with protein factors in addition cohesion. The logic is similar to that used by DNA topoisomerase II to transport a DNA segment through to cohesin) while the lower band contained noncohesed two oppositely located entrance and exit gates during (NC) plasmid molecules. This inference was subjected to DNA relaxation (34). The diﬀerence in the case further veriﬁcation (see below). The fact that the of cohesin is that the second gate opening event involves pSG6(ARS) proﬁle was almost entirely free of the upper proteolytic cleavage of a protein subunit. Notwithstanding band implies little contribution from catenated or covalent the evidence favoring cohesion by topological DNA– plasmid dimers to the ‘cohesed’ DNA fraction. protein association, the possibility of cohesion by Digestion by EcoRI at 4 C did not alter the DNA physical interaction remains a viable alternative mobility of the S (non-cohesed) fractions of pSG6(STB) (21–24,35). We have now shown that 2 micron plasmid in electrophoresis under native conditions (Figure 5C). molecules, or at least those amongst them that remain However, electrophoresis in presence of SDS without cohesed under the assay conditions, also conform to EcoRI treatment revealed monomeric plasmid DNA, pri- cohesion by the topological dictum. marily as the supercoiled form (70–80%) and the remainder as nicked circles in S, I and F fractions. Sister plasmid cohesion in the 2 micron circle EcoRI treatment resulted in conversion of 60–70% of the DNA into linear molecules which migrated below (but The stable propagation of the 2 micron plasmid is con- almost overlapping with) the nicked circle under SDS- ferred by a partitioning system consisting of the plasmid electrophoresis. Consistent with the extent of linearization coded Rep1 and Rep2 proteins and the cis-acting STB of plasmid by EcoRI, native gel electrophoresis revealed a locus (36,37). Requirement of an active partitioning similar conversion of the cohesed form of pSG6 from the I system is mandated by the organization of the multi- and F fractions to the noncohesed form (Figure 5D). copy plasmid into a tight cluster of 3–5 foci, the cluster Conversion from the cohesed to the non-cohesed being the unit of segregation (38). The REP-STB system form was also promoted by TEV protease cleavage appears to couple plasmid segregation to chromosome (Figure 5E). Breaching of cohesion either by linearization segregation either by appropriating chromosome segrega- of pSG6(STB) or by Mcd1 cleavage was as expected for tion factors or tethering the plasmid cluster to a chromo- topology mediated cohesion. The outcomes of EcoRI and some (5). Association of cohesin with STB, assisted by the TEV protease digestions were quite similar between Rep proteins and an intact mitotic spindle (5,8,39), pSG6(STB) and pSG6(CEN) (Supplementary Figure establishes cohesion between plasmid sisters that S5A–C). Furthermore, the sedimentation assay revealed subsequently segregate one-to-one from each other (6). little cohesed dimers of pSG6 in galactose grown but Our present analyses favor the embrace model for nocodazole treated cells (Supplementary Figure S5D, plasmid cohesion, and suggest a stoichiometry of one bottom). This result attests to the complete inactivation cohesin ring per two sister plasmids (or STBs). of CEN by galactose-induced transcription, and veriﬁes The fraction of cohesin-associated STB reporter the authenticity of STB-mediated cohesion when the plasmid obtained in pull-down and sedimentation mitotic spindle is intact (Supplementary Figure S5D, assays is <20%, comparable to 10–30% reported for top; Figure 5B). minichromosomes (20). In contrast, cohesion of STB The data for plasmid sedimentation followed by ex vivo reporter plasmids as assayed by cell biological methods cleavage assays support the presence of two plasmid in metaphase populations is much higher, >70% (6). monomers per cohesed unit. Results from the previous The loss of cohesion during biochemical manipulations pull-down analyses are most easily explained by the occu- could be due to a physical, and less stable, mode of pancy of this unit by one monomeric cohesin ring. cohesin–DNA interaction. However, mutually concordant Together they are consistent with the embrace model for outcomes from distinct aﬃnity interaction and velocity plasmid cohesion, in which a monomeric cohesin ring sedimentation assays are consistent with a uniform, and holds two sister rings of the STB reporter plasmid in a topological, mode of cohesion in a ﬁnite fraction of tripartite catenane. cohesin-associated plasmids. Molar ratio of DNA rings and cohesin within cohesed DISCUSSION plasmid species: a 2:1 DNA–cohesin tri-link? The topology model for sister chromatid cohesion derives Chromatin immunoprecipitation assays reveal a periodic its support from experiments that convert DNA from distribution of cohesin at 10–15 Kb intervals along chro- circular to linear form or break a polypeptide chain in mosome arms in S. cerevisiae with a higher density cohesin or non-covalently or covalently seal borders at of localization at and around centromeres (40–42). critical points where cohesin subunits interface with each Fluorescent intensity of Smc3-GFP at kinetochores other (16–20). Closure of the hinge gate in cohesin appar- normalized to one copy of the histone H3 variant Cse4 ently blocks entry of a chromosome into its interior (16); per centromere (43), suggests the presence of one cohesin and circular minichromosomes already associated with molecule for every 4 Kb of centric/pericentric DNA (22). cohesin remain trapped until an opening is intro- Our ﬁnding that a pair of sister STB reporter plasmids is duced either in the DNA or in the protein (18–20). likely held together by one cohesin ring agrees well with 582 Nucleic Acids Research, 2010, Vol. 38, No. 2 Figure 6. A single ring formed by a monomeric cohesin complex as the unit of cohesion at STB.(A) The results from Haering et al. (18) for CEN cohesion ruling out ‘double’ rings of cohesin are schematically represented. In their experimental design, covalent protein ring closure required crosslinking two neighboring pairs of cysteines at two locations (green circles) to form a pair of chemical bridges (green triangles). Cross-linking eﬃciency (or probability ‘p’ of circle to triangle conversion) was 55% in their assays. Experiments agreed with entrapment probability of DNA 2 4 sisters ‘P’ equal to p (30%) and not p (9%). Note that, upon SDS treatment during the assay, a physical hand-cuﬀ (but not a topological one) would fall apart to yield monomeric cohesin rings, each with a single trapped plasmid molecule. (B) In the pre-cohesed state of the 2 micron circle, multiple cohesin molecules may interact physically and dynamically at or near STB. Such interactions could be promoted by the cohesin loading factors Scc2 and Scc4, which are required for cohesin assembly on the plasmid (5). Transition to the stable topological association may be mediated by passage of the replisome and closure of a single cohesin ring around a pair of STB sisters. this estimate. It is also consistent with in vivo FRET surrounding the sister DNAs (Supplementary Figure results indicating lack of interaction between more than S6A). In the case of two rings, the expected P is 2 2 2 4 one cohesin complex (44) and biochemical outcomes 1[1p ] =2p p = 51%. However, double ring from induced cohesin ring closure within cohesed models in which the DNA sisters reside within one ring minichromosomes (18). cannot be ruled out (Supplementary Figure S6B). Based on the extent of cohesed minichromosomes A biased hand-cuﬀ, physical or topological, predicts entrapped by chemically crosslinking cohesin subunits, P=p =30%, since two crosslinks would suﬃce to trap Haering et al. (18) argue in favor of cohesion by both sisters. monomeric cohesin rings (embrace model) over dimeric Although multiple cohesin molecules could be involved cohesin rings (hand-cuﬀ and bracelet models). In their in precohesive interactions, likely mediated through assays, covalent protein closure in a monomeric ring DNA-bound cohesin loading factors Scc2 and Scc4 (45). required crosslinking of two pairs of cysteine neighbors Only a subset of such interactions may be consummated resident at distinct locations (green circles) to form a to stable topological cohesion by passage of the replisome. pair of chemical bridges (green triangles) [Figure 6A; In the case of the 2 micron plasmid, only one cohesin ring, adapted from (18)]. For a probability ‘p’ of forming a in most cases, may be closed to encircle the nascent STB single cross-link (p = eﬃciency of the crosslinking duplexes (Figure 6B). agent), the expectation for entrapment of DNA sisters by a monomeric ring is p , since two crosslinks have to Conserved mode of cohesion at CEN and STB: be formed to seal the ring. For a dimeric ring of the evolutionary implications bracelet or hand-cuﬀ type, this value is p . For a value of ‘p’ =55%, experimentally determined by Haering The genetically deﬁned ‘point’ centromere of S. cerevisiae, et al., the observed extent of entrapment ‘P’ matches contrasted by the epigenetically speciﬁed centromeres of 2 4 p = 30%, and not p = 9%. The Haering et al. result is fungi in general, poses a rather puzzling transitional also contrary to two or more monomeric cohesin rings oddity in centromere evolution (46). It has been suggested Nucleic Acids Research, 2010, Vol. 38, No. 2 583 2. Onn,I., Heidinger-Pauli,J.M., Guacci,V., Unal,E. and that the point centromere is a domesticated version of the Koshland,D.E. (2008) Sister chromatid cohesion: a simple partitioning locus of an ancestral 2 micron-like plasmid concept with a complex reality. Annu. Rev. Cell Dev. Biol., 24, that functionally replaced the canonical epigenetic fungal 105–129. centromere. Miniaturization of the centromere and 3. Uhlmann,F. (2004) The mechanism of sister chromatid cohesion. loss of the machinery for establishing pericentric hetero- Exp. Cell Res., 296, 80–85. 4. Watanabe,Y. (2005) Sister chromatid cohesion along arms and at chromatin and RNA interference (47) appear to have been centromeres. Trends Genet., 21, 405–412. related events. It is noteworthy in this regard that the exis- 5. Mehta,S., Yang,X.M., Chan,C.S., Dobson,M.J., Jayaram,M. and tence of 2 micron-related plasmids is limited to fungal Velmurugan,S. (2002) The 2 micron plasmid purloins the yeast lineages belonging to Saccharomycetaceae (46,48). The cohesin complex: a mechanism for coupling plasmid partitioning and chromosome segregation? J. Cell Biol., 158, 625–637. emergence of a novel centromere had necessarily to engen- 6. Ghosh,S.K., Hajra,S. and Jayaram,M. (2007) Faithful segregation der mechanisms for integrating it into the established of the multicopy yeast plasmid through cohesin-mediated chromosome segregation pathway. Principally, it had to recognition of sisters. Proc. Natl Acad. Sci. USA, 104, 13034–13039. be rendered competent in recruiting components of the 7. Ghosh,S.K., Hajra,S., Paek,A. and Jayaram,M. (2006) Mechanisms kinetochore complex. Several subunits (Ndc10 and for chromosome and plasmid segregation. Annu. Rev. Biochem., 75, 211–241. Ctf13, for example) of the CBF3 complex, which binds 8. Mehta,S., Yang,X.M., Jayaram,M. and Velmurugan,S. 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Ivanov,D. and Nasmyth,K. (2007) A physical assay for sister nical assistance. We appreciate the insightful critique from chromatid cohesion in vitro. Mol. Cell, 27, 300–310. the reviewers that helped improve the article in style and 20. Ivanov,D. and Nasmyth,K. (2005) A topological interaction between cohesin rings and a circular minichromosome. Cell, 122, content. 849–860. 21. Guacci,V. (2007) Sister chromatid cohesion: the cohesin cleavage model does not ring true. Genes Cells, 12, 693–708. FUNDING 22. Yeh,E., Haase,J., Paliulis,L.V., Joglekar,A., Bond,L., Bouck,D., Salmon,E.D. and Bloom,K.S. (2008) Pericentric chromatin is National Institutes of Health (GM064363); Robert F organized into an intramolecular loop in mitosis. Curr. Biol., 18, Welch Foundation award (F-1274, partial). Funding 81–90. for open access charge: National Institutes of Health 23. Surcel,A., Koshland,D., Ma,H. and Simpson,R.T. (2008) (GM064363). 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Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex

Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex

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Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex

Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex

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