Centromeres: Getting a grip of chromosomes

Current Opinion in Cell Biology

Volumn 12, Issue 3, 2000, Pages 308-319

  Pidoux, Alison L ^a   Allshire, Robin C ^a  

^a WESTERN GENERAL HOSPITAL   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

FUNGAL PROTEIN;

CELL ULTRASTRUCTURE; CENTROMERE; CHROMATIN STRUCTURE; CHROMOSOME STRUCTURE; MEIOSIS; MICROTUBULE; MITOSIS; NONHUMAN; PRIORITY JOURNAL; REVIEW; YEAST;

EID: 0034019644     PISSN: 09550674     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0955-0674(00)00094-6     Document Type: Review

References (76)

1. Albertson D.G., Thomson J.N. Segregation of holocentric chromosomes at meiosis in the nematode, Caenorhabditis elegans. Chromosome Res. 1:1993;15-26.
2. Goday C., Pimpinelli S. Centromere organization in meiotic chromosomes of Parascaris univalens. Chromosoma. 98:1989;160-166.
3. Pimpinelli S., Goday C. Unusual kinetochores and chromatin diminution in Parascaris. Trends Genet. 5:1989;310-315.
4. Amon A. The spindle checkpoint. Curr Opin Genet Dev. 9:1999;69-75.
5. Zachariae W. Progression into and out of mitosis. Curr Opin Cell Biol. 11:1999;708-716.
6. Karpen G.H., Allshire R.C. The case for epigenetic effects on centromere identity and function. Trends Genet. 13:1997;489-496.
7. Csink A.K., Henikoff S. Something from nothing: the evolution and utility of satellite repeats. Trends Genet. 14:1998;200-204.
8. Cambareri E.B., Aisner R., Carbon J. Structure of the chromosome VII centromere region in Neurospora crassa: degenerate transposons and simple repeats. Mol Cell Biol. 18:1998;5465-5477.
9. Russell I.D., Grancell A.S., Sorger P.K. The unstable F-box protein p58-Ctf13 forms the structural core of the CBF3 kinetochore complex. J Cell Biol. 145:1999;933-950. Intricate in vitro analyses provide evidence of the order of events that result in the assembly of a stable CBF3 complex. The unstable protein Ctf13 appears to be pivotal in limiting the amount of CBF3 assembled. Ctf13 activation by Skp1 allows association with Cep3, and subsequent binding to Ndc10 protects Ctf13 and results in a stable CBF3 complex. The authors speculate that this elaborate assembly pathway might restrict the number of active centromeres that can be formed and guard against unwanted, and potentially lethal, ectopic kinetochore assembly.
10. Kitagawa K., Skowyra D., Elledge S.J., Harper J.W., Hieter P. SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex. Mol Cell. 4:1999;21-33. A high dosage of SGT1 rescues alleles of skp1 that are defective in S. cerevisiae kinetochore function. Like SKP1, specific alleles of SGT1 display defects in kinetochore function. The Sgt1 protein is conserved and is shown to physically interact with Skp1 and to participate in the activation of Ctf13 and formation of the CBF3 complex.
11. Pietrasanta L.I., Thrower D., Hsieh W., Rao S., Stemmann O., Lechner J., Carbon J., Hansma H. Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)-binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy. Proc Natl Acad Sci USA. 96:1999;3757-3762.
12. Hyland K.M., Kingsbury J., Koshland D., Hieter P. Ctf19p: a novel kinetochore protein in Saccharomyces cerevisiae and a potential link between the kinetochore and mitotic spindle. J Cell Biol. 145:1999;15-28. Ctf19 is a non-essential protein that specifically associates with S. cerevisiae centromere DNA. Cells lacking Ctf19 have a high rate of chromosome mis-segregation, and released minichromsomes displayed a weakened ability to interact with MTs in vitro. The localisation of Ctf19 is consistent with its association with centromeres, however it might also associate with spindle poles.
13. Ortiz J., Stemmann O., Rank S., Lechner J. A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore. Genes Dev. 13:1999;1140-1155. A one-hybrid screen with S. cerevisiae CEN3 DNA is used to identify novel centromere-interacting proteins. Three new putative kinetochore proteins (Ctf19, Mcm21 and Okp1) were fused to the Gal4-activation domain and induced strong expression of the HIS3 reporter only when functional centromere DNA was used. Mutations in all three genes resulted in elevated rates of chromosome loss. Two-hybrid and/or co-immunoprecipitation analyses indicated that Ctf19, Mcm21 and Okp1 proteins interact with each other: Ctf19 interacts with Ndc10, Cep3 and Cse4; Mcm21 interacts with Skp1, Mif2, Cbf1 and itself; Okp1 associates with Ndc10 and Cep3. Chromatin immunoprecipitation shows that Ctf19, Mcm21 and Okp1 associate with centromere DNA via wild-type CDEII DNA in a CBF3-dependent manner. It is proposed that CDEII contacts various centromere proteins (perhaps Mif2, Cse4/nucleosome) resulting in a DNA-protein complex.
14. Yoon H.J., Carbon J. Participation of Bir1p, a member of the inhibitor of apoptosis family, in yeast chromosome segregation events. Proc Natl Acad Sci USA. 96:1999;13208-13213.
15. Goshima G., Yanagida M. Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast. Cell. 100:2000;619-634. Prior to this paper, budding yeast researchers have assumed that GFP-binding Lac or Tet operator arrays placed 23-35 kb distal from a centromere report the state of cohesion at the centromere. Surprisingly, here it is demonstrated that similar arrays within 4 kb of a centromere physically separate from each other in the very early stages of spindle assembly and significantly in advance of the release of arm cohesion sites. The authors also demonstrate that Mtw1, the S. cerevisiae homologue of S. pombe Mis12 protein, is localised at budding yeast centromeres. the natural precocious separation of S. cerevisiae centromeres raises additional questions about how the spindle checkpoint operates and the high concentration of cohesion at these centromeres and its temporally regulated release during mitosis.
16. Meluh P.B., Yang P., Glowczewski L., Koshland D., Smith M.M. Cse4p is a component of the core centromere of Saccharomyces cerevisiae. Cell. 94:1998;607-613. The first direct demonstration that the S. cerevisiae counterpart of the histone H3 variant CENP-A is associated with centromere DNA in vivo. The authors present a model to accommodate the folding of centromere DNA around a centromere-specific nucleosome and its interactions with known centromere-associated proteins.
17. Dobie K.W., Hari K.L., Maggert K.A., Karpen G.H. Centromere proteins and chromosome inheritance: a complex affair. Curr Opin Genet Dev. 9:1999;206-217.
18. Maney T., Ginkel L.M., Hunter A.W., Wordeman L. The kinetochore of higher eucaryotes: a molecular view. Int Rev Cytol. 194:2000;67-131.
19. Smith M.M., Yang P., Santisteban M.S., Boone P.W., Goldstein A.T., Megee P.C. A novel histone H4 mutant defective in nuclear division and mitotic chromosome transmission. Mol Cell Biol. 16:1996;1017-1026.
20. Keith K.C., Baker R.E., Chen Y., Harris K., Stoler S., Fitzgerald-Hayes M. Analysis of primary structural determinants that distinguish the centromere-specific function of histone variant Cse4p from histone H3. Mol Cell Biol. 19:1999;6130-6139.
21. Partridge J.F., Borgstrom B., Allshire R.C. Distinct protein interaction domains and protein spreading in a complex centromere. Genes Dev. 2000;. in press. Using chromatin immunoprecipitation, the authors show that Swi6 and Chp1 are associated with the outer repeat regions of fission yeast centromere 1, whereas Mis6 is confined to the inner repeats and the central core. Swi6 and Chp1 are required to mediate silencing within the outer centromere regions but defective Mis6 specifically alleviates silencing in the central core. Thus, there are at least two types of silent chromatin at fission yeast centromeres and both are required for the assembly of a fully functional kinetochore. In addition, Swi6 and Mis6 can spread over and silence genes inserted within cen1, and Swi6 can efficiently coat more than 3 kb of exogenous DNA inserted within this centromere. tRNA genes reside in the region of transition between Swi6 and Mis6 associated centromeric chromatin. The ability of these centromere-associated proteins to spread may have relevance to the processes that lead to neocentromere function.
22. Saitoh S., Takahashi K., Yanagida M. Mis6, a fission yeast inner centromere protein, acts during G1/S and forms specialized chromatin required for equal segregation. Cell. 90:1997;131-143.
23. + encodes a protein conserved amongst fungi. Mis12 localises to S. pombe centromeres and is associated with the central regions of centromere 1 where it is required for the maintenance of the specialised chromatin. Centromeres appear to separate prematurely and spindle morphogenesis is abnormal in cells defective in either Mis6 or Mis12 function. Spindles are elongated at metaphase and may be explained by defective centromere-spindle attachment and associated unbalancing of forces.
24. Aagaard L., Laible G., Selenko P., Schmid M., Dorn R., Schotta G., Kuhfittig S., Wolf A., Lebersorger A., Singh P.B.et al. Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M31. EMBO J. 18:1999;1923-1938. Suvar39 has an amino-terminal chromodomain and carboxy-terminal SET domain, which is also found in the S. pombe Clr4 protein required for normal centromere structure and function. Mammalian Suvar39H1 localises to centromeres at metaphase and interacts with heterochromatin protein 1 (HP1) in vitro.
25. Aagaard L., Schmid M., Warburton P., Jenuwein T. Mitotic phosphorylation of SUV39H1, a novel component of active centromeres, coincides with transient accumulation at mammalian centromeres. J Cell Sci. 113:2000;817-829. The centromeric localisation of SUV39H1 is retained in dicentric human chromosomes and SUV39H1 is recruited to neocentromeres at novel sites. This suggests that it may have a role in determining centromere activity. SUV39H1 accumulates at centromeres during prometaphase and dissociates at the metaphase to anaphase transition. SUV39H1 displays a mitosis-specific pattern of phosphorylation.
26. Ekwall K., Cranston G., Allshire R.C. Fission yeast mutants that alleviate transcriptional silencing in centromeric flanking repeats and disrupt chromosome segregation. Genetics. 153:1999;1153-1169. Transcriptional repression at S. pombe centromeres appears to be closely associated with full centromere function. Mutations at 12 loci that specifically alleviate centromere, but not mating locus, silencing were identified. Many of these mutants display sensitivity to spindle destabilising drugs, elevated rates of chromosome segregation and high rates of lagging chromosomes in late anaphase. None of these loci is allelic with the previously identified components chp1, clr4, rik1, or swi6.
27. Javerzat J.-P., McGurk G., Cranston G., Barreau C., Bernard P., Gordon C., Allshire R.C. Defects in components of the proteasome enhance transcriptional repression at fission yeast centromeres and impair chromosome segregation. Mol Cell Biol. 19:1999;5155-5165.
28. McEwen B.F., Hsieh C.E., Mattheyses A.L., Rieder C.L. A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution. Chromosoma. 107:1998;366-375. Kinetochores of newt lung cells are examined by EM after high pressure freezing followed by freeze substitution with chemical fixative. These analyses provide a different view of vertebrate kinetochore ultrastructure.
29. Buchwitz B.J., Ahmad K., Moore L.L., Roth M.B., Henikoff S. A histone H3-like protein in C. elegans. Nature. 401:1999;547-548. The histone H3 variant CENP-A is conserved in C. elegans and localises to the kinetochore of these holocentric chromosomes. Injection of hcp-3 into C. elegans (CENP-A) dsRNA resulted in embryonic death, with a high incidence of nuclei of unequal size, presumably due to chromosome mis-segregation. This confirms that histone H3 variants are fundamental determinants of kinetochore assembly. How are kinetochores assembled along the length of such chromosome is the next question. These initial observations suggest that separate nucleation centres seen in interphase nuclei may be gathered together into linear structures in mitosis.
30. Moore L.L., Morrison M., Roth M.B. HCP-1, a protein involved in chromosome segregation, is localized tothe centromere of mitotic chromosomes in caenorhabditis elegans. J Cell Biol. 147:1999;471-480. Monoclonal antibody mAbC4 was found to recognise the poleward-facing side of C. elegans mititic chromosome. The epitope recognised was found to reside in the HCP-1 gene encoding a homologue of mammalian CENP-F/mitosin. A second protein, HCP-2, which is 54% similar to HCP-1, was identified in the database. RNA interference was used to disrupt expression in C. elegans of hcp-1 and hcp-2. Only co-injection of dsRNAs for both CENP-F homologues, hcp-1 and hcp-2, resulted in an altered phenotype; 99% embryonic lethality with 80% of anaphase cells displaying chromosome-segregation defects. Thus HCP-1 and HCP-2 must act together to form functional kinetochores. This represents the first comprehensive analysis of a kinetochore protein on holocentric chromosomes.
31. Esteban M.R., Giovinazzo G., de la Hera A., Goday C. PUMA1: a novel protein that associates with the centrosomes, spindle and centromeres in the nematode Parascaris. J Cell Sci. 111:1998;723-735. PUMA1 is a coiled-coil protein that localises with the holocentric kinetochores on mitotic Parascaris chromosomes. It also associates with the sites of direct MT attachment at telomeres observed during meiosis. PUMA1 is probably not a structural component at kinetochores since this association is disrupted by microtubule-destabilising drugs. In addition it localises to centrosomes and kinetochore microtubules.
32. Yang C.H., Tomkiel J., Saitoh H., Johnson D.H., Earnshaw W.C. Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C. Mol Cell Biol. 16:1996;3576-3586.
33. Dawe R.K., Reed L.M., Yu H.G., Muszynski M.G., Hiatt E.N. A maize homolog of mammalian CENPC is a constitutive component of the inner kinetochore. Plant Cell. 11:1999;1227-1238. Three separate loci encode maize CENP-C homologues. Staining with anti-CENPCA antibodies shows that maize CENP-C is constitutively associated with centromeres. Surprisingly, this CENP-C antibody does not stain the knob neocentromeres. It is possible that knobs associate with microtubules by a different mechanism, or perhaps other CENP-C homologues, not recognised by this antibody, are associated with these structure.
34. Fukagawa T., Pendon C., Morris J., Brown W. CENP-C is necessary but not sufficient to induce formation of a functional centromere. EMBO J. 18:1999;4196-4209. Withdrawal of CENP-C results in accumulation of chicken DT40 cells in metaphase due to disruption of kinetochore assembly (indicated by loss of ZW10 staining). Overexpression of CENP-C inhibits mitotic progression, leading to chromosome mis-segregation, but it does not disrupt ZW10 association with kinetochores. Although excess CENP-C is distributed over chromosome arms it does not lead to the ectopic assembly of kinetochore structures.
35. Everett R.D., Earnshaw W.C., Findlay J., Lomonte P. Specific destruction of kinetochore protein CENP-C and disruption of cell division by herpes simplex virus immediate-early protein Vmw110. EMBO J. 18:1999;1526-1538.
36. He D., Zeng C., Woods K., Zhong L., Turner D., Busch R.K., Brinkley B.R., Busch H. CENP-G: a new centromeric protein that is associated with the alpha-1 satellite DNA subfamily. Chromosoma. 107:1998;189-197.
37. Sugata N., Munekata E., Todokoro K. Characterization of a novel kinetochore protein, CENP-H. J Biol Chem. 274:1999;27343-27346.
38. Dang V.D., Benedik M.J., Ekwall K., Choi J., Allshire R.C., Levin H. A new member of the Sin3 family of corepressors is essential for cell viability and required for retroelement propagation in fission yeast. Mol Cell Biol. 19:1999;2351-2365.
39. Shelby R.D., Vafa O., Sullivan K.F. Assembly of CENP-A into centromeric chromatin requires a cooperative array of nucleosomal DNA contact sites. J Cell Biol. 136:1997;501-513.
40. Ekwall K., Olsson T., Turner B.M., Cranston G., Allshire R.C. Transient inhibition of histone deacetylation alters the structural and functional imprint at fission yeast centromeres. Cell. 91:1997;1021-1032.
41. Biggins S., Murray A.W. Sister chromatid cohesion in mitosis. Curr Opin Genet Dev. 9:1999;230-236.
42. Zou H., McGarry T.J., Bernal T., Kirschner M.W. Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis. Science. 285:1999;418-422.
43. Chan G.K., Schaar B.T., Yen T.J. Characterization of the kinetochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and hBUBR1. J Cell Biol. 143:1998;49-63.
44. Chan G.K., Jablonski S.A., Sudakin V., Hittle J.C., Yen T.J. Human BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions at kinetochores and binds the cyclosome/APC. J Cell Biol. 146:1999;941-954. Using antibody injection experiments and overexpression of a kinase-defective version of BUBR1 it is shown that BUBR1 is required for nocodazole induced mitotic arrest and for passage through a normal mitosis. It appears to monitor the activities of the CENP-E motor protein, and it is also shown that BUBR1 associates with the cyclosome/APC. See also [40].
45. Dujardin D., Wacker U.I., Moreau A., Schroer T.A., Rickard J.E. De, Mey, J. R.: Evidence for a role of CLIP-170 in the establishment of metaphase chromosome alignment. J Cell Biol. 141:1998;849-862.
46. Starr D.A., Williams B.C., Hays T.S., Goldberg M.L. ZW10 helps recruit dynactin and dynein to the kinetochore. J Cell Biol. 142:1998;763-774.
47. Scaerou F., Aguilera I., Saunders R., Kane N., Blottiere L., Karess R. The rough deal protein is a new kinetochore component required for accurate chromosome segregation in Drosophila. J Cell Sci. 112:1999;3757-3768.
48. Martinez-Exposito M.J., Kaplan K.B., Copeland J., Sorger P.K. Retention of the BUB3 checkpoint protein on lagging chromosomes. Proc Natl Acad Sci USA. 96:1999;8493-8498.
49. Jablonski S.A., Chan G.K.T., Cooke C.A., Earshaw W.C., Yen T.J. The hBUB1 and hBUBR1 kinases sequentially assemble onto kinetochores with hBUBR1 concentrating at the kinetochore plates in mitosis. Chromosoma. 107:1998;386-396.
50. •] these studies provide evidence that kinetochore-MT attachment is regulated by a balance between the activities of a kinase (Ipl1p) and a protein phosphatase (Glc7p).
51. •] these studies provide evidence that kinetochore-MT attachment is regulated by a balance between the activities of a kinase (Ipl1p) and a protein phosphatase (Glc7p).
52. •].
53. Giet R., Prigent C. Aurora/Ipl1p-related kinases, a new oncogenic family of mitoticserine-threonine kinases. J Cell Sci. 112:1999;3591-3601.
54. Shapiro P.S., Vaisberg E., Hunt A.J., Tolwinski N.S., Whalen A.M., McIntosh J.R., Ahn N.G. Activation of the MKK/ERK pathway during somatic cell mitosis: direct interactions of active ERK with kinetochores and regulation of the mitotic 3F3/2 phosphoantigen. J Cell Biol. 142:1998;1533-1545.
55. Zecevic M., Catling A.D., Eblen S.T., Renzi L., Hittle J.C., Yen T.J., Gorbsky G.J., Weber M.J. Active MAP kinase in mitosis: localization at kinetochores and association with the motor protein CENP-E. J Cell Biol. 142:1998;1547-1558.
56. 1 through to anaphase. Scc2 mediates this chromatin binding, and Eco1/Ctf7 establishes sister-chromatid cohesion during replication.
57. Skibbens R.V., Corson L.B., Koshland D., Hieter P. Ctf7p is essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinery. Genes Dev. 13:1999;307-319. The protein Ctf7/Eco1 acts during S phase to establish sister chromatid cohesion in S. cerevisiae. Once cohesion has been established Ctf7/Eco1 is no longer required. Genetic interactions between ctf7 and components of the replication machinery suggest that it may act at replication forks.
58. Uhlmann F., Nasmyth K. Cohesion between sister chromatids must be established during DNA replication. Curr Biol. 8:1998;1095-1101.
59. Uhlmann F., Lottspeich F., Nasmyth K. Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature. 400:1999;37-42. It is demonstrated in vitro that an Esp1-dependent activity is unleashed that cleaves the S. cerevisiae cohesin Scc1/Mcd1 at two sites, resulting in its release from chromatin. This cleavage by Esp1 is inhibited by Pds1, consistent with maximal cleavage and dissociation of cohesin at anaphase onset when Pds1 is degraded. Mutation of the Esp1-dependent cleavage sites in Scc1/Mcd1 results in the establishment of cohesion but inhibition of sister-chromatid separation.
60. Losada A., Hirano M., Hirano T. Identification of Xenopus SMC protein complexes required for sister chromatid cohesion. Genes Dev. 12:1998;1986-1997. Homologs of Smc1, Smc3 and Scc1/Mcd1 are identified as part of the isolated Xenopus cohesin complex. This complex associates with chromatin during S phase and is required for chromatid cohesion. It dissociates from chromatin during mitotic prophase.
61. Darwiche N., Freeman L.A., Strunnikov A. Characterization of the components of the putative mammalian sister chromatid cohesion complex. Gene. 233:1999;39-47.
62. Rieder C.L., Cole R. Chromatid cohesion during mitosis: lessons from meiosis. J Cell Sci. 112:1999;2607-2613. The authors present the available data and the arguments for two distinct forms of sister-chromatid cohesion: arm and centromere.
63. Klein F., Mahr P., Galova M., Buonomo S.B., Michaelis C., Nairz K., Nasmyth K. A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis. Cell. 98:1999;91-103. A comprehensive analysis of the role of cohesin components in sister chromatid cohesion and recombination during S. cerevisiae meiosis. Smc3 and the meiosis specific Scc1/Mcd1 homologue Rec8 are required for sister chromatid cohesion and synatonemal complex formation. Meiotic recombination was reduced in cells lacking Smc3 or Rec8, however, double-strand breaks were formed with normal timing but was hyper-resected, consistent with abnormal processing. Rec8 and Smc1 coat the entire chromosomal axis during pachytene and subsequently become restricted to a few foci, which coincide with centromeric regions at anaphase I. By anaphase II, association of Rec8 and Smc3 with chromosome spreads is not detected.
64. Watanabe Y., Nurse P. Cohesin Rec8 is required for reductional chromosome segregation at meiosis. Nature. 400:1999;461-464. In S. pombe ectopic expression of the meiotic cohesin component Rec8 can rescue mitotic defects in cells lacking the mitotic cohesin Rad21, but Rad21 cannot substitute for Rec8 during meiosis. In cells lacking Rec8, sister centromere regions undergo equational rather than reductional division and separate to opposite poles in meiosis I. In meiosis II random, unequal chromosome segregation takes place, resulting in low spore viability. During meiotic prophase, Rec8 associates with chromosomes in the nuclear domain occupied by centromeres but not telomeres. After meiosis I chromatin-associated Rec8 is retained only at centromeres. Rec8 disappears completely at anaphase of meiosis II. Expression of Rec8 in place of Rad21 in mitotic diploid cells induces a reductional chromosome segregation pattern. The authors argue that Rec8 is required to ensure cohesion of chromatids in centromeric regions and to orient sister-kinetochores so that they act as a single functional unit and move to the same pole during meiosis I.
65. Tang T.T.L., Bickel S.E., Young L.M., Orr-Weaver T.L. Maintenance of sister-chromatid cohesion at the centromere by the Drosophila MEI-S332 protein. Genes Dev. 12:1998;3843-3856. Drosophila Mei-S332 is recruited to centromeres during prometaphase, and this association is independent of intact microtubules. The carboxyl terminus is essential for chromosomal association. The authors suggest that MEI-S332 is required to maintain sister chromatid cohesion specifically at centromeres, so as to counteract the poleward pulling forces exerted by kinetochores attached to microtubules.
66. Megee P.C., Koshland D. A functional assay for centromere associated sister chromatid cohesion. Science. 285:1999;254-257. An inducible recombination system combined with visual monitoring of chromatid separation is used to demonstrate that active centromeres are sites of sister chromatid cohesion in S. cerevisiae. The assay is performed on cells released into nocodazole, thus maintenance of centromere cohesion and chromatid separation are scored while the spindle checkpoint is activated.
67. Blat Y., Kleckner N. Cohesins bind to preferential sites along yeast chromosome III, with differential regulation along arms versus the centric region. Cell. 98:1999;249-259. The distribution of cohesin was examined over the entire length of S. cerevisiae chromosome III by hybridisation of DNA recovered from chromatin immunoprecipitations to ordered arrays of chromosome II clones. Scc1/Mcd1 and Smc1 associated preferential with 23 sites but particularly strong association with a 50 kb region around the centromere was found. The DNA sequences associated with cohesin tended to have a high AT composition.
68. 1 or M phase and demonstrate that centromeric DNA is required to establish and maintain cohesin association with centromere flanking chromatin. The high AT (60%) content of the sequences adjacent to the centromere promotes efficient recruitment of cohesin. A centromere-associated protein may be responsible for the initial recruitment of cohesin, which might then spread outwards along neighbouring nucleosomes. The functional significance of centromeric cohesin must be reassessed with the finding that native centromeres separate early during an unadulterated cell cycle (G Goshima, M Yanagida, personal communication).
69. Tanaka T., Cosma M.P., Wirth K., Nasmyth K. Identification of cohesin association sites at centromeres and along chromosome arms. Cell. 98:1999;847-858. Extensive analyses using chromatin immunoprecipitation in S. cerevisiae indicates that Scc1/Mcd1 is associated with specific sites on chromosome arms from S phase until anaphase onset. Short cis-acting sequences were identified that confer cohesin association. Scc1/Mcd1 was found to strongly associate with CEN3 and CEN6. The entire 6 kb region flanking CEN3 appeared to be associated with cohesin. A functional centromere was shown to be required to efficiently recruit cohesin. Prior inactivation of a centromere was found to be required to abolish recruitment of cohesin in cells with defective kinetochore components. Minichromosomes with functional centromeres apparently separate prematurely in cells arrested at metaphase compared with chromosomal centromeres. In the light of new data (G Goshima, M Yanagida, personal communication) this may reflect the fact that endogenous centromeres were visualised with Tet operator arrays 35 kb from CEN5. Addition of multiple strong-arm cohesion sites delayed separation of minichromosome chromatids. Thus the cohesin-associated sites located along chromosome arms confer sister chromatid cohesion. The functional significance of centromeric cohesin must be reassessed with the finding that native centromeres separate early during a normal cell cycle (G Goshima, M Yanagida, personal communication).
70. Winey M., Mamay C.L., O'Toole E.T., Mastronarde D.N., Giddings T.H.Jr, McDonald K.L., McIntosh J.R. Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle. J Cell Biol. 129:1995;1601-1615.
71. Parisi S., McKay M.J., Molnar M., Thompson M.A., van der Spek P.J., van Drunen-Schoenmaker E., Kanaar R., Lehmann E., Hoeijmakers J.H., Kohli J. Rec8p, a meiotic recombination and sister chromatid cohesion phosphoprotein of the Rad21p family conserved from fission yeast to humans. Mol Cell Biol. 19:1999;3515-3528.
72. Bernard P., Hardwick K., Javerzat J.P. Fission yeast bub1 is a mitotic centromere protein essential for the spindle checkpoint and the preservation of correct ploidy through mitosis. J Cell Biol. 143:1998;1775-1787. Mutations in the S. pombe HP1 homologue Swi6 are synthetically lethal when the mitotic checkpoint protein Bub1 is deleted. As in multicellular eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis. However, a pool of Bub1 remains centromere associated at metaphase and even at telophase.
73. Zeng X., Kahana J.A., Silver P.A., Morphew M.K., McIntosh J.R., Fitch I.T., Carbon J., Saunders W.S. Slk19p is a centromere protein that functions to stabilize mitotic spindles. J Cell Biol. 146:1999;415-425.
74. Coweison N.P., Partridge J.F., Allshire R.C., McLaughlin P.J. Dimerisation of chromo shadow domain and distinction from chromo domain revealed by structural analysis. Curr Biol. 2000;. in press. The crystal structure of the chromo shadow domain of fission yeast Swi6 (HP1 homologue) is resolved. These analyses reveal a novel dimeric structure formed by two chromo shadow domains. Dimerisation is confirmed by in vitro studies. It is proposed that the deep cleft formed upon the assembly of this dimeric structure may accomodate an interaction with pentapeptide motifs found in interacting proteins. The implications of this structure for the formation of silent chromatin are discussed.
75. Saffery R., Irvine D.V., Griffiths B., Kalistis P., Wordeman L., Choo K.H. Human centromeres and neocentromeres show identical distribution patterns of >20 functionally important kinetochore-associated proteins. Hum Mol Genet. 200:9;175-185. Fluorescence in situ hybridisation using probes that unambiguously decorate a neocentromere was combined with immunostaining with reagents that specifically recognise the centromere/kinetochore associated proteins CENP-A, CENP-B, CENP-C, CENP-E, CENP-F, INCENP, CLIP-170, dynein, dynactin subunits p150 (Glued) and Arp1, MCAK, Tsg24, p55CDC, HZW10, HBUB1, HBUBR1, BUB3, MAD2, ERK1, 3F3/2, topoisomerase II and a murine HP1 homologue, M31. All proteins apart from CENP-B were present at the neocentromeres. Thus, the components at these extraordinary centromeres are very similar to normal human centromeres. Thus, the primary DNA sequence at active centromeres seems unimportant once a functional kinetochore has been established.
76. Henikoff S., Ahmad K., Platero J.S., van Steensel B. Heterochromatic deposition of centeromeric histone H3-like proteins. Proc Natl Acad Sci USA. 97:2000;716-721. Cid1, the Drosophila homologue of the histone H3-like protein CENP-A, is shown to localise at fly centromeres. Expression of the human, mouse, and budding yeast centromeric H3-like proteins in flies from the Cid1 promoter results in their enrichment in pericentric heterochromatin. Remarkably, the yeast and worm proteins also accumulate at pericentric heterochromatin when expressed in human cells. The authors propose that centric heterochromatin assists in the deposition of these centromere specific histone H3-like proteins.