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Separating sister chromatids
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Separating sister chromatids Kim Nasmyth Loss of cohesion between sister chromatids triggers their segregation during anaphase. Recent work has identified both a cohesin complex that holds sisters together and a sister-separating protein, separin, that destroys cohesion. Separins are bound by inhibitory proteins whose proteolysis at the metaphase–anaphase transition is mediated by the anaphase-promoting complex and its activator protein CDC20 (APCCDC20). When chromosomes are misaligned, a surveillance mechanism (checkpoint) blocks sister separation by inhibiting APCCDC20. Defects in this apparatus are implicated in causing aneuploidy in human cells. THE SIMULTANEOUS SEPARATION of 46 pairs of sister chromatids at the metaphase–anaphase transition is one of the most dramatic events of the human cell cycle. As long ago as 1879, Flemming noticed that ‘the impetus causing nuclear threads to split longitudinally acts simultaneously on all of them’1. Chromosome splitting is an irreversible event and must therefore be highly regulated. Once sister chromatids separate from one another, damage to the genome cannot easily be repaired by recombination nor can mistakes in chromosome alignment be corrected. Sister chromatids are pulled to opposite halves of the cell by microtubules that emanate from spindle poles at opposite sides of the cell. The microtubules emanating from opposite poles interdigitate. Their role is to keep (and drive) the two poles apart. Meanwhile, a second set of microtubules attaches to chromosomes through specialized structures called kinetochores and pulls them towards the poles. Sister chromatids segregate and move away from each other because their kinetochores attach to microtubules that emanate from opposite poles2. Chromosomes are not mere passengers during this process. During metaphase, the tendency of microtubules to move sisters apart is counteracted by the cohesion that holds sisters together. Cohesion therefore generates the tension by which cells align sister chromatids on the metaphase plate. Were sisters to separate before spindle formation, cells would have trouble distinguishing sisters from chromatids that K. Nasmyth is at the IMP Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria. Email: [email protected]
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were merely homologous. The sudden loss of cohesion, rather than an increase in the exertion of microtubules, probably triggers sister separation during anaphase3. Cohesion also prevents chromosomes falling apart because of doublestranded breaks and facilitates their repair by recombination. Stable sister-chromatid cohesion might be unique to eukaryotic cells. Recent observations on the movement of bacterial origins of DNA replication suggest that sister origins move to opposite poles of the cell soon after initiation of DNA replication4. Thus, anaphase in bacteria commences long before chromosome replication is complete. The development of a cohesion apparatus might have been a pre-condition for the temporal separation of DNA replication and chromosome segregation in eukaryotes. What holds sister chromatids together after chromosome replication? What is Flemming’s impetus that triggers loss of cohesion, and how do cells ensure that sister separation never occurs before all pairs of sister chromatids have been aligned on the metaphase plate? Such questions are equally pertinent to meiosis, where loss of sister-chromatid cohesion within chromosome arms and centromeres must take place at different times.
The anaphase-promoting complex or cyclosome The first clue to the molecular basis for sister-chromatid separation arose from the discovery of mitotic cyclins as proteins whose abundance fluctuates during embryonic-cleavage divisions5. Mitotic cyclins are regulatory subunits of a cyclindependent kinase (CDK1) whose activation in late G2 phase triggers mitosis. Their sudden degradation as cells commence anaphase has an important role
in the destruction of CDK1 activity, but it is not required for the separation of sister chromatids6. Nevertheless, overloading the cyclin-degradation system blocks separation of sister chromatids in Xenopus laevis extracts; this suggests that proteolysis of proteins other than cyclins is involved in the separation of sister chromatids7. The apparatus responsible for targeting cyclin degradation is a highly conserved multisubunit particle that possesses ubiquitin-protein-ligase activity8,9. Initially named the cyclosome10, the ligase particle is often called the anaphase-promoting complex (APC), because it mediates destruction of many proteins other than cyclins and is essential for the separation of sister chromatids11,12. Ubiquitination mediated by the APC requires activator proteins, whose abundance or activity is rate limiting and which bind to the APC. At least two such APC activators exist in yeast, flies and vertebrate cells: related proteins called CDC20 (also known as Fizzy) and CDH1 (also known as Fizzy related)13. These activators specify both substrate specificity and the timing of proteolysis. In yeast, Cdc20p mediates destruction of an anaphase inhibitor called Pds1p, whereas Cdh1p mediates destruction of the mitotic cyclin Clb2p (Fig. 1). The APC therefore exists in at least three forms: one bound by CDC20 (APCCDC20); one bound by CDH1 (APCCDH1); and one bound by neither14–16.
APCCDC20 destroys anaphase inhibitors In the absence of APCcdc20 function, yeast cells arrest in metaphase; sister chromatids fail to separate owing to the persistence of the anaphase inhibitor Pds1p (Ref. 17). Pds1p accumulates within nuclei during late G1 phase, persists during G2 phase and early M phase, but is abruptly degraded by APCcdc20 shortly before the onset of anaphase18–20. A non-degradable version of Pds1p blocks cells in metaphase. Remarkably, Pds1p seems to be the only buddingyeast protein whose destruction by APCcdc20 is necessary for sister separation17,21. The fission-yeast Cut2p protein has similar, although not identical, properties22: Cut2p is essential23, whereas Pds1p is necessary only for proliferation at high temperatures. Despite the low sequence similarity shared by Pds1p and Cut2p, they nevertheless appear to have similar functions (see below). Proteins that have similar properties to these yeast proteins have recently been found in animal cells (M. Kirschner, pers. commun.).
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Cohesins Abundance
Pds1p (Cut2p) Cyclin-B–Cdk1p
APCcdc20
Activity
Might Pds1p and Cut2p be sisterchromatid cohesion proteins whose destruction permits sister separation? This seems unlikely for several reasons: Pds1p is not essential; the majority of Pds1p does not appear to bind chromosomes tightly21; and Pds1p clearly regulates cell-cycle progression as well as sister-chromatid separation17. Cut2p is also not associated with chromosomes. Indeed, some, if not most, of it is associated with the mitotic spindle23. What then are the chromosomal proteins that hold sisters together between replication and the onset of anaphase? Do such proteins even exist? Might sisters be held together simply by their intercatenation, which results from collision of neighbouring replication forks24? The recent discovery of chromosomal proteins that are essential for sister-chromatid cohesion during G2 and M phases25,26 suggests that cohesion could be due to proteinaceous bridges. By looking for mutants that lose chromosomes at a high frequency and separate sister chromatids in the absence of APC function, my group and others25,27–29 have identified six essential yeast genes that are needed for sister cohesion: SMC1, SMC3, SCC1 (also known as MCD1), SCC2, SCC3 and ECO1 (also known as CTF7). The products of four of these genes (Smc1p, Smc3p, Scc1p and Scc3p) bind as a cohesin complex (Fig. 2a) to multiple sites along chromosomes25,29. An Scc2p homologue, Mis4p, is needed for sister-chromatid cohesion in fission yeast30. Although not a stoichiometric subunit of cohesin, Scc2p is needed for the association between cohesin and chromosomes29. Scc1p, Scc2p and Scc3p are unrelated proteins whose sequences have thus far provided no clue to their function. Smc1p and Smc3p, by contrast, belong to the SMC family of proteins, members of which contain an NTP-binding domain at their N-terminus, two long stretches of coiled coil that are separated by a hinge region at their centre, and a conserved C-terminal domain that binds DNA31,32. SMC proteins exist both in bacterial and in eukaryotic lineages, which suggests that they are ancient modulators of chromosome structure. Unlike bacteria, whose genomes encode at most one SMC protein, all eukaryotic organisms express at least four members of the SMC family: Smc1p, Smc2p, Smc3p and Smc4p. Smc2p and Smc4p are components of a 13S complex that contains three other subunits and is essential for
APCCdh1
M
A
G1
S
Phase
Figure 1 APCcdc20 and APCCdh1 mediate proteolysis at different stages of the cell cycle. Proteolysis of Pds1p (Cut2p) and cyclin B is driven by ubiquitination and is necessary for sister separation and exit from mitosis, respectively. The anaphase-promoting complex (APC) requires activator proteins, either Cdc20p or Cdh1p. Pds1p, an anaphase inhibitor, is ubiquitinated by APCcdc20, which is activated by cyclin-B–Cdk1p. Cyclins are ubiquitinated, at least during G1 phase, by APCCdh1, which is inhibited by cyclin-B–Cdk1p. Thus, the activity of APCcdc20 peaks during mitosis, whereas that of APCCdh1 peaks during G1 phase.
chromosome condensation in yeast and in Xenopus, and has therefore been called condensin33. Condensin can supercoil DNA in an ATP-dependent manner. It binds to chromosomes and mediates their condensation only upon activation of cyclin-B–CDK1 (Ref. 34). Smc1 and
Smc3 from Xenopus are found in a cohesin complex with three other proteins, one of which is the Xenopus homologue of Scc1p (Ref. 26). Depletion of the Xenopus cohesin complex, by using antibodies directed against either Smc1 or Smc3, has little or no effect on chromosome
(a) Scc2p (Mis4p)
Eco1p (Ctf7p)
Cohesin
Centromere Microtubules attempting to pull sister chromatids apart (b) Replication fork Eco1p (Ctf7p) Cohesin
(c) Replication apparatus
N C or
C N Smc1p Smc3p
Figure 2 Multisubunit complexes that contain Smc proteins mediate sister-chromatid cohesion and condensation. A cohesin complex (a), containing Smc1p, Smc3p, Scc1p/Mcd1p and Scc3p and possibly other proteins, is necessary for sister-chromatid cohesion, which is established during DNA replication and depends on Eco1p/Ctf7p (b). Cohesin might contain an Smc1p–Smc3p heterodimer in which the coiled coils of each partner are antiparallel (c). The result is a V-shaped molecule that possesses two similar, but not identical, globular domains at each end, which are composed of an N-terminal domain from one member and a C-terminal domain from the other.
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REVIEWS condensation but causes sister chromatids to separate prematurely. This suggests that eukaryotic cells use a conserved cohesin complex to hold sister chromatids together. The involvement of SMC proteins in cohesion and condensation suggests that these two processes are mechanistically related, although we do not know how. Bacterial SMC proteins form homodimers whose coiled coils are antiparallel, and N- and C-teminal domains are paired together35. The implication is that cohesin contains an Smc1–Smc3 heterodimer in which the coiled coils of Smc1 run antiparallel to those of Smc3 (Fig. 2b). It is not known where accessory proteins such as Scc1p and Scc3p bind to the Smc1–Smc3 heterodimer or indeed whether cohesin is even part of the link between sister chromatids. Sisters might be joined together by a single Smc1–Smc3 heterodimer whose two globular domains are bound to each sister chromatid or instead by a pair of heterodimers, each of which is bound to a single chromatid, that are somehow linked together.
Establishment and maintenance of cohesion In yeast, Scc1p can bind to chromosomes from late G1 phase until metaphase, but it can establish cohesion only when present during DNA replication36. Mis4p (the Scc2p homologue in fission yeast) likewise functions during S phase30. The formation of links between sisters might be a function associated with replication forks. Most, if not all, yeast cohesin subunits remain bound to chromosomes until the onset of anaphase and are required for maintenance of cohesion during metaphase. Eco1p (also known as Ctf7p), by contrast, is required to establish cohesion during S phase but not to maintain it during G2 phase28,29, and presumably acts at replication forks (Fig. 2a). The nature of the link between sisters produced by cohesin during DNA replication and whether it is formed at specific DNA sequences are not known.
Dissolution of cohesion What triggers sister separation? Might changes in the state of cohesin – for example, in its association with chromatids – be responsible for the loss of cohesion at the metaphase–anaphase transition? This could be the case in yeast, in which cohesins remain bound to chromosomes until sisters separate; whereupon, Scc1p and Scc3p disappear from chromosomes25,29. The disappearance of Scc1p depends on APCcdc20,
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TIBS 24 – MARCH 1999 whose role in this regard is to destroy Pds1p (Ref. 21). However, cohesin behaves rather differently in animal cells (Fig. 3). In Xenopus, cohesin binds to chromosomes at the time that they replicate (or even before); it remains associated throughout G2 phase but dissociates during prophase as cyclinB–Cdk1 is activated26. Cohesin’s dissociation coincides with, but does not depend on, condensin’s association with chromosomes. This suggests that animal cells use at least two types of cohesion: (1) cohesin-mediated cohesion, which is established during DNA replication and persists until entry into mitosis; and (2) a mechanism that holds sister chromatids together during chromosome alignment and is destroyed only at the metaphase–anaphase transition. The molecules involved in the second form of cohesion have not yet been identified. Yeast might also possess both types of cohesion – in which case, the main difference between yeast and animals might be only in the timing with which the two types of cohesion are destroyed. Differences in the degree of chromosome condensation between yeast and animal cells might account for the different timing of cohesin’s dissociation from chromosomes. In animal cells, cohesin’s prior dissociation, which presumably loosens connections between sisters, might be required for condensation, during which most sister sequences are resolved from each other37. Yeast chromosomes undergo far less condensation. Indeed, they maintain high rates of transcription throughout mitosis, and sister DNA sequences remain closely connected even during metaphase38. Thus, the absence of a clearly defined resolution/condensation step in yeast might permit cohesin to remain associated with chromosomes until the metaphase– anaphase transition. Cohesin’s abundance on G2-phase chromosomes ensures that sisters remain tightly associated, even when cells (such as oocytes) spend long periods in this stage of the cell cycle, whereas its dissociation from chromosomes as cells enter mitosis might be a pre-condition for the rapid separation of sister chromatids at the metaphase–anaphase transition.
Sister-separating proteins and their inhibitors Pds1p blocks dissociation of Scc1p from yeast chromosomes. How does it do this? Unlike cohesins, the bulk of Pds1p does not appear to associate with chromosomes but instead forms stable complexes with a soluble 180-kd protein,
Esp1p (Ref. 21), whose fission-yeast homologue, Cut1p, forms a similar complex with Cut2p (Ref. 23). When incubated at the restrictive temperature, esp1 mutant cells replicate DNA, form bipolar spindles and even destroy Pds1p (and also eventually cyclins), but they fail to separate sister chromatids, and Scc1p never dissociates from chromatids21. Thus, unlike the APC, which is required both for sister-chromatid separation and for cell-cycle progression, Esp1p is needed specifically for sister separation. Esp1p and Cut1p might be dedicated to the separation of sister chromatids; it has therefore been suggested that they be called separins. Separin homologues exist in humans, Xenopus and Caenorhabditis elegans. An appealing hypothesis to explain the role of APCcdc20 in the separation of sisters in budding yeast emerges from these findings: Pds1p blocks sister separation by binding to Esp1p, which inhibits its separating activity. According to this model, sister separation depends on APCcdc20 because it destroys the separin inhibitor Pds1p. Thus, the main role of APCcdc20 in the separation of sister chromatids in yeast might be to destroy inhibitory proteins that bind to separins rather than to destroy cohesion proteins themselves (Fig. 3). The recent identification of human and Xenopus anaphase-inhibitory proteins that have the properties of Pds1p, and bind to Esp1p and are degraded by APCCDC20, suggests that the yeast model applies also to animal cells (M. Kirschner, pers. commun.). Whether Pds1p-like proteins are the main targets for APCCDC20 in animals remains to be determined. Certainly, the APC might also contribute to sister separation by destroying chromosomal proteins that are involved in maintaining cohesion during metaphase. A key issue is how separins promote sister separation. Most Cut1p resides in the cytoplasm until cells enter mitosis; whereupon, it accumulates on the mitotic spindle until late anaphase. Cut1p might modulate the activity of anaphase motor proteins or microtubule dynamics in a manner that would increase the spindle’s pull on kinetochores39. Other evidence, however, suggests that Esp1p/Cut1p separins act directly on chromosomes. In cells that lack a mitoticsurveillance mechanism, Esp1p can induce Scc1p to disappear from chromosomes in cells treated with nocodazole (which destroys spindles) and in mutants that lack functional kinetochores21. Thus, separins might act both on spindles and
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TIBS 24 – MARCH 1999 on chromosomes. How Esp1p triggers dissociation of Scc1p is not understood. It does not simply replace cohesins: it is never found stably associated with chromosomes. Sister separation in animal cells can also occur during anaphase in the absence of chromosomes being attached to spindles40,41. It will clearly be of interest to establish whether separins are required for the dissociation of cohesin from chromatids as animal cells enter mitosis or whether they are required only for their eventual separation during anaphase. Other proteins besides Esp1/Cut1 are involved in separation of sister chromatids. In Drosophila melanogaster, for example, pimples and three rows, neither of which resemble Esp1p/Cut1p, are necessary for separating centromeres but not chromosome arms42.
Regulating loss of cohesion
Yeast
Esp1
Cyclin-B–Cdk1
APCcdc20
Pds1uuu
Esp1–Pds1
Cohesin joint Chromatid Microtubule Condensin
Vertebrates
Figure 3
What determines the onset Dissolution of sister-chromatid cohesion. In budding yeast, sister separation might be driven by Esp1p of Pds1p destruction and, (separin) activity, which causes the dissociation of Scc1p from chromosomes. Esp1p is inhibited by the thereby, activation of Esp1p? binding of Pds1p. Cut1p and Cut2p perform these two functions in fission yeast. Esp1p is activated by proteolysis of Pds1p by APCcdc20, which is thought to be activated by cyclin-B–Cdk1p. Vertebrates also Accumulation of Cdc20p dur20,43 possess separin homologues, but less is known about their action. In vertebrates, a condensin complex ing late G2 phase presumthat contains SMC2, SMC4, X-CAPG, X-CAPD and Barren associates with chromosomes as cells enter ably has some role; however, mitosis, at which point most, but possibly not all, cohesin dissociates from chromosomes. Yellow Cdc20p alone cannot be rechevrons represent the pulling force exerted by microtubules. u, ubiquitin. sponsible, because advancing its synthesis does not immediately precipitate Pds1p destruction44. Cells detect either tension between sis- that depends on Mad2p. Furthermore, APCcdc20 might be activated by cyclin-B– ter centromeres46 or their occupancy by both Mad2p and Pds1p are essential for Cdk1p (Fig. 3). Either Cdc20p itself or core spindles47. Remarkably, the kinetochore preventing the disappearance of Scc1 APC subunits such as Cdc27p, which of a single lagging chromosome emits a from chromosomes (G. Alexandru et al., become hyperphosphorylated during signal capable of blocking separation of unpublished) and for arresting sister sepmitosis, might be targets of Cdk1p. all other sister pairs48. It also blocks all aration17,57 when spindles are damaged. Surprisingly, Pds1p is not necessary further cell-cycle progression, including This implies that the checkpoint blocks for cell proliferation at low temperatures45. proteolysis of B-type but not A-type cy- sister separation exclusively by inhibiting Indeed, there is little or no difference in clins, cytokinesis, and chromosome de- proteolysis of Pds1, whose persistence the timing of sister separation or disso- condensation and re-replication. Five ensures that Esp1p remains inactive and, ciation of Scc1p from chromosomes in nonessential proteins (Mad1p, Mad2p, as a consequence, cohesin links remain the wild type and pds1-deletion mutants Mad3p, the protein kinase Bub1p and intact. Whether animal cells use a similar (G. Alexandru, pers. commun.). Why, if Bub3p) are necessary for this response mechanism for halting sister separation Pds1p proteolysis is necessary for Esp1p in yeast49,50. In addition Mps1, a protein is unclear. Because the bulk of cohesin activation, is there no advancement of kinase that is also required for spindle dissociates from vertebrate chromosister separation when Pds1p is missing? pole duplication51, and Ndc10p, a sub- somes as cells enter mitosis as opposed Presumably, dissociation of cohesins unit of the centromere-binding complex to when sister chromatids actually from chromosomes is regulated by a CBF321,52, are needed. Animal hom- separate, the vertebrate checkpoint second, Pds1p-independent, mechanism. ologues of Mad1p, Mad2p, Mad3p and probably does not halt dissociation of Bub1p associate with the kinetochores cohesin, as it appears to do in yeast. Stopping sister separation of lagging chromosomes but not with How do Mad proteins inhibit proMost eukaryotic cells possess surveil- those of chromosomes correctly aligned teolysis of Pds1p? Three observations lance mechanisms (checkpoints) that on the metaphase plate53–56. indicate that they do so by inhibiting prevent sister-chromatid separation when How do Mad/Bub proteins block sister APCcdc20 (Fig. 4). First, Mad2p, possibly spindles are damaged or chromosomes separation? In yeast, spindle damage along with other Mad proteins, associfail to form bivalent spindle attachments. blocks Pds1p proteolysis by a process ates with both Cdc20p and APCcdc20
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are pulled towards, opposite spindle poles. However, as long as at least one reciprocal exchange between homologous chromatids has occurred, cohesion between sister chromatid arms will oppose disjunction of homologues and their segregation to opposite poles of the cell62. Thus, loss of sister-chromatid cohesion along Esp1p (Cut1p) chromosome arms is essential for chromosome segregation during meiosis I. Meanwhile, Active Inactive however, cohesion between sister centromeres persists so APCcdc20 Mad–APCcdc20 that it can be used later to align sisters on the meiosis-II metaphase plate. The differEsp1p–Pds1p (Cut1p–Cut2p) ence in the timing of sisterchromatid cohesion loss in chromosome arms and centromeres is therefore a crucial aspect of meiosis (Fig. 5). Some evidence suggests that Cohesin joint complexes related to cohesin Chromatid are required for meiotic sisterMicrotubule chromatid cohesion. The genomes of budding and fission yeast both encode two Scc1plike proteins: Scc1p (Rad21p in fission yeast) and Rec8p. Figure 4 Scc1p/Rad21p is essential for Stopping sister-chromatid separation. Lagging chromosomes emit a signal that induces the inactivation of mitosis, whereas Rec8p is esAPCcdc20 by a complex containing Mad1p, Mad2p and Mad3p. In yeast, this prevents destruction of Pds1p sential for meiosis and, along and prevents Esp1p from displacing Scc1p from chromosomes, which is thought to play a key role in loss with Smc3p, for prevention of of cohesion. Yellow chevrons represent the pulling force exerted by microtubules. premature separation of sister chromatids63 (F. Klein and (Refs 14, 15, 58). Second, Mad2p can in- construct that encodes an N-terminal K. Nasmyth, unpublished). Rec8p and hibit cyclin ubiquitination mediated by Bub1p fragment54,61 reduces the interval Smc3p are found all along the longitudinal APCcdc20 in vitro58. Third, overproduction between nuclear-membrane breakdown axis of chromosomes during pachytene. of Cdc20p, or forms of Cdc20p that fail and the onset of sister separation; this Both proteins largely disappear from to bind Mad2p, allows cells whose mi- raises the possibility (but does not chromosome arms during the first meiotic totic checkpoint has been activated to prove) that signals that emanate from division but persist around centromeres escape mitotic arrest59,60. How kineto- lagging chromosomes during normal mi- until metaphase II. They then disappear chores of lagging chromosomes, which toses are essential to prevent premature completely as cohesion between sister are bound by Mad and Bub proteins, sister separation. centromeres is lost at the onset of anaseed formation of Mad2p-containing phase II (Y. Watanabe and P. Nurse, pers. complexes that inhibit APCcdc20 through- Meiosis commun.; F. Klein and K. Nasmyth, unout the cell is still mysterious. Mad2p Meiosis is a variation on the theme of published). Scc2p (Mis4p) is homologous tetramers are more effective than Mad2p mitosis. It produces haploid gametes to Rad9 from Coprinus cinereus64, whereas dimers at inhibiting APCcdc20 in vitro; this that contain recombinant chromosomes Scc3p is homologous to Rec11p from fisraises the possibility that lagging chromo- – parts of which are derived from ‘mum’, sion yeast; both Rad9p and Rec11p are somes catalyse a conformational change and parts of which are derived from necessary for meiosis. In Drosophila, in Mad2p (Ref. 15). Activation of APCCdh1 ‘dad’. As in the case of mitosis, meiosis MeiS332 also has an important role in is also inhibited by the checkpoint, but starts with a round of DNA replication in preservation of sister cohesion at centrothe mechanism is not clear. diploid germ cells, which produces con- meres until the second meiotic division. The genes that encode Mad and nected sister chromatids. Recombination It binds to centromeric heterochromatin Bub proteins are dispensable in yeast. between homologous chromatids (and during metaphase of meiosis I, persists Furthermore, sister separation does not possibly other pairing mechanisms) there until sisters separate during anaeven occur early in mad2 mutants. brings homologues together. During the phase of meiosis II and is essential for Whether the same is true for animal first meiotic division, sister kinetochores prevention of premature separation of siscells is still unclear. Injection of Mad2p- are treated as a single unit, and homolo- ter centromeres65. It is not known whether specific antibodies or expression of a gous kinetochore pairs attach to, and MeiS332 interacts with cohesins.
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Medical relevance Chromosome-segregation defects uncover recessive mutations, including those that permit the uncontrolled proliferation of tumour cells. Sister-chromatidcohesion defects could therefore contribute to tumorigenesis. Most tumour cells are indeed highly aneuploid, and many, including most colorectal cancers, actually lose and gain chromosomes at a high frequency as tumour cells divide66. But, is this chromosome instability a cause or a consequence of oncogenic mutations? Some evidence suggests that mitotic-checkpoint defects contribute to colorectal aneuploidy. Most, if not all, colorectal-carcinoma cell lines that lose chromosomes with a high frequency appear to be defective in their halting of the cell cycle when treated with nocodazole67. Moreover, one particular cell line contains a BUB1 mutation. Viruses also target mitotic surveillance. Human T-cell leukaemia virus (HTLV) causes the formation of multinucleate cells and encodes an oncoprotein, Tax, that binds to and possibly inhibits MAD1 (Ref. 55). Tax might facilitate proliferation induced by HTLV by abrogating mitotic arrest. Mutations in cohesins cause very high levels of chromosome loss in yeast, but whether similar mutations in humans contribute to tumour-cell aneuploidy is not known. Defects in the maintenance of sisterchromatid cohesion could also contribute to congenital aneuploidy in humans. The best example of this is Down’s syndrome, which is caused by the presence of an extra copy of chromosome 21. Most human aneuploidy is caused by mistakes that occur during meiosis in females. A high fraction of meiosis-IImetaphase oocytes obtained from infertility clinics have an incidence and pattern of extra chromosomes that are similar to those of aneuploid foetuses68. Remarkably, all extra chromosomes in these abnormal oocytes are single chromatids, not sister chromatid pairs, and very possibly arose because of a failure to maintain sister-chromatid cohesion during meiosis I. Oocytes spend long periods arrested in the G2 phase of the first meiotic division; once lost during this period, cohesion might be irreparable.
Concluding remarks Recent research has identified a crucial component of the sister-chromatidcohesion apparatus (cohesin), a class of sister separating proteins (the separins), the proteolytic system responsible for destroying separin inhibitors (APCCDC20),
(a)
(b)
Meiosis I
Meiosis II
Cohesin joint Chromatid Microtubule
Figure 5 Sister-chromatid cohesion and its destruction during meiosis. (a) Meiosis I. (b) Meiosis II. Homologous chromosomes (shown in blue and red) are held together by chiasmata (sites of reciprocal recombination between homologous chromatids) and cohesion between sister chromatids within chromosome arms. Loss of arm cohesion and cosegregation of sister centromeres reduces the chromosome number at meiosis I, whereas loss of sister cohesion at centromeres at meiosis II produces gametes that have a haploid chromosome number.
and a mitotic-surveillance mechanism that regulates APCCDC20. The study of sister-chromatid separation has therefore moved from the phenomenological to the molecular level, a transition that not only poses a new set of questions but also promises to shed insight into human pathology. Each round of sister separation is fraught with potential errors that, in somatic cells, can lead to cancer and, in the germline, are the raw material for natural selection. We are largely a product of our chromosomes. We are finally beginning to understand the complexities of the apparatus by which we inherit a complete set.
Acknowledgements I thank Mitsohiro Yanagida, Paul Nurse and Marc Kirscher for communicating unpublished results, Michael Glotzer and Jan-Michael Peters for their comments on the manuscript, Hannes Tkadletz for help with the figures, and Tim Hunt, who encouraged me to write this article.
References 1 Flemming, W. (1879) Archiv f. mikrosk. Anatomie 18, 151–259 2 Rieder, C. L. and Salmon, E. D. (1998) Trends Cell Biol. 8, 310–317 3 Miyazaki, W. Y. and Orr-Weaver, T. L. (1994) Annu. Rev. Genet. 28, 167–187 4 Lin, D. C. and Grossman, A. D. (1998) Cell 92, 675–685 5 Evans, T. et al. (1983) Cell 33, 389–396 6 Surana, U. et al. (1993) EMBO J. 12, 1969–1978 7 Holloway, S. L., Glotzer, M., King, R. W. and Murray, A. W. (1993) Cell 73, 1393–1402 8 Yu, H. et al. (1998) Science 279, 1219–1222
9 Zachariae, W. et al. (1998) Science 279, 1216–1219 10 Sudakin, V. et al. (1995) Mol. Biol. Cell 6, 185–198 11 King, R. W. et al. (1995) Cell 81, 279–288 12 Irniger, S., Piatti, S., Michaelis, C. and Nasmyth, K. (1995) Cell 81, 269–278 13 Townsley, F. M. and Ruderman, J. V. (1998) Trends Cell Biol. 8, 238–244 14 Kallio, M. et al. (1998) J. Cell Biol. 141, 1393–1406 15 Fang, G., Yu, H. and Kirschner, M. W. (1998) Genes Dev. 12, 1871–1883 16 Zachariae, W., Schwab, M., Nasmyth, K. and Seufert, W. (1998) Science 282, 1721–1724 17 Yamamoto, A., Guacci, V. and Koshland, D. (1996) J. Cell Biol. 133, 99–110 18 Cohen-Fix, O., Peters, J-M., Kirschner, M. W. and Koshland, D. (1996) Genes Dev. 10, 3081–3093 19 Visintin, R., Prinz, S. and Amon, A. (1997) Science 278, 460–463 20 Shirayama, M., Zachariae, W., Ciosk, R. and Nasmyth, K. (1998) EMBO J. 17, 1336–1349 21 Ciosk, R. et al. (1998) Cell 93, 1067–1076 22 Funabiki, H. et al. (1996) Nature 381, 438–441 23 Funabiki, H., Kumada, K. and Yanagida, M. (1996) EMBO J. 15, 6617–6628 24 Murray, A. W. and Szostak, J. W. (1985) Annu. Rev. Cell Biol. 1, 289–315 25 Michaelis, C., Ciosk, R. and Nasmyth, K. (1997) Cell 91, 35–45 26 Losada, A., Hirano, M. and Hirano, T. (1998) Genes Dev. 12, 1986–1997 27 Guacci, V., Koshland, D. and Strunnikov, A. (1997) Cell 91, 47–57 28 Skibbens, R. V., Corson, L. B., Koshland, D. and Hieter, P. Genes Dev. (in press) 29 Toth, A. et al. Genes Dev. (in press) 30 Furuya, K., Takahashi, K. and Yanagida, M. (1998) Genes Dev. 12, 3408–3418 31 Koshland, D. and Strunnikov, A. (1996) Annu. Rev. Cell Dev. Biol. 12, 305–313 32 Akhemedov, A. T. et al. (1998) J. Biol. Chem. 273, 24088–24094 33 Hirano, T. (1998) Curr. Opin. Cell Biol. 10, 317–322
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34 Hirano, T., Kobayashi, R. and Hirano, M. (1997) Cell 89, 511–521 35 Melby, T. E., Ciampaglio, C. N., Briscoe, G. and Erickson, H. P. (1998) J. Cell Biol. 142, 1595–1604 36 Uhlmann, F. and Nasmyth, K. (1998) Curr. Biol. 8, 1095–1101 37 Hirano, T. (1995) Trends Biochem. Sci. 20, 357–361 38 Guacci, V., Hogan, E. and Koshland, D. (1994) J. Cell Biol. 125, 517–530 39 Kumada, K. et al. (1998) Curr. Biol. 8, 633–641 40 Liang, H. et al. (1993) Exp. Cell Res. 204, 110–120 41 Sluder, G. et al. (1997) J. Cell Sci. 110, 421–429 42 Stratmann, R. and Lehner, C. F. (1996) Cell 84, 25–35 43 Weinstein, J. (1997) J. Biol. Chem. 272, 28501–28511 44 Prinz, S., Hwang, E. S., Visintin, R. and Amon, A. (1998) Curr. Biol. 8, 750–760
45 Yamamoto, A., Guacci, V. and Koshland, D. (1996) J. Cell Biol. 133, 85–97 46 Nicklas, R. B. (1997) Science 275, 632–637 47 Waters, J. C., Chen, R. H., Murray, A. W. and Salmon, E. D. (1998) J. Cell Biol. 141, 1181–1191 48 Wells, W. A. and Murray, A. W. (1996) J. Cell Biol. 133, 75–84 49 Li, R. and Murray, A. W. (1991) Cell 66, 519–531 50 Hoyt, M. A., Trotis, L. and Roberts, B. T. (1991) Cell 66, 507–517 51 Hardwick, K. G. et al. (1996) Science 273, 953–956 52 Tavormina, P. A. and Burke, D. J. (1998) Genetics 148, 1701–1713 53 Chen, R. H., Waters, J. C., Salmon, E. D. and Murray, A. W. (1996) Science 274, 242–246 54 Taylor, S. S. and McKeon, F. (1997) Cell 89, 727–735 55 Jin, D. Y., Spencer, F. and Jeang, K. T. (1998) Cell 93, 81–91 56 Taylor, S. S., Ha, E. and McKeon, F. (1998) J. Cell Biol. 142, 1–11
Bacterial solutions to the iron-supply problem Volkmar Braun and Helmut Killmann The insolubility of Fe31 necessitates special mechanisms for iron acquisition in most organisms. Bacteria use siderophores to chelate Fe31 and iron in heme, hemoglobin, transferrin and lactoferrin, and employ novel mechanisms for receptor-dependent iron transport and iron-regulated gene expression. These mechanisms involve transfer of energy from the cytoplasmic membrane to the outer membrane to drive active transport and might induce transcription of transport genes by transmitting a signal from the cell surface. IRON PLAYS A central role in many redox enzymes that function in electrontransport chains of intermediary metabolism. It is particularly suited to participating in a wide range of electron-transfer reactions because, depending on the ligand and protein environment, the redox potential of Fe31/Fe21 spans 1300 mV to 2500 mV. Although iron is abundant in nature, the extremely low solubility (10218 M) of Fe31 at pH 7 means that most organisms face the problem of obtaining enough iron from their environment. After absorption of iron through the gut, V. Braun and H. Killmann are at Lehrstuhl Mikrobiologie/Membranphysiologie, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany. Email: volkmar.braun@mikrobio. uni-tuebingen.de
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humans use proteins to keep iron in solution: transferrin is the iron carrier in the blood; lactoferrin is the carrier in secretory fluids; and ferritin stores iron within cells. Disturbances of iron metabolism result in severe diseases, such as congenital hemochromatosis. Several iron-supply strategies are available to bacteria. Under anaerobic conditions, Fe21 is present. It is sufficiently soluble that it can be taken up by anaerobic bacteria without the help of iron chelators. Similarly, acid-tolerant bacteria might find enough Fe31, which has a solubility of 1028 M at pH 3, to cover their iron needs. Alternatively, bacteria (and fungi) synthesize a wide variety of low-molecular-weight iron ligands that are called siderophores. These form three major structural types: hydroxamates,
57 Straight, A. F., Belmont, A. S., Robinett, C. C. and Murray, A. W. (1996) Curr. Biol. 6, 1599–1608 58 Li, Y. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12431–12436 59 Kim, S. H. et al. (1998) Science 279, 1045–1047 60 Hwang, L. H. et al. (1998) Science 279, 1041–1044 61 Gorbsky, G. J., Chen, R. H. and Murray, A. W. (1998) J. Cell Biol. 141, 1193–1205 62 Moore, D. P. and Orr-Weaver, T. L. (1998) Curr. Top. Dev. Biol. 37, 263–299 63 Molnar, M., Bahler, J., Sipiczki, M. and Kohli, J. (1995) Genetics 141, 61–73 64 Seitz, L. C., Tang, K., Cummings, W. J. and Zolan, M. E. (1996) Genetics 142, 1105–1117 65 Kerrebrock, A. W., Moore, D. P., Wu, J. S. and Orr-Weaver, T. L. (1995) Cell 83, 247–256 66 Lengauer, C., Kinzler, K. W. and Vogelstein, B. (1997) Nature 386, 623–627 67 Cahill, D. P. et al. (1998) Nature 392, 300–303 68 Angell, R. (1997) Am. J. Hum. Genet. 61, 23–32
catecholates and α-hydroxycarboxylates1. The siderophores, as in the cases of the transferrins and lactoferrins, bind Fe31 very strongly; the free Fe31 concentration is in the order of 10224 M (the calculated pM value {2log[M(H2O)n]m1, where M is the metal ion, m the metal valence and n the number of water molecules bound} for 1 mM Fe31 and 10 mM ligand at pH 7.4)2. Certain pathogenic bacteria employ iron sources that occur in their hosts, and do not use siderophores. For example, Serratia marcescens, Yersinia enterocolitica, Yersinia pestis, Shigella dysenteriae, Escherichia coli O157 and Vibrio cholerae use heme; Neisseria gonorrhoeae, Neisseria meningitidis and Haemophilus influenzae use hemoglobin – both alone and bound to haptoglobin; H. influenzae also uses heme bound to hemopexin, and Neisseria and H. influenzae also use iron bound to transferrin and lactoferrin (Fig. 1)3–5. We do not know how iron is released from the iron proteins or whether it is transported in free or complexed form into the bacteria. A protein that is secreted into the culture medium of S. marcescens releases heme from hemoglobin6, and heme bound to hemopexin is released by a secreted protein of H. influenzae type b (Ref. 3).
Initial binding of Fe31 chelates to receptor proteins Fe31 siderophores bind to highly specific receptor proteins and are then transported into the cytoplasm (Fig. 1). In Gram-positive bacteria, which lack an outer membrane, the receptors are bind-
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Separating sister chromatids Kim Nasmyth Loss of cohesion between sister chromatids triggers their segregation during anaphase. Recent work has identified both a cohesin complex that holds sisters together and a sister-separating protein, separin, that destroys cohesion. Separins are bound by inhibitory proteins whose proteolysis at the metaphase–anaphase transition is mediated by the anaphase-promoting complex and its activator protein CDC20 (APCCDC20). When chromosomes are misaligned, a surveillance mechanism (checkpoint) blocks sister separation by inhibiting APCCDC20. Defects in this apparatus are implicated in causing aneuploidy in human cells. THE SIMULTANEOUS SEPARATION of 46 pairs of sister chromatids at the metaphase–anaphase transition is one of the most dramatic events of the human cell cycle. As long ago as 1879, Flemming noticed that ‘the impetus causing nuclear threads to split longitudinally acts simultaneously on all of them’1. Chromosome splitting is an irreversible event and must therefore be highly regulated. Once sister chromatids separate from one another, damage to the genome cannot easily be repaired by recombination nor can mistakes in chromosome alignment be corrected. Sister chromatids are pulled to opposite halves of the cell by microtubules that emanate from spindle poles at opposite sides of the cell. The microtubules emanating from opposite poles interdigitate. Their role is to keep (and drive) the two poles apart. Meanwhile, a second set of microtubules attaches to chromosomes through specialized structures called kinetochores and pulls them towards the poles. Sister chromatids segregate and move away from each other because their kinetochores attach to microtubules that emanate from opposite poles2. Chromosomes are not mere passengers during this process. During metaphase, the tendency of microtubules to move sisters apart is counteracted by the cohesion that holds sisters together. Cohesion therefore generates the tension by which cells align sister chromatids on the metaphase plate. Were sisters to separate before spindle formation, cells would have trouble distinguishing sisters from chromatids that K. Nasmyth is at the IMP Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria. Email: [email protected]
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were merely homologous. The sudden loss of cohesion, rather than an increase in the exertion of microtubules, probably triggers sister separation during anaphase3. Cohesion also prevents chromosomes falling apart because of doublestranded breaks and facilitates their repair by recombination. Stable sister-chromatid cohesion might be unique to eukaryotic cells. Recent observations on the movement of bacterial origins of DNA replication suggest that sister origins move to opposite poles of the cell soon after initiation of DNA replication4. Thus, anaphase in bacteria commences long before chromosome replication is complete. The development of a cohesion apparatus might have been a pre-condition for the temporal separation of DNA replication and chromosome segregation in eukaryotes. What holds sister chromatids together after chromosome replication? What is Flemming’s impetus that triggers loss of cohesion, and how do cells ensure that sister separation never occurs before all pairs of sister chromatids have been aligned on the metaphase plate? Such questions are equally pertinent to meiosis, where loss of sister-chromatid cohesion within chromosome arms and centromeres must take place at different times.
The anaphase-promoting complex or cyclosome The first clue to the molecular basis for sister-chromatid separation arose from the discovery of mitotic cyclins as proteins whose abundance fluctuates during embryonic-cleavage divisions5. Mitotic cyclins are regulatory subunits of a cyclindependent kinase (CDK1) whose activation in late G2 phase triggers mitosis. Their sudden degradation as cells commence anaphase has an important role
in the destruction of CDK1 activity, but it is not required for the separation of sister chromatids6. Nevertheless, overloading the cyclin-degradation system blocks separation of sister chromatids in Xenopus laevis extracts; this suggests that proteolysis of proteins other than cyclins is involved in the separation of sister chromatids7. The apparatus responsible for targeting cyclin degradation is a highly conserved multisubunit particle that possesses ubiquitin-protein-ligase activity8,9. Initially named the cyclosome10, the ligase particle is often called the anaphase-promoting complex (APC), because it mediates destruction of many proteins other than cyclins and is essential for the separation of sister chromatids11,12. Ubiquitination mediated by the APC requires activator proteins, whose abundance or activity is rate limiting and which bind to the APC. At least two such APC activators exist in yeast, flies and vertebrate cells: related proteins called CDC20 (also known as Fizzy) and CDH1 (also known as Fizzy related)13. These activators specify both substrate specificity and the timing of proteolysis. In yeast, Cdc20p mediates destruction of an anaphase inhibitor called Pds1p, whereas Cdh1p mediates destruction of the mitotic cyclin Clb2p (Fig. 1). The APC therefore exists in at least three forms: one bound by CDC20 (APCCDC20); one bound by CDH1 (APCCDH1); and one bound by neither14–16.
APCCDC20 destroys anaphase inhibitors In the absence of APCcdc20 function, yeast cells arrest in metaphase; sister chromatids fail to separate owing to the persistence of the anaphase inhibitor Pds1p (Ref. 17). Pds1p accumulates within nuclei during late G1 phase, persists during G2 phase and early M phase, but is abruptly degraded by APCcdc20 shortly before the onset of anaphase18–20. A non-degradable version of Pds1p blocks cells in metaphase. Remarkably, Pds1p seems to be the only buddingyeast protein whose destruction by APCcdc20 is necessary for sister separation17,21. The fission-yeast Cut2p protein has similar, although not identical, properties22: Cut2p is essential23, whereas Pds1p is necessary only for proliferation at high temperatures. Despite the low sequence similarity shared by Pds1p and Cut2p, they nevertheless appear to have similar functions (see below). Proteins that have similar properties to these yeast proteins have recently been found in animal cells (M. Kirschner, pers. commun.).
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Cohesins Abundance
Pds1p (Cut2p) Cyclin-B–Cdk1p
APCcdc20
Activity
Might Pds1p and Cut2p be sisterchromatid cohesion proteins whose destruction permits sister separation? This seems unlikely for several reasons: Pds1p is not essential; the majority of Pds1p does not appear to bind chromosomes tightly21; and Pds1p clearly regulates cell-cycle progression as well as sister-chromatid separation17. Cut2p is also not associated with chromosomes. Indeed, some, if not most, of it is associated with the mitotic spindle23. What then are the chromosomal proteins that hold sisters together between replication and the onset of anaphase? Do such proteins even exist? Might sisters be held together simply by their intercatenation, which results from collision of neighbouring replication forks24? The recent discovery of chromosomal proteins that are essential for sister-chromatid cohesion during G2 and M phases25,26 suggests that cohesion could be due to proteinaceous bridges. By looking for mutants that lose chromosomes at a high frequency and separate sister chromatids in the absence of APC function, my group and others25,27–29 have identified six essential yeast genes that are needed for sister cohesion: SMC1, SMC3, SCC1 (also known as MCD1), SCC2, SCC3 and ECO1 (also known as CTF7). The products of four of these genes (Smc1p, Smc3p, Scc1p and Scc3p) bind as a cohesin complex (Fig. 2a) to multiple sites along chromosomes25,29. An Scc2p homologue, Mis4p, is needed for sister-chromatid cohesion in fission yeast30. Although not a stoichiometric subunit of cohesin, Scc2p is needed for the association between cohesin and chromosomes29. Scc1p, Scc2p and Scc3p are unrelated proteins whose sequences have thus far provided no clue to their function. Smc1p and Smc3p, by contrast, belong to the SMC family of proteins, members of which contain an NTP-binding domain at their N-terminus, two long stretches of coiled coil that are separated by a hinge region at their centre, and a conserved C-terminal domain that binds DNA31,32. SMC proteins exist both in bacterial and in eukaryotic lineages, which suggests that they are ancient modulators of chromosome structure. Unlike bacteria, whose genomes encode at most one SMC protein, all eukaryotic organisms express at least four members of the SMC family: Smc1p, Smc2p, Smc3p and Smc4p. Smc2p and Smc4p are components of a 13S complex that contains three other subunits and is essential for
APCCdh1
M
A
G1
S
Phase
Figure 1 APCcdc20 and APCCdh1 mediate proteolysis at different stages of the cell cycle. Proteolysis of Pds1p (Cut2p) and cyclin B is driven by ubiquitination and is necessary for sister separation and exit from mitosis, respectively. The anaphase-promoting complex (APC) requires activator proteins, either Cdc20p or Cdh1p. Pds1p, an anaphase inhibitor, is ubiquitinated by APCcdc20, which is activated by cyclin-B–Cdk1p. Cyclins are ubiquitinated, at least during G1 phase, by APCCdh1, which is inhibited by cyclin-B–Cdk1p. Thus, the activity of APCcdc20 peaks during mitosis, whereas that of APCCdh1 peaks during G1 phase.
chromosome condensation in yeast and in Xenopus, and has therefore been called condensin33. Condensin can supercoil DNA in an ATP-dependent manner. It binds to chromosomes and mediates their condensation only upon activation of cyclin-B–CDK1 (Ref. 34). Smc1 and
Smc3 from Xenopus are found in a cohesin complex with three other proteins, one of which is the Xenopus homologue of Scc1p (Ref. 26). Depletion of the Xenopus cohesin complex, by using antibodies directed against either Smc1 or Smc3, has little or no effect on chromosome
(a) Scc2p (Mis4p)
Eco1p (Ctf7p)
Cohesin
Centromere Microtubules attempting to pull sister chromatids apart (b) Replication fork Eco1p (Ctf7p) Cohesin
(c) Replication apparatus
N C or
C N Smc1p Smc3p
Figure 2 Multisubunit complexes that contain Smc proteins mediate sister-chromatid cohesion and condensation. A cohesin complex (a), containing Smc1p, Smc3p, Scc1p/Mcd1p and Scc3p and possibly other proteins, is necessary for sister-chromatid cohesion, which is established during DNA replication and depends on Eco1p/Ctf7p (b). Cohesin might contain an Smc1p–Smc3p heterodimer in which the coiled coils of each partner are antiparallel (c). The result is a V-shaped molecule that possesses two similar, but not identical, globular domains at each end, which are composed of an N-terminal domain from one member and a C-terminal domain from the other.
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REVIEWS condensation but causes sister chromatids to separate prematurely. This suggests that eukaryotic cells use a conserved cohesin complex to hold sister chromatids together. The involvement of SMC proteins in cohesion and condensation suggests that these two processes are mechanistically related, although we do not know how. Bacterial SMC proteins form homodimers whose coiled coils are antiparallel, and N- and C-teminal domains are paired together35. The implication is that cohesin contains an Smc1–Smc3 heterodimer in which the coiled coils of Smc1 run antiparallel to those of Smc3 (Fig. 2b). It is not known where accessory proteins such as Scc1p and Scc3p bind to the Smc1–Smc3 heterodimer or indeed whether cohesin is even part of the link between sister chromatids. Sisters might be joined together by a single Smc1–Smc3 heterodimer whose two globular domains are bound to each sister chromatid or instead by a pair of heterodimers, each of which is bound to a single chromatid, that are somehow linked together.
Establishment and maintenance of cohesion In yeast, Scc1p can bind to chromosomes from late G1 phase until metaphase, but it can establish cohesion only when present during DNA replication36. Mis4p (the Scc2p homologue in fission yeast) likewise functions during S phase30. The formation of links between sisters might be a function associated with replication forks. Most, if not all, yeast cohesin subunits remain bound to chromosomes until the onset of anaphase and are required for maintenance of cohesion during metaphase. Eco1p (also known as Ctf7p), by contrast, is required to establish cohesion during S phase but not to maintain it during G2 phase28,29, and presumably acts at replication forks (Fig. 2a). The nature of the link between sisters produced by cohesin during DNA replication and whether it is formed at specific DNA sequences are not known.
Dissolution of cohesion What triggers sister separation? Might changes in the state of cohesin – for example, in its association with chromatids – be responsible for the loss of cohesion at the metaphase–anaphase transition? This could be the case in yeast, in which cohesins remain bound to chromosomes until sisters separate; whereupon, Scc1p and Scc3p disappear from chromosomes25,29. The disappearance of Scc1p depends on APCcdc20,
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TIBS 24 – MARCH 1999 whose role in this regard is to destroy Pds1p (Ref. 21). However, cohesin behaves rather differently in animal cells (Fig. 3). In Xenopus, cohesin binds to chromosomes at the time that they replicate (or even before); it remains associated throughout G2 phase but dissociates during prophase as cyclinB–Cdk1 is activated26. Cohesin’s dissociation coincides with, but does not depend on, condensin’s association with chromosomes. This suggests that animal cells use at least two types of cohesion: (1) cohesin-mediated cohesion, which is established during DNA replication and persists until entry into mitosis; and (2) a mechanism that holds sister chromatids together during chromosome alignment and is destroyed only at the metaphase–anaphase transition. The molecules involved in the second form of cohesion have not yet been identified. Yeast might also possess both types of cohesion – in which case, the main difference between yeast and animals might be only in the timing with which the two types of cohesion are destroyed. Differences in the degree of chromosome condensation between yeast and animal cells might account for the different timing of cohesin’s dissociation from chromosomes. In animal cells, cohesin’s prior dissociation, which presumably loosens connections between sisters, might be required for condensation, during which most sister sequences are resolved from each other37. Yeast chromosomes undergo far less condensation. Indeed, they maintain high rates of transcription throughout mitosis, and sister DNA sequences remain closely connected even during metaphase38. Thus, the absence of a clearly defined resolution/condensation step in yeast might permit cohesin to remain associated with chromosomes until the metaphase– anaphase transition. Cohesin’s abundance on G2-phase chromosomes ensures that sisters remain tightly associated, even when cells (such as oocytes) spend long periods in this stage of the cell cycle, whereas its dissociation from chromosomes as cells enter mitosis might be a pre-condition for the rapid separation of sister chromatids at the metaphase–anaphase transition.
Sister-separating proteins and their inhibitors Pds1p blocks dissociation of Scc1p from yeast chromosomes. How does it do this? Unlike cohesins, the bulk of Pds1p does not appear to associate with chromosomes but instead forms stable complexes with a soluble 180-kd protein,
Esp1p (Ref. 21), whose fission-yeast homologue, Cut1p, forms a similar complex with Cut2p (Ref. 23). When incubated at the restrictive temperature, esp1 mutant cells replicate DNA, form bipolar spindles and even destroy Pds1p (and also eventually cyclins), but they fail to separate sister chromatids, and Scc1p never dissociates from chromatids21. Thus, unlike the APC, which is required both for sister-chromatid separation and for cell-cycle progression, Esp1p is needed specifically for sister separation. Esp1p and Cut1p might be dedicated to the separation of sister chromatids; it has therefore been suggested that they be called separins. Separin homologues exist in humans, Xenopus and Caenorhabditis elegans. An appealing hypothesis to explain the role of APCcdc20 in the separation of sisters in budding yeast emerges from these findings: Pds1p blocks sister separation by binding to Esp1p, which inhibits its separating activity. According to this model, sister separation depends on APCcdc20 because it destroys the separin inhibitor Pds1p. Thus, the main role of APCcdc20 in the separation of sister chromatids in yeast might be to destroy inhibitory proteins that bind to separins rather than to destroy cohesion proteins themselves (Fig. 3). The recent identification of human and Xenopus anaphase-inhibitory proteins that have the properties of Pds1p, and bind to Esp1p and are degraded by APCCDC20, suggests that the yeast model applies also to animal cells (M. Kirschner, pers. commun.). Whether Pds1p-like proteins are the main targets for APCCDC20 in animals remains to be determined. Certainly, the APC might also contribute to sister separation by destroying chromosomal proteins that are involved in maintaining cohesion during metaphase. A key issue is how separins promote sister separation. Most Cut1p resides in the cytoplasm until cells enter mitosis; whereupon, it accumulates on the mitotic spindle until late anaphase. Cut1p might modulate the activity of anaphase motor proteins or microtubule dynamics in a manner that would increase the spindle’s pull on kinetochores39. Other evidence, however, suggests that Esp1p/Cut1p separins act directly on chromosomes. In cells that lack a mitoticsurveillance mechanism, Esp1p can induce Scc1p to disappear from chromosomes in cells treated with nocodazole (which destroys spindles) and in mutants that lack functional kinetochores21. Thus, separins might act both on spindles and
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TIBS 24 – MARCH 1999 on chromosomes. How Esp1p triggers dissociation of Scc1p is not understood. It does not simply replace cohesins: it is never found stably associated with chromosomes. Sister separation in animal cells can also occur during anaphase in the absence of chromosomes being attached to spindles40,41. It will clearly be of interest to establish whether separins are required for the dissociation of cohesin from chromatids as animal cells enter mitosis or whether they are required only for their eventual separation during anaphase. Other proteins besides Esp1/Cut1 are involved in separation of sister chromatids. In Drosophila melanogaster, for example, pimples and three rows, neither of which resemble Esp1p/Cut1p, are necessary for separating centromeres but not chromosome arms42.
Regulating loss of cohesion
Yeast
Esp1
Cyclin-B–Cdk1
APCcdc20
Pds1uuu
Esp1–Pds1
Cohesin joint Chromatid Microtubule Condensin
Vertebrates
Figure 3
What determines the onset Dissolution of sister-chromatid cohesion. In budding yeast, sister separation might be driven by Esp1p of Pds1p destruction and, (separin) activity, which causes the dissociation of Scc1p from chromosomes. Esp1p is inhibited by the thereby, activation of Esp1p? binding of Pds1p. Cut1p and Cut2p perform these two functions in fission yeast. Esp1p is activated by proteolysis of Pds1p by APCcdc20, which is thought to be activated by cyclin-B–Cdk1p. Vertebrates also Accumulation of Cdc20p dur20,43 possess separin homologues, but less is known about their action. In vertebrates, a condensin complex ing late G2 phase presumthat contains SMC2, SMC4, X-CAPG, X-CAPD and Barren associates with chromosomes as cells enter ably has some role; however, mitosis, at which point most, but possibly not all, cohesin dissociates from chromosomes. Yellow Cdc20p alone cannot be rechevrons represent the pulling force exerted by microtubules. u, ubiquitin. sponsible, because advancing its synthesis does not immediately precipitate Pds1p destruction44. Cells detect either tension between sis- that depends on Mad2p. Furthermore, APCcdc20 might be activated by cyclin-B– ter centromeres46 or their occupancy by both Mad2p and Pds1p are essential for Cdk1p (Fig. 3). Either Cdc20p itself or core spindles47. Remarkably, the kinetochore preventing the disappearance of Scc1 APC subunits such as Cdc27p, which of a single lagging chromosome emits a from chromosomes (G. Alexandru et al., become hyperphosphorylated during signal capable of blocking separation of unpublished) and for arresting sister sepmitosis, might be targets of Cdk1p. all other sister pairs48. It also blocks all aration17,57 when spindles are damaged. Surprisingly, Pds1p is not necessary further cell-cycle progression, including This implies that the checkpoint blocks for cell proliferation at low temperatures45. proteolysis of B-type but not A-type cy- sister separation exclusively by inhibiting Indeed, there is little or no difference in clins, cytokinesis, and chromosome de- proteolysis of Pds1, whose persistence the timing of sister separation or disso- condensation and re-replication. Five ensures that Esp1p remains inactive and, ciation of Scc1p from chromosomes in nonessential proteins (Mad1p, Mad2p, as a consequence, cohesin links remain the wild type and pds1-deletion mutants Mad3p, the protein kinase Bub1p and intact. Whether animal cells use a similar (G. Alexandru, pers. commun.). Why, if Bub3p) are necessary for this response mechanism for halting sister separation Pds1p proteolysis is necessary for Esp1p in yeast49,50. In addition Mps1, a protein is unclear. Because the bulk of cohesin activation, is there no advancement of kinase that is also required for spindle dissociates from vertebrate chromosister separation when Pds1p is missing? pole duplication51, and Ndc10p, a sub- somes as cells enter mitosis as opposed Presumably, dissociation of cohesins unit of the centromere-binding complex to when sister chromatids actually from chromosomes is regulated by a CBF321,52, are needed. Animal hom- separate, the vertebrate checkpoint second, Pds1p-independent, mechanism. ologues of Mad1p, Mad2p, Mad3p and probably does not halt dissociation of Bub1p associate with the kinetochores cohesin, as it appears to do in yeast. Stopping sister separation of lagging chromosomes but not with How do Mad proteins inhibit proMost eukaryotic cells possess surveil- those of chromosomes correctly aligned teolysis of Pds1p? Three observations lance mechanisms (checkpoints) that on the metaphase plate53–56. indicate that they do so by inhibiting prevent sister-chromatid separation when How do Mad/Bub proteins block sister APCcdc20 (Fig. 4). First, Mad2p, possibly spindles are damaged or chromosomes separation? In yeast, spindle damage along with other Mad proteins, associfail to form bivalent spindle attachments. blocks Pds1p proteolysis by a process ates with both Cdc20p and APCcdc20
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are pulled towards, opposite spindle poles. However, as long as at least one reciprocal exchange between homologous chromatids has occurred, cohesion between sister chromatid arms will oppose disjunction of homologues and their segregation to opposite poles of the cell62. Thus, loss of sister-chromatid cohesion along Esp1p (Cut1p) chromosome arms is essential for chromosome segregation during meiosis I. Meanwhile, Active Inactive however, cohesion between sister centromeres persists so APCcdc20 Mad–APCcdc20 that it can be used later to align sisters on the meiosis-II metaphase plate. The differEsp1p–Pds1p (Cut1p–Cut2p) ence in the timing of sisterchromatid cohesion loss in chromosome arms and centromeres is therefore a crucial aspect of meiosis (Fig. 5). Some evidence suggests that Cohesin joint complexes related to cohesin Chromatid are required for meiotic sisterMicrotubule chromatid cohesion. The genomes of budding and fission yeast both encode two Scc1plike proteins: Scc1p (Rad21p in fission yeast) and Rec8p. Figure 4 Scc1p/Rad21p is essential for Stopping sister-chromatid separation. Lagging chromosomes emit a signal that induces the inactivation of mitosis, whereas Rec8p is esAPCcdc20 by a complex containing Mad1p, Mad2p and Mad3p. In yeast, this prevents destruction of Pds1p sential for meiosis and, along and prevents Esp1p from displacing Scc1p from chromosomes, which is thought to play a key role in loss with Smc3p, for prevention of of cohesion. Yellow chevrons represent the pulling force exerted by microtubules. premature separation of sister chromatids63 (F. Klein and (Refs 14, 15, 58). Second, Mad2p can in- construct that encodes an N-terminal K. Nasmyth, unpublished). Rec8p and hibit cyclin ubiquitination mediated by Bub1p fragment54,61 reduces the interval Smc3p are found all along the longitudinal APCcdc20 in vitro58. Third, overproduction between nuclear-membrane breakdown axis of chromosomes during pachytene. of Cdc20p, or forms of Cdc20p that fail and the onset of sister separation; this Both proteins largely disappear from to bind Mad2p, allows cells whose mi- raises the possibility (but does not chromosome arms during the first meiotic totic checkpoint has been activated to prove) that signals that emanate from division but persist around centromeres escape mitotic arrest59,60. How kineto- lagging chromosomes during normal mi- until metaphase II. They then disappear chores of lagging chromosomes, which toses are essential to prevent premature completely as cohesion between sister are bound by Mad and Bub proteins, sister separation. centromeres is lost at the onset of anaseed formation of Mad2p-containing phase II (Y. Watanabe and P. Nurse, pers. complexes that inhibit APCcdc20 through- Meiosis commun.; F. Klein and K. Nasmyth, unout the cell is still mysterious. Mad2p Meiosis is a variation on the theme of published). Scc2p (Mis4p) is homologous tetramers are more effective than Mad2p mitosis. It produces haploid gametes to Rad9 from Coprinus cinereus64, whereas dimers at inhibiting APCcdc20 in vitro; this that contain recombinant chromosomes Scc3p is homologous to Rec11p from fisraises the possibility that lagging chromo- – parts of which are derived from ‘mum’, sion yeast; both Rad9p and Rec11p are somes catalyse a conformational change and parts of which are derived from necessary for meiosis. In Drosophila, in Mad2p (Ref. 15). Activation of APCCdh1 ‘dad’. As in the case of mitosis, meiosis MeiS332 also has an important role in is also inhibited by the checkpoint, but starts with a round of DNA replication in preservation of sister cohesion at centrothe mechanism is not clear. diploid germ cells, which produces con- meres until the second meiotic division. The genes that encode Mad and nected sister chromatids. Recombination It binds to centromeric heterochromatin Bub proteins are dispensable in yeast. between homologous chromatids (and during metaphase of meiosis I, persists Furthermore, sister separation does not possibly other pairing mechanisms) there until sisters separate during anaeven occur early in mad2 mutants. brings homologues together. During the phase of meiosis II and is essential for Whether the same is true for animal first meiotic division, sister kinetochores prevention of premature separation of siscells is still unclear. Injection of Mad2p- are treated as a single unit, and homolo- ter centromeres65. It is not known whether specific antibodies or expression of a gous kinetochore pairs attach to, and MeiS332 interacts with cohesins.
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Medical relevance Chromosome-segregation defects uncover recessive mutations, including those that permit the uncontrolled proliferation of tumour cells. Sister-chromatidcohesion defects could therefore contribute to tumorigenesis. Most tumour cells are indeed highly aneuploid, and many, including most colorectal cancers, actually lose and gain chromosomes at a high frequency as tumour cells divide66. But, is this chromosome instability a cause or a consequence of oncogenic mutations? Some evidence suggests that mitotic-checkpoint defects contribute to colorectal aneuploidy. Most, if not all, colorectal-carcinoma cell lines that lose chromosomes with a high frequency appear to be defective in their halting of the cell cycle when treated with nocodazole67. Moreover, one particular cell line contains a BUB1 mutation. Viruses also target mitotic surveillance. Human T-cell leukaemia virus (HTLV) causes the formation of multinucleate cells and encodes an oncoprotein, Tax, that binds to and possibly inhibits MAD1 (Ref. 55). Tax might facilitate proliferation induced by HTLV by abrogating mitotic arrest. Mutations in cohesins cause very high levels of chromosome loss in yeast, but whether similar mutations in humans contribute to tumour-cell aneuploidy is not known. Defects in the maintenance of sisterchromatid cohesion could also contribute to congenital aneuploidy in humans. The best example of this is Down’s syndrome, which is caused by the presence of an extra copy of chromosome 21. Most human aneuploidy is caused by mistakes that occur during meiosis in females. A high fraction of meiosis-IImetaphase oocytes obtained from infertility clinics have an incidence and pattern of extra chromosomes that are similar to those of aneuploid foetuses68. Remarkably, all extra chromosomes in these abnormal oocytes are single chromatids, not sister chromatid pairs, and very possibly arose because of a failure to maintain sister-chromatid cohesion during meiosis I. Oocytes spend long periods arrested in the G2 phase of the first meiotic division; once lost during this period, cohesion might be irreparable.
Concluding remarks Recent research has identified a crucial component of the sister-chromatidcohesion apparatus (cohesin), a class of sister separating proteins (the separins), the proteolytic system responsible for destroying separin inhibitors (APCCDC20),
(a)
(b)
Meiosis I
Meiosis II
Cohesin joint Chromatid Microtubule
Figure 5 Sister-chromatid cohesion and its destruction during meiosis. (a) Meiosis I. (b) Meiosis II. Homologous chromosomes (shown in blue and red) are held together by chiasmata (sites of reciprocal recombination between homologous chromatids) and cohesion between sister chromatids within chromosome arms. Loss of arm cohesion and cosegregation of sister centromeres reduces the chromosome number at meiosis I, whereas loss of sister cohesion at centromeres at meiosis II produces gametes that have a haploid chromosome number.
and a mitotic-surveillance mechanism that regulates APCCDC20. The study of sister-chromatid separation has therefore moved from the phenomenological to the molecular level, a transition that not only poses a new set of questions but also promises to shed insight into human pathology. Each round of sister separation is fraught with potential errors that, in somatic cells, can lead to cancer and, in the germline, are the raw material for natural selection. We are largely a product of our chromosomes. We are finally beginning to understand the complexities of the apparatus by which we inherit a complete set.
Acknowledgements I thank Mitsohiro Yanagida, Paul Nurse and Marc Kirscher for communicating unpublished results, Michael Glotzer and Jan-Michael Peters for their comments on the manuscript, Hannes Tkadletz for help with the figures, and Tim Hunt, who encouraged me to write this article.
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Bacterial solutions to the iron-supply problem Volkmar Braun and Helmut Killmann The insolubility of Fe31 necessitates special mechanisms for iron acquisition in most organisms. Bacteria use siderophores to chelate Fe31 and iron in heme, hemoglobin, transferrin and lactoferrin, and employ novel mechanisms for receptor-dependent iron transport and iron-regulated gene expression. These mechanisms involve transfer of energy from the cytoplasmic membrane to the outer membrane to drive active transport and might induce transcription of transport genes by transmitting a signal from the cell surface. IRON PLAYS A central role in many redox enzymes that function in electrontransport chains of intermediary metabolism. It is particularly suited to participating in a wide range of electron-transfer reactions because, depending on the ligand and protein environment, the redox potential of Fe31/Fe21 spans 1300 mV to 2500 mV. Although iron is abundant in nature, the extremely low solubility (10218 M) of Fe31 at pH 7 means that most organisms face the problem of obtaining enough iron from their environment. After absorption of iron through the gut, V. Braun and H. Killmann are at Lehrstuhl Mikrobiologie/Membranphysiologie, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany. Email: volkmar.braun@mikrobio. uni-tuebingen.de
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humans use proteins to keep iron in solution: transferrin is the iron carrier in the blood; lactoferrin is the carrier in secretory fluids; and ferritin stores iron within cells. Disturbances of iron metabolism result in severe diseases, such as congenital hemochromatosis. Several iron-supply strategies are available to bacteria. Under anaerobic conditions, Fe21 is present. It is sufficiently soluble that it can be taken up by anaerobic bacteria without the help of iron chelators. Similarly, acid-tolerant bacteria might find enough Fe31, which has a solubility of 1028 M at pH 3, to cover their iron needs. Alternatively, bacteria (and fungi) synthesize a wide variety of low-molecular-weight iron ligands that are called siderophores. These form three major structural types: hydroxamates,
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catecholates and α-hydroxycarboxylates1. The siderophores, as in the cases of the transferrins and lactoferrins, bind Fe31 very strongly; the free Fe31 concentration is in the order of 10224 M (the calculated pM value {2log[M(H2O)n]m1, where M is the metal ion, m the metal valence and n the number of water molecules bound} for 1 mM Fe31 and 10 mM ligand at pH 7.4)2. Certain pathogenic bacteria employ iron sources that occur in their hosts, and do not use siderophores. For example, Serratia marcescens, Yersinia enterocolitica, Yersinia pestis, Shigella dysenteriae, Escherichia coli O157 and Vibrio cholerae use heme; Neisseria gonorrhoeae, Neisseria meningitidis and Haemophilus influenzae use hemoglobin – both alone and bound to haptoglobin; H. influenzae also uses heme bound to hemopexin, and Neisseria and H. influenzae also use iron bound to transferrin and lactoferrin (Fig. 1)3–5. We do not know how iron is released from the iron proteins or whether it is transported in free or complexed form into the bacteria. A protein that is secreted into the culture medium of S. marcescens releases heme from hemoglobin6, and heme bound to hemopexin is released by a secreted protein of H. influenzae type b (Ref. 3).
Initial binding of Fe31 chelates to receptor proteins Fe31 siderophores bind to highly specific receptor proteins and are then transported into the cytoplasm (Fig. 1). In Gram-positive bacteria, which lack an outer membrane, the receptors are bind-
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