YEASTBOOK Chromosome Segregation in Budding Yeast: Sister Chromatid Cohesion and Related Mechanisms Adele L. Marston The Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3JR, United Kingdom ABSTRACT Studies on budding yeast have exposed the highly conserved mechanisms by which duplicated chromosomes are evenly distributed to daughter cells at the metaphase–anaphase transition. The establishment of proteinaceous bridges between sister chromatids, a function provided by a ring-shaped complex known as cohesin, is central to accurate segregation. It is the destruction of this cohesin that triggers the segregation of chromosomes following their proper attachment to microtubules. Since it is irreversible, this process must be tightly controlled and driven to completion. Furthermore, during meiosis, modifications must be put in place to allow the segregation of maternal and paternal chromosomes in the first division for gamete formation. Here, I review the pioneering work from budding yeast that has led to a molecular understanding of the establishment and destruction of cohesion. TABLE OF CONTENTS Abstract 31 Introduction 32 Building Mitotic Chromosomes 32 Structure and function of the cohesin complex 32 Discovery of cohesion: 32 The cohesin ring: 33 How does cohesin hold chromosomes together?: 34 Loading cohesin onto chromosomes 34 The pattern of cohesin localization on chromosomes: 35 The cohesin loading reaction: 36 Replication–coupled establishment of cohesion 37 Eco1-directed cohesion establishment: 37 Other factors involved in cohesion establishment: 38 DNA damage-induced cohesion 39 Other structural components of chromosomes 39 The condensin complex: 40 Topoisomerase II: 40 Establishment of Biorientation 40 Role of kinetochore geometry and centromere structure 41 Continued Copyright © 2014 by the Genetics Society of America doi: 10.1534/genetics.112.145144 Manuscript received July 24, 2013; accepted for publication September 18, 2013 Available freely online through the author-supported open access option. Address for correspondence: School of Biological Sciences, Michael Swann Building, Mayfield Rd., Edinburgh EH9 3JR, United Kingdom. E-mail: [email protected] Genetics, Vol. 196, 31–63 January 2014 31 CONTENTS, continued Error correction 42 Destruction of Sister Chromatid Cohesion and Anaphase Onset 43 Cleavage of cohesin by separase 43 The spindle checkpoint 43 Inhibition of APC–Cdc20: 43 Checkpoint silencing: 45 Other factors regulating anaphase onset 45 Anaphase Progression 46 Separase initiates mitotic exit 46 Committing to anaphase: 47 Nuclear position: 47 Completion of chromosome segregation: 47 Maintaining spindle integrity: 48 Chromosome Segregation During Meiosis 48 Establishment of links between homologs during meiosis I 49 Homologous chromosome pairing: 49 Meiotic recombination: 49 Monoorientation of sister chromatids during meiosis I 50 Stepwise loss of cohesion 51 Biorientation of homologs 51 Alteration of cell cycle controls in meiosis 52 Meiotic prophase I to meiosis I transition: 52 Meiosis I to meiosis II transition: 52 Perspectives 53 URING cell division, chromosomes must be replicated yeast that led to an understanding of the molecular biology Dexactly and accurately distributed into daughter cells. of chromosome segregation together with the exquisite con- The regulated sequence of events that leads to cell division is trols that ensure its accuracy and the modifications that take known as the cell cycle. In S phase of the cell cycle, DNA place to generate gametes. synthesis and the establishment of sister chromatid cohesion generates two identical sister chromatids that are held Building Mitotic Chromosomes tightly together by a conserved protein complex, known as Structure and function of the cohesin complex cohesin. In mitosis, the cohesin linkages provide resistance and generate tension to facilitate the attachment of sister Discovery of cohesion: Pioneering studies in budding yeast chromatids to microtubules emanating from opposite poles. were instrumental in the discovery of the chromosome Once all the chromosomes have properly attached to micro- segregation machinery that is conserved in all eukaryotes. tubules, an enzyme known as separase becomes active and Early on, it was recognized that the two sister chromatids cleaves cohesin, thereby triggering the separation of sister must be held tightly together at metaphase to resist spindle chromatids to opposite poles (Figure 1). This process is forces, thereby allowing their attachment to microtubules modified during meiosis, which produces haploid gametes from opposite poles. However, the nature of this cohesion from a diploid progenitor cell. During meiosis, two rounds of was not known and two general models were put forward. chromosome segregation follow a single S phase. In meiosis One model postulated that the intertwining of sister DNA I, the maternal and paternal chromosomes, called homologs, molecules, perhaps due to the persistence of catenations are segregated, whereas sister chromatids are segregated after DNA replication, might provide cohesion (Murray and during meiosis II, which resembles mitosis (Figure 2). To Szostak 1985). Another, not mutually exclusive, model pro- achieve this, an additional layer of regulation must be in- posed the existence of proteins that generate molecular troduced. While conserved in eukaryotes, what we know bridges between sister chromatids. However, testing these about the molecular biology of chromosome segregation is models relied on the establishment of an assay for sister derived largely from work on the budding yeast Saccharo- chromatid cohesion. This was initially achieved by the de- myces cerevisiae. Here I review the discoveries in budding velopment of a fluorescence in situ hybridization assay 32 A. L. Marston Figure 1 Chromosome segregation during mito- sis. Schematic diagram showing the key features of chromosome segregation during budding yeast mitosis. (FISH) in yeast (Koshland and Hartwell 1987). Using this tains a signature motif that is required for the activity of ABC assay, it was shown that minichromosomes are cohesed at family ATPases. Smc1 NBD crystallized as a dimer with ATP metaphase even though they lack catenations, dispelling the sandwiched between the Walker A motif of one monomer idea that DNA catenation was sufficient to provide the and the signature motif on the other. In reality, Smc1 and “glue” (Koshland and Hartwell 1987; Guacci et al. 1994). Smc3 heterodimerize at their hinge domains to create This prompted the search for proteins that might mediate a V-shaped structure (Anderson et al. 2002; Haering et al. cohesion. A key technical development was the ingenious 2002). Therefore, the most likely arrangement is that two development of a method to label a single chromosome by molecules of ATP are sandwiched between the Smc1 and integration of tandem repeats of bacterial lacO, to which Smc3 NBDs. Consistently, fluorescence resonance energy ectopically produced LacI–GFP binds (Straight et al. 1996). transfer (FRET) experiments indicated that Smc1 and A similar system was developed using tetO and TetR–GFP Smc3 NBD domains are in close proximity (Mc Intyre et al. (Michaelis et al. 1997). The availability of these methods to 2007). label single chromosomes enabled the first cohesion proteins The kleisin subunit, Scc1, forms a bridge between the to be identified (Guacci et al. 1997; Michaelis et al. 1997). NBDs of the Smc1–Smc3 heterodimer, making contacts with These elegant studies isolated mutants incapable of main- Smc3 at its N terminus and Smc1 at its C terminus (Haering taining sister chromatid cohesion when arrested in mitosis. et al. 2002). A crystal structure revealed that the Scc1 C Subsequent studies revealed that sister chromatid cohesion terminus forms a winged helix domain that contacts the genes fall into functional classes (Table 1). One class of Smc1 NBD and mutations in this interface demonstrated genes encodes the proteins that make up the structural com- that this interaction is essential (Haering et al. 2004). In- ponent of cohesion, called cohesin. Others are accessory, terestingly, prior binding of the Scc1 C terminus to the Smc1 loading, or establishment factors. Remarkable progress has NBD is required for the Scc1 N terminus to bind the Smc3 been made in understanding how these many gene products NBD (Arumugam et al. 2003; Haering et al. 2004). This may interact to generate sister chromatid cohesion. ensure that a single molecule of Scc1 binds to the Smc1– Smc3 heterodimer. Although ATP binding to Smc1’s NBD is The cohesin ring: The core structural component of cohesin required for binding to the Scc1 C terminus, the interaction forms a ring, composed of two structural maintenance of of Scc1’s N terminus with Smc3 does not require ATP chromosome (SMC) proteins, Smc1 and Smc3, and a “klei- (Arumugam et al. 2003; Gruber et al. 2003). An explanation sin” (from the Greek for closure) subunit, Scc1/Mcd1 for this observation is offered by the arrangement of a bacte- (Guacci et al. 1997; Michaelis et al. 1997; Losada et al. rial Smc–kleisin complex. While the C terminus of a bacterial 1998) (Figure 3). A meiosis-specific kleisin, Rec8, replaces kleisin contacts the ATPase head of one Smc protein, its N Scc1 in meiotic cells and plays several roles important for terminus associates with the coil-coiled domain of the other the segregation of homologous chromosomes (see below). Smc subunit (Bürmann et al.