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Centromeres and telomeres
Centromeres
and telomeres
Carolyn University
of Nebraska,
M. Price Lincoln,
Nebraska,
USA
Centromeres and telomeres are both composed of specific DNA sequences and unique chromosomal proteins. Isolation and characterization of some of these sequences and proteins has greatly increased our knowledge of centromere and telomere structure. This information is allowing us to determine how centromeres and telomeres perform their various roles in a cell.
Current
Opinion
in Ceil
Biology
Introduction
Saccharomyces
centromeres
Two centromere-binding proteins have been identified: CBFl (also known ;LSCPFI or CPI), which binds to CDE I [F-11 1, and CBD, which binds to CDE III [ 12.1. CBFl is an abundant multifunctional helix-loop-helix protein that recognizes the palindrome within the CDE I element. Although CBFl does bind CDE I in l*ir,o, this protein does not seem to be crucial for centromere function. CBF3 is a 240.kD multisubunit protein that can only bind DNA when phosphotylated [12*] CBF3 binds well to the wild type CDE III element but does not bind a point mutant of CDE III that causes inactivation of centromere function i)z lkn It is noteworthy that a suppressor gene (JRXI) that can specifically overcome the detrimental effect of mutations in CDE III, encodes a protein kinase [ 13.1. Perhaps the activity of CBF3, and hence the activity of the whole centromere. is regulated via kinases such as MCKl.
Centromeres Centromere structure has primarily been studied in SLJSchcuomyces cereelvlsiut: ScfJir~~~lcchrrror~~~~ce.~ pombe and mammals. S. cereik&ze centromeres exist as a nucleoprotein complex that encompasses only 200 bp of unique sequence DNA [ 11. In contrast, mammalian centromeres contain many megabases of repeated sequence DNA and are composed of a series of distinctive chromatin domains [2,3]. The kinetochore, the site of spindle microtubule attachment, comprises one of these domains [7]. .S.pombe centromeres lie between S. cerezlisiue and mammalian centromeres in terms of complexity as they contain kilobases of repeated sequence DNA [ 11. The relative simplicity of S. cerervbiue centromeres may reflect the lack of chromosome condensation during mitosis, the existence of only one kinetochore microtubule, and Biology
cerevisiae
The region that specifies full centromere function in S. cerezMze consists of a 125.bp DNA segment that includes three elements: CDE I, an 8-bp conserved sequence; CDE II, a 7%86-bp AT-rich region; and CDE III, a 25.bp conserved sequence [l]. CDE Ill is essential for all centromere functions whereas CDE I and CDE II are important for optimal mitotic chromosome stability. CDE I is also required for meiotic function. Mutations in CDE I and CDE III that alter the level of centromere activiv also alter the itz llillo dimethyl sulfate footprint of the centromeric DNA-protein complex. This suggests that sequence-specilic interactions occur between protein and CDE I and CDE III [ S,9].
Both centromeres and telomeres are complex structures composed of specifc DNA sequences and the associated chromosomal proteins. This review covers recent studies that have focussed on identifying or characterizing the DNA and protein components and on how these components contribute to centromere or telomere function.
Current
4:37%384
the persistence of this microtubule throughout much of the cell c)rle [ 11. A large complex kinetochore may simply not be needed.
Centromeres and telomeres are structural elements of eukaryotic chromosomes which ensure that the correct complement of intact full-length chromosomes is maintained within a cell. The centromere is a complex multifunctional region that mediates attachment of a chromosome to the mitotic or meiotic spindle [l-3]. It also holds the sister chromatids together during meiosis I and mitotic metaphase, and at anaphase it participates in movement of the chromosomes towards the spindle poles. Telomeres, the natural ends of chromosomes, stabilize chromosomes by preventing telomere-telomere joining and degradation by nucleases [+6]. They also ensure that chromosome length is maintained by preventing loss of DNA from the end of the chromosome following DNA replication. They may link chromosomes into the nuclear architecture and thus help position chromosomes within a cell.
@
1992,
Future investigation into the role that centromere-binding proteins play in centromere function should be greatly zsisted by a new in llitro assay for microtubule binding by functional centromeres [ 14=*] In this assay, isolated S. cerellisiae minichromosomes are incubated with bovine microtubules; minichromosomes that have a functional centromere become attached to the microtubules. The Ltd
ISSN
0955-0674
379
380
Nucleus
and
gene
expression
assay has been used to show that cells arrested in mitosis have more microtubule-binding capacity than cells in G,.
Schizosaccharomyces
pombe
centromeres
Centromeres from the three chromosomes of S. pombe encompass 40-100 kb and contain multiple classes of repeated sequence DNA [ 11. Although the centromeres each have a different organization and even a diRerent composition of repeated sequences, there is a common structural theme. At each centromere, various classes of repeated sequences are organized into one large inverted repeat that flanks a central core of non-repeated sequence DNA [ 15,16*]. Clusters of tRNA genes are embedded within some of the repeated sequences [ 17*,18*]. At least seven tRNA genes are present in Cenl, 22 in Cen2 and nine in Cen3. Thus, over 10% of the S. ponzbe tRNA genes are clustered within a region that constitutes onl). 1.8% of the genome. Functional dissection of Cenl and Cen2 has been achieved by transforming S. pon&e with plasmid vectors containing defined regions of centromeric DNA [ I6*.19]. Subsequent measurement of the plasmid centromere activi~~ revealed that different aspects of centromere function are fulfilled by different regions of the centromere. Only a portion of the inverted repeat is required for normal mitotic chromosome segregation. For Cenl. the critical region spans 17 kb and includes the conserved core and some adjacent repeated sequences [ I6*]. In contrast, all the centromeric DNA is required for full meiotic centromere activiv and removal of even relatively small sections of Cenl or Cen2 causes precocious sister chromatid separation during meiosis 1 [16*,19]. In light of these findings it is interesting to note that in both CenI and Cen2 the central core and some of the adjacent repeated sequence DNA is not packaged into a standard nucleosome array [20]. The reduction or absence of nucleosomes in this region may reflect the binding of centromere-specific proteins that cause assembly of the chromatin into a kinetochore and mediate attachment of the chromosome to the spindle.
Mammalian
centromeres
In primates, the centromeric DNA consists primaril!, (if not solely) of 0.5-10 megabase blocks of a-satellite DNA. Each block of satellite DNA contains tandem repeats of a unique version of the basic 170-bp a-satellite sequence [21]. The centromeres of other mammals also contain satellite DNA, although the composition of the satellite is species-specific [3,22,23]. The abundance of repeated sequence DNA within mammalian centromeres has given rise to a model for centromere structure in which the centromere-kinetochore complex has a modular organization that mirrors the repeated arrangement of the DNA [ 3,24*,25**]. Specifically, proteins that make up the kinetochore (microtubule-binding proteins, motor proteins, etc.) are postulated to assemble on certain blocks of satellite DNA and hence form a series of functional kinetochore units along the DNA. Association of these functional units would then result in formation of the plate-like kinetochore complex.
One prediction of this model is that deletion of a substantial portion of the centromeric DNA would reduce the size of the centromere but would not destroy centromere function. In fact, this is what has been observed for human chromosome 17, when 2500 kb of the 3200 kb of a-satellite DNA are deleted [ 24.1. Visible etidence for a modular centromere-kinetochore structure has been obtained with caffeine-treated or physically stretched chromosomes. Following either treatment, the centromere appears to separate into a series of smaller units that lie along the chromatin fibre [25**]. Evidence that centromere assembly does occur in specific stages has been obtained from microinjection experiments using anti-centromere antibodies from CREST scleroderma patients [ 260*], Depending on what time in the cell cycle the antibodies are injected, both kinetochore assembly and centromere function are completely or partially inhibited. Identification of centromere proteins is progressing quite rapidly and 15-20 have now been identified ( [ 27e,28*,2y31,32*] and reviewed in [ 2,7] ). CENP-B and CENP-A are the best characterized structural proteins. CENP-A is a 17.kD centromere-specific histone that is homologous to histone H3 in select regions [27-l. As it is a component of nucleosonie core particles, it ma! pla!. a role in centrometic chromatin packaging. CENPB is a highly conserved helix-loop-helix protein that is present in the dense heterochromatin that underlies the kinetochore [ 21*,28*]. Human CENP-B binds specilically to a 17.bp sequence that is found in some, but not all, monomers of a-satellite DNA, as well as in the centromeric minor satellite DNA from mice. CENP-B may participate in centromere assembly either b!r mediating association between blocks of a-satellite DNA or 1~) promoting assembl!, of other centromere proteins into functional centromere-kirietochore units. The function of man!. of the other centromere proteins is still unclear. While some remain associated with the centromere throughout the cell cycle, others become associated with the centromere only during specific stages of mitosis [2931.32*]. For example, CENP-E appears at the centromere during prometaphase, remains there throughout metaphase, but then redistributes to the midplate at the onset of anaphase [32*]. Microinjection of antibodies against CENP-E into metaphase cells blocks progression into anaphase. suggesting that CENP-E function is required for the metaphase-anaphase transition. Telomeres Telomeric DNA from organisms as diverse as Gimdiu. ciliates, mammals and plants consists of a tandemly repeated &8-bp sequence [-i,5,33,34]. Although the toti length of the telomeric DNA varies dramatically from species to species (from 36 bp to more than 15 kbp) there are welt defined upper and lower limits for each species indicating that telomere length is quite closel). regulated. The telomeric repeated sequences are added to the 3’ end of the chromosome by the RNA-containing enzyme telomerase. The RNA component of telomerase is a structurally consenTed molecule that has a short region complimentary to the telomeric DNA; this region
Centromeres
acts as a template for synthesis of the telomeric repeats [4,5,35]. Although telomerase is processive in rlitro and adds hundreds of telomeric repeats to a primer, there is evidence that the telomerase activity is modulated in llizjo to make it distributive [ 36,37**]. This finding may explain why telomere length normally only increases by 3-10 bp per generation, and in some organisms the length is constant. Although identification of telomere-binding proteins has lagged behind characterization of the telomeric DNA, six or seven different telomere-binding proteins have now been isolated. These proteins seem to function in many different processes, including telomere length regulation, packaging of telomeric DNA and attachment of chromosomes to the nuclcdr matrix [-i,5,38*]. Teiomeric
DNA
sequence
and
structure
For many years, 1111the nuclear telomeric DNAs examined were found to have a very well defined organization with strict segregation of G and C residues to the 3’ and 5’ strands of the telomere and clustering of at least three GC base pairs per repeat eg.(CCCCAAM),.(GGGGTTTT),, or (AATCCCC),,.(TT’A GGGG), [4,5]. Because the G bias and G-rich nature of the 3’ strand appeared to be tightly conserved, these fe:ltures were thought to be somehow important for telomere function. This idea gained further support when an assortment of telomeric DNA sequences were found to form novel structures that contain non-Watsonxrick G.G base pairs [ 5,39,-iO]. The four-strdnded G-quartet is an example of such a structure. It was suggested that the G-containing structures might form at nahld telomeres and promote various aspects of telomere function, eg. regulation of telomerase or stabilization of chromosomes. A number of recent findings have brought into question the importance of the G-strand bias and the G-richness that is found in so many telomeric DNA sequences. First, strict segregation of the Gs and Cs to opposite strands is not found in the telomeric DNA from some organisms. eg. Ascal Iumbricoides (TTAGGC) [il.*] , Parascaris unirulens (ITGCA) [ 421, Plusmodium hergbei [ t3] and S.pombe (cited in [44] 1. Second, many telomeric repeats are not G-rich, eg. those of A Illmbricoides [+I**], P. unilulens [ 42], and CIJl~It?!))dot)1o11us reinbardtii (TTTTAGGG) [-45]. The CtX~~~~~~ionlorzm telomeric DNA cannot readily form a G-quartet structure under conditions that are likely to occur in llizjo [45]. Finally, neither the telomere-binding protein nor the telomerase that has been is&ted from Owyytricha tzolu recognize OSyfricha telomeric DNA when the DNA is folded into a G-quartet structure [46,47*3. As both the telomere-binding prctein and telomerase are known to interact with O.xyricha telomeres iu llizlo, these findings suggest that a G-quartet may not occur at native telomeres. De
nova
synthesis
of telomeres
H&ing of broken chromosomes by de noz’o telomere synthesis has been observed as a rare spontaneous event in cells that have suffered chromosome damage and as a routine event during the life cycle of cili-
and
telomeres
Price
ates [37*=,48*,49*]. Recent experiments have shown that routine chromosome healing also occurs in the nematode R Iumbricoides when the sequence (TTAGGC), is added to the ends of the somatic chromosomes following fragmentation and elimination of much of the germlinespecific chromatin [41**]. In all three cases, telomeric repeats are added to non-telomeric sequences present at the end of a broken chromosome. While it has been suspected that telomerase is responsible for addition of the telomeric sequences, the mechanism of addition posed a dilemma because early experiments had suggested that telomerase uses only telomeric DNA sequences as a primer. Two recent papers have demonstrated that the primer requirements for telomerase recognition are quite relaxed [ 48*,49*]. Telomerase from humans ( HeLa cells) and Tefral$anena cdn add telomeric repeats to primers that have very little homology to the telomerase template. In fact, human telomerase can add telomeric repeats to a sequence that corresponds to a naturally healed break in human chromosome 16 (49*]. Only three base pairs can fomi between the 3’ terminus of this sequence and the proposed telomerase RNA template. It seems that telomerase interacts both with sequences at the 3’ end of the primer and sequences that are internal to the primer [ 48*49*], Strong evidence that telomerase adds telomeric repeats directly onto the ends of broken chromosomes in rlitlo has been obtained from studies of developmentally programmed de t?ozlo telomere synthesis in Tenzlh?,mena [37**]. TetraLynena were transformed with telomerase RNA genes encoding an altered template sequence and the cells were then mated so that chromosome fragmentation and de tzoz’o telomere sythesis would occur. When the newly synthesized telomeres were sequenced, many of them were found to have the sequence corresponding to the mutant telomerase template added directlv to the end of the fragmented chromosome. As both itz rlillo and ill ritro experiments indicate that telomerase can use a non-telomeric sequence to prime addition of telomeric repeats, it now seems almost certain that telomerase is generally responsible for the healing of broken chromosomes. Telomere-binding
proteins
Two groups of telomere-binding proteins have so far been isolated. These include proteins that bind only to the extreme end of the telomeric DNA, and proteins that bind along the length of the telomeric DNA. Proteins that bind specifically to the end of the DNA have been isolated from the ciliates Owyyhichn nova and ffuplotes cru;cu.s [ 50*,51]. The proteins from both species bind the T,G,-containing extension on the 3’ strand. The O.xytricba protein is composed of two subunits (CLand p). Although contact with the DNA is predominantly \ia the a-subunit, both subunits are required to form the full DNA-binding site. End-binding proteins such s the Oqftricha and Euplotes telomere proteins are thought to promote chromosome stability by forming a protective cap over the end of the chromosome. However, they may also regulate telomere length either by directly competing with telomerase for the end of the chromosome, or by modulating telomerase activity.
381
382
Nucleus
and gene expression
Proteins that bind internal stretches of telomeric DNA have been isolated from Pkysurum (PPT) [52] and yeast (TEiFa and RAP1 )[5355,56*]. PPT is a small (10 kD) heat-stable protein that is thought to coat the length of the double-stranded (T2AG& telomeric DNA (521. TE%Fa appears to bind at the junction between the GImjT telomeric repeats and the subtelomeric X sequence 1531. RAP1 is a multifunctional protein that binds a consensus sequence which occurs within the telomeric Gl.jT repeats as well as in the upstream activating site of many genes and the silencer elements flanking the silent matingtype loci HMR and HML. The role played by RAP1 in telomere length regulation appears to be complex as mutation or over-expression of RAP1 can make telomeres grow either longer or shorter [ 54,55,56**]. Various experiments suggest that mutations in RAP1 affect both telomere length and silencing by altering chromatin structure. RAP1 appears to interact with various proteins via the carboxyi-terminus, thus mutations that affect this terminus may alter the interactions between RAP1 and other chromosomal proteins [ 56**]. This in turn could alter the telomeric chromatin structure so that the telomeric DNA becomes more or less accessible to telomerase or nucleases. Telomeric chromatin structure appears to influence not only telomere length but also expression of telomere proximal genes. When genes are placed near a telomere their transcription is often repressed [ 57,581. However, in yeast this telomere position effect is relieved by mutations in a variety of proteins that are known to influence chromatin structure [ 59~01. It seems likely that derepression of transcription arises because the mutant proteins alter the chromatin structure in the telomere-proximal region of the chromosome.
plex than was previously thought. It is becoming apparent that the extent to which telomeric DNA is elongated by telomerase is influenced by telomere-binding proteins. Recent experiments with yeast suggest that this regulation of telomerase activity may occur via alteration of the telomeric chromatin structure. However, direct interactions between telomerase and telomere-binding proteins may also occur. Acknowledgements I wish to thank this manuscript
References Papers of view. have . of .. of
Research on telomeres published over the past year has demonstrated that we need to reconsider some ideas about the type of sequence that constitutes telomeric DNA and about how this sequence is recognized by telomerase. As not all telomeric DNAs exhibit G-richness or segregation of the Gs to one strand, these features are unlikely to be important. Moreover, it has been demonstrated that telomerase can add telomeric repeats directly to the ends of broken chromosomes that lack pre-existing telomeric DNA. This finding indicates that primer recognition by telomerase is clearly more com-
and recommended
particular interest, published been highlighted as: special interest outstanding interest
for reading
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of Budding
the annual
and Fission
period
Yeast.
of re.
1.
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PLUTA AF, CCXXE man Centromere 15:1Rl-185.
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WILLARD H: Centromeres of Trends Gener 1990, 6:4l(kl5.
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BOLIRGAIN FM, KA~NKA MD: Telomeres Inhibit End to End Fusion and Enhance Maintenance of Linear DNA Molecules Injected into the Paramecium prfmaureliu Macronucleus. n~~dc~ic AcidsRes 1991, 19:15.i&l5+7.
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NIEDE~IHAL R. STOIL R, HEGEI~ANN J: In Viva Characterization of the Succhuronzyces cerevisiue Centromere DNA Element I, a Binding Site for the Helix-Loop-Helix Protein CPFI. nlol Cell Biol 1991. 11:354!%3553.
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Conclusions Many of the past studies of centromere structure and function have, of necessity, concentrated on iden@ ing the DNA sequences and the proteins that make up a functional centromere. However, now that many of the essential components have been identified, it has become possible to move on and ask what role these proteins and DNA sequences play in centromere function. The in rlitro assay for centromere activity that has been developed b) Kingsbury and Koshland [14-l should greatly facilitate such studies in the S. cmez&zze system. While microinjection of antibodies to centromere proteins is proving a useful tool in the mammalian system, in t&r0 assays for centromere function still need to be developed.
J. Berman, S. Darr and D. Shippen.Lentz and making helpful comments.
Trends
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of Telomeres. of Telomeres.
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LECHNER J, CARBON J: A 240 kd Multisubunit Protein Complex, CBF3 is a Major Component of the Budding Yeast Centromere. Cell 1991, 641717.725. Reports the purification and characterization of a novel centromere. binding protein CBF3. This protein binds to the essential element CDE III and hence is probably an important determinant of centromere function. 13. .
SHERO J, HIETER PA: Suppressor of a Centromere DNA Mutation Encodes a Putative Protein Kinase (MCKI). Ce;erzes Dev 1991, 5:54+560.
Centromeres Links protein phosphorylation to regulation of centromere activity. In. creased dosage of MCKl specifically suppresses mutations in CDE II! but not mutations in CDE I and CDL; II. Disruption of MCKl results in decreased growth and increased chromosome instability when cells are grown in conditions that destabilize microtubules. MCKl shows homol0~ to multiple protein kinases and appears to be a member of the .serine/threonine kinase group. 14. ..
KINGSBURY J, KOSHLWD D: Centromere-dependent Binding of Yeast Minichromosomes to Microtubules in Vitro. Cell 1991, rX483-495. Reports the development of an in (vitro assay for the binding of centromeres to microtubules, one aspect of centromere function. The data clearly show that the assay can discriminate between active and inactive centromeres. The great utility of the arsay is demonstrated in studies of cell cycle related variation in centromere activity. 15.
CHIKA‘~IIIGE Y, KINOSHITA N. NAKASEKO Y, Mmwh~o’ro MLIKAKAMI T, NIWA S, YANACII~A M: Composite Motifs Repeat Symmetry in S. Porn&e Centromeres: Direct ysis by Integration of Not1 Restriction Sites. Cell 57:73%751.
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HAHNENHERGER K, CARHON J, CIARKE L: Identification of DNA Regions Required for Mitotic and Meiotic Functions Within the Centromere of Schizosaccharomyces porn&e Chromosome I. Afol &II Rio/ 1991, 11:22062215. Describes the structural organization of the different classes of repe;lted DNA sequences within Crnl. Maps the regions of Cerzl that are re quired for full centromere activity during mitosis and meiosis. Shows that different aspects of centromere function are fulfilled by different regions of the centromere. 17. .
KLIH~’ R, CIARKE I, CARBON J: Clustered tRNA Genes in Schizosaccharomyces pombe Centromeric DNA Sequence Repeats. Proc Nail Acad Sci (ISA 1991. 88:13061310. Identities 22 tRNA genes that are clustered within the B’ sequence of Cen2 These genes are structurally normal and may be transcribed 18. .
TAKAHASHI K, MAUAKANI S C~~IKAS~IICX Y, N~A 0. YANAGIIIA M: A Large Number of tRNA Genes are Symmetrically Located in Fission Yeast Centromeres. J rllol Rio/ 1991. 218:13-17. Identities 36 tRNA genes within the three ?; pot&e centromeres; seven genes from Cenl. 20 genes from Cefr-7 and 9 genes from Cen.3. These genes encode 11 different tRNAs and they all appear to be structurally normal. 19.
CIAIUZ 1 BAO~~ M: Functional Analysis of a Centromere t?om Fission Yeast: a Role for Centromere-specific Repeated DNA Sequences. 11101 Cell Hiol 1990, 103186%3872.
20.
POLIZI C, Cm L: The Chromatin Structure of tromeres from Fission Yeast: Differentiation of the tral Core that Correlates with Function. / CeN Rio1 112:191-201.
21.
WE~RICK R, WIIL.\RI) I-1: Physical Map of the Centromeric Region of Human Chromosome 7: Relationship between Two Distinct Alpha Satellite Arrays. Nlicleic Acid Res 1991. 19:2295-2301.
22.
V~Y J, MACC~GOR H. BARXI.X-~ L Characterization Short, Highly Repeated and CentromericaUy Localized Sequence in Crested and Marbled Newts of the Genus urus. chonracoff?ci 1990. lot: 15-3 1.
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RICHARDS E. CkXIDhLW H. A~IXI~I:I. F: The CentrOmere Of Arubidopsis thafiana Chromosome I Contains Telomeresimilar Sequences. Ntrciek Acid Rev 1991, 19:3351-3357.
24. .
WEVKlCK R, EARNSHAW’ W. HuwARI~-PI:EH~S P, WIU.4RD H: Partial Deletion of Alpha Satellite DNA Associated with Reduced Amounts of the Centromere Protein CENP-B in a MitoticaUy Stable Human Chromosome Rearrangement. Mel Cell Biol 1990, 10:6374380. A rearrangement Ltithin chromosome 17 result4 in deletion of 20.30% of the a-satellite DNA This rearranged chromosome was inherited b! two offspring and appeared to retain full mitotic centromere activity. The data provide tlidence that CENP-B is associated with a.satellitc DNA in ttrlo and that human centromeres are structurally and functionally repetitive.
Price
25. . .
ZINKOW%I R. ME~ENE J, BRINKLEY BR: The CentromereXinetochore Complex: a Repeat Subunit Model. / Cell Biol1991. 113:1091-l 110. Examination of the 3-dimensional structure of the kinetochore. The kinetochore was separated into small subdomains following detachment of the kinetochore from the chromosome with caffeine, or physi. cal stretching of the chromosome by hypotonic or shear forces. Pro. vides visual evidence that the kinetochore is assembled from repeating DNA-protein complexes that are tandemly arranged along the length of the centromeric DNA 26. . .
BERNAT R, DE~ANNOY M, ROTHFIEID N, EARNSHAW W: Dirup tion of Centromere Assembly During Interphase Inhibits Kinetochore Motphogenesis and Function in Mitosis. Cell 1991, 66:122’+1238. A successful attempt to identify some of the steps in klnetochore as sembly. Microinjection of anti-centromere antibodies (predominandy anti-CENP.A and anti-CENP.B) into G1- or S-phase cells prevents assem. bly of any visible kinetochore structure and results in cell cycle arrest at prometaphase. Injection of the antibodies during Gz prevents assembly of the inner plate of the kinetochore and compaction of the heterochromatin directly beneath the kinetochore, and results in inhibition of the metaphaseanaphase transition. 27. .
PAIMER D, O’DAY K. TRONC H, CHAR~ONNEALI H: Purification of the Centromere-specific Protein CENP-A and Demonstration that it is a Distinctive Histone. Proc Null Acud Sci USA 1991, 88:3734-3738. Purificadon and amino acid sequence analysis of CENP-A Demonstration that segments of CENP-A are very similar in sequence to histone H3 although other segmenLs show no such sequence conservation. 28. .
SLIIUVAN K, GLA? C: CENP-B is a Highly Conserved Mammalian Centromere Protein with Homology to the Helix-Loop-Helix Family of Proteins. aromasomu 1991, 100:36G370. Cloning and sequencing of CENF-B gene from mouse cells. Demonstration that CENP.B is highly conserved in mammalian cells and thus is likely to be important for centromere function. 29.
COM~TON D. YEN T. CLEVELAND D: Identification of Novel Centromere/Kinetochore-associated Proteins Using Monoclonal Antibodies Generated Against Human Mitotic Cluomosome Sctiolds. / Cell Biol 1991, 112:1083-1097.
30.
BALCZON R. ACCA~I~ MA. BKINKLEV BR Identification of a 40,000 Molecular Weight Centromere-associated Protein in Cultured Mammalian Cells. J Cell Sci 1990, 97:705-713.
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EARNSHAW W, COOKE C: Analysis of the Distribution of the INCENP’s throughout Mitosis Reveals the Existence of a Pathway of Structural Changes in the Chromosomes during Metaphase and Early Events in Cleavage Furrow Formation. J Cell Sci 1991, 98:4+461.
CenCen1991,
of a DNA ttit
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YEN T. COLOPHON D, WISE D, ZINKOWSKI R, Bw B, E,~~NSHAW’ W, CLEVEIAND D: CENP-E, a Novel Human Centromere-associated Protein Required for Progression from Metaphase to Anaphase. EMBO J 1991, 10:124%1254. - . Monoclonal antibodies made against human chromosome scattold proteins recognize a new centromere protein (CENP-E) of 250-300kD. CENP-E appears at the centromeres at prometaphase, remains there during metaphase, and dissociates to the midplate at anaphase. Micro. injection of anti-CENP-E antibodies inhibits the metaphase-anaphase transition. 32.
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GANM Tomato
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RohlERo DP, BLAC~LIRN EH: ture for Telomerase RNA
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Yu GL, BWCKBLIRN ing of Chromosomes 1991. 67:823-832.
M, WITAN Telomeres.
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N, TANKSL!ZY S: Macrostructure Pkan~ Cell 1991, 587-94.
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A Conserved Secondary Cell 1991, 67:343-353 Processive.
E: Developmentally by Telomerase
‘MO/
in
CeN Biol
Programmed Tekuhymenu.
of
the
Stfuc1991,
HealCell
383
384
Nucleus’ and gene expression
. Demonstrates that telomerase can add telomeric repeats directly onto the ends of broken chromosomes that lack any pre.existing telomeric DNA This provides strong evidence that telomerase is responsible for healing of broken chromosomes. The data also demonstrate that telomerase is not processive i?, rdrto. DELUGE T: Human Telomeres are Attached to the Nuclear Matrbt. EMBO J 1992, 11:717-724. ;rovides the first real biochemical evidence that telomeres are attached to the nuclear matrix via their TTAGGG repeats. Internal TTAGGG repeats do not attach chromosomes to the nuclear matrix hence the telomeric attachment must be mediated by a telomere-specilic nuclroprotein complex.
38.
39.
HAR~IN cation Folding
C, HENDERV)N E, WATSON T PROSSER J: Monovalent structural transitions in telomeric DNAs: G-DNA Intermediates. Biochem 1991, 30:1460+472.
40.
ACEVED~ 0. herence of 19:34w3419.
DICUNSON Synthetic
L. MACKE Telomeres.
T. THO~W C: The CoNfdcleic Acid< Kes 1991.
Shows that a sequence corresponding to a naturally hcdlrd break in human chromosome 16 senres as a primer for human telomerace. This sequence hns little homoloR to the putative telomerase RNA template. The dara suggest that telomerase has the capacit) to heal chromosomes by the de ~01’0 addition of telomeric DNA to broken ends that have no preexisting telomeric sequences. 50. .
GRAS JT. CEL~~DER DW, PRICE CM, CECH TR: Cloning and Expression of Genes for the O.\ytricba Telomere-binding Protein: Specific Subunit Interactions in the Telomeric Complex. Cc,// 199 I. 67,H07-8 11. Examines the relative conrributions of the (1. and P-subunits of the O.yVricha t&mere protein 10 DNA-binding.
51.
PRICE CM: Telomere Structure in Errplores crassus Characterization of DNA-protein Interactions and Isolation of a Telomere-binding Protein. .1/o/ Cell Rioi 1990, lo:.+12 l-343 I.
52.
COREN J. EI%~IN E. \‘CXX \‘. Characterization of a TelomereBinding Protein from Pbysancm polycephalum. .1/o/ Cell Riol 1991. 11:12832-2290.
.
41.
MUUER F. WICHI’ C. SPICHER A. TO~XER H: New Telomere Formation after Developmentally Regulated Chromosomal Breakage during the Process of Chromatin Diminution in Ascaris lumbricoides Cell 1991, 67ki15-822. Shows that telomeres in somatic cells are synthesized de ?1or*o folloning fragmentation of the germline chromosomes. This synthesis im’olves addition of the novel telomeric sequence ITAAGGC to the broken ends of the somatic chromosomes. A good model Tstem for exam ining chromosome healing. ..
42.
TISCHW C. tilJIDt% G. MOIUT?! K: The Highly Variable Pentameric Repeats of the AT-rich Germline Limited DNA in Parascarik univalens are the Telomeric Repeats of Somatic Chromosomes. Nucleic .&xi.% Res 1991, 19:2677-26%
43.
PON~Z~ M, PACE T, DORE Telomeric DNA Sequences J 1985, 4:2291-1995.
E, FRO~TAU C: in Plasmodium
Identification bergbei.
44.
ALLSHIRE RC, GOSDEN JR. CROSS SH. CRAM-ON G. Rotl-r D. SUCAWARA N, SZOSTAK JW, Fm P, HAYIIE ND: Telomeric Repeat from 7: thermophila Cross Hybridizes with Human Telomeres. N&ire 1988. 332:65&X159.
45.
PBIRACEK ME, B~ahm J: Cblamydomonas reinhartii Telomere Repeats form Unstable Structures Involving Guanineguanine Base Pairs. Nucleic Acids Res 1992, 20:89-95.
46.
RACHllRAh!.&~ MK, CECH TR: Effect of Monovalent Cationinduced Telomeric DNA Structure on the Binding of Oxytricha Telomeric Protein. Nlrcleic .4cids f&.v 1990. 18454h551.
ZAHER A, WIIUAAISON J, CECH T, PRESCOTT D: Inhibition of Telomerase by G-quartet DNA Structures. Narrrre 1991. 350:718-720. Demonstrates that oligonucleotides with the same sequence ;LS Oqlricba telomeres (T,t&), only act as primers for Oq~rid~u relomeraSe when the terminal T,G, repeat is not part of a Gquartet structure. 48. HARRINCTON L, GREIDER C: Telomerase Primer Specificity and . Chromosome Healing. Nature 1991. 353:451&453. Demonstrates that Tetrabymena telomerase can add TTGGGG repeats to the end of primers that are not homologous at the 3’ end to the telomerase template RNA However, an internal region that is homologous to the template is required. The data suggest that telomemse has the capacity to heal chromosomes by the de ?ror’o addition of telomeric DNA to broken ends that have no preexisting telomeric sequences.
49.
111’ %. RI! 13: A Yeast Telomeres and Conserved l?,cV~ 1991. 5:4%59.
54.
Lr’srlc; .\I. KlXlX s. SHAKY D: Involvement of the Silencer and llAS Binding Protein RAPI in Regulation of Telomere Length. Scie~cr 1990. 750:5-19-ii?.
55.
CONIUI) MN. WKICHT JI 1. w’0lP Al, T>KuI\: Interacts with Yeast Telomeres itz lttla Alters T&mere Structure and Decreases bility. Ccl/ 1990. 63:‘jSr-50.
of 61180
47. .
.
53.
MORIN G: Recognition of a Chromosome Truncation Site Associated with Alpha-thalassemia by Human Telomerase. Nature 1991, 353:45+-%56.
Protein Yeast
that Binds to Vertebrate Telomeric Junctions. &f/e.<
\‘A: RAP1 Protein Over Production Chromosome Sta-
56 ..
SLhSlil. I.. SHOKI; D: Separation of Transcriptional Activation and Silencing Functions of the RAPl-encoded Repressor/Activator Protein 1: Isolation of Viable Mutants Affecting Both Silencing and Telomere Length. Pra,Vcrfl Acad Sci 1 ‘SA 1991. 88:T4~+753. Shows that mutlitions at specitic amino ac~cls in the carbo~~l-trrrninlls of RAP1 holh suppress silencing of rhr silent mating npr Ioci and illcrease telomcrr Irngdi. Sugg:r.srs that mutations in /2-11’1 m&c t&m eric chromatin structure and hence the accesslbili~ of telomeric DNA IO t&nl~ns~. 57.
GOTTSCHIJN~; D. AI’.AtuCIO 0. MIJJNWON B. %WAN \‘: Position Effect at S. ceretpisiae Telomeres: Reversible Repression of Pol 111 Transcription. Cell 1990. 63:751-762
5X.
7x1.~ Tretrth
VA. SAVDEIJ. L: Telomeric Cell Hiol 1992. 210-14.
Position
Effect
in Yeast.
59. ..
APARICIO 0. HIUJNGTON H. G~ITSCHUNG D: Modifiers of Position Effects are Shared between Telomeric and Silent Mating-type Loci in S. cerec:isiae. Cell 1991, i&1273-1288 Shows that transcriptional repression of tekmlere pr0xinlal genvs is derepressed by mutations in S/K-, Sl&. SIR4, X’TI. Ah’DI wd HI~IFZ. AIl the gene prtducts are required for silencing of the mating ripe Ioci and they are all bt+rved to modify chromatin structure. Suggests that the telomere position effect is caused by the telomeric chromatin strucfure.
CM Price, Nebraska,
Department of Chemistr), Lincoln, Nebraska 68588,
610 Hamilton LISA
Hall.
liniversity
of
and telomeres
Carolyn University
of Nebraska,
M. Price Lincoln,
Nebraska,
USA
Centromeres and telomeres are both composed of specific DNA sequences and unique chromosomal proteins. Isolation and characterization of some of these sequences and proteins has greatly increased our knowledge of centromere and telomere structure. This information is allowing us to determine how centromeres and telomeres perform their various roles in a cell.
Current
Opinion
in Ceil
Biology
Introduction
Saccharomyces
centromeres
Two centromere-binding proteins have been identified: CBFl (also known ;LSCPFI or CPI), which binds to CDE I [F-11 1, and CBD, which binds to CDE III [ 12.1. CBFl is an abundant multifunctional helix-loop-helix protein that recognizes the palindrome within the CDE I element. Although CBFl does bind CDE I in l*ir,o, this protein does not seem to be crucial for centromere function. CBF3 is a 240.kD multisubunit protein that can only bind DNA when phosphotylated [12*] CBF3 binds well to the wild type CDE III element but does not bind a point mutant of CDE III that causes inactivation of centromere function i)z lkn It is noteworthy that a suppressor gene (JRXI) that can specifically overcome the detrimental effect of mutations in CDE III, encodes a protein kinase [ 13.1. Perhaps the activity of CBF3, and hence the activity of the whole centromere. is regulated via kinases such as MCKl.
Centromeres Centromere structure has primarily been studied in SLJSchcuomyces cereelvlsiut: ScfJir~~~lcchrrror~~~~ce.~ pombe and mammals. S. cereik&ze centromeres exist as a nucleoprotein complex that encompasses only 200 bp of unique sequence DNA [ 11. In contrast, mammalian centromeres contain many megabases of repeated sequence DNA and are composed of a series of distinctive chromatin domains [2,3]. The kinetochore, the site of spindle microtubule attachment, comprises one of these domains [7]. .S.pombe centromeres lie between S. cerezlisiue and mammalian centromeres in terms of complexity as they contain kilobases of repeated sequence DNA [ 11. The relative simplicity of S. cerervbiue centromeres may reflect the lack of chromosome condensation during mitosis, the existence of only one kinetochore microtubule, and Biology
cerevisiae
The region that specifies full centromere function in S. cerezMze consists of a 125.bp DNA segment that includes three elements: CDE I, an 8-bp conserved sequence; CDE II, a 7%86-bp AT-rich region; and CDE III, a 25.bp conserved sequence [l]. CDE Ill is essential for all centromere functions whereas CDE I and CDE II are important for optimal mitotic chromosome stability. CDE I is also required for meiotic function. Mutations in CDE I and CDE III that alter the level of centromere activiv also alter the itz llillo dimethyl sulfate footprint of the centromeric DNA-protein complex. This suggests that sequence-specilic interactions occur between protein and CDE I and CDE III [ S,9].
Both centromeres and telomeres are complex structures composed of specifc DNA sequences and the associated chromosomal proteins. This review covers recent studies that have focussed on identifying or characterizing the DNA and protein components and on how these components contribute to centromere or telomere function.
Current
4:37%384
the persistence of this microtubule throughout much of the cell c)rle [ 11. A large complex kinetochore may simply not be needed.
Centromeres and telomeres are structural elements of eukaryotic chromosomes which ensure that the correct complement of intact full-length chromosomes is maintained within a cell. The centromere is a complex multifunctional region that mediates attachment of a chromosome to the mitotic or meiotic spindle [l-3]. It also holds the sister chromatids together during meiosis I and mitotic metaphase, and at anaphase it participates in movement of the chromosomes towards the spindle poles. Telomeres, the natural ends of chromosomes, stabilize chromosomes by preventing telomere-telomere joining and degradation by nucleases [+6]. They also ensure that chromosome length is maintained by preventing loss of DNA from the end of the chromosome following DNA replication. They may link chromosomes into the nuclear architecture and thus help position chromosomes within a cell.
@
1992,
Future investigation into the role that centromere-binding proteins play in centromere function should be greatly zsisted by a new in llitro assay for microtubule binding by functional centromeres [ 14=*] In this assay, isolated S. cerellisiae minichromosomes are incubated with bovine microtubules; minichromosomes that have a functional centromere become attached to the microtubules. The Ltd
ISSN
0955-0674
379
380
Nucleus
and
gene
expression
assay has been used to show that cells arrested in mitosis have more microtubule-binding capacity than cells in G,.
Schizosaccharomyces
pombe
centromeres
Centromeres from the three chromosomes of S. pombe encompass 40-100 kb and contain multiple classes of repeated sequence DNA [ 11. Although the centromeres each have a different organization and even a diRerent composition of repeated sequences, there is a common structural theme. At each centromere, various classes of repeated sequences are organized into one large inverted repeat that flanks a central core of non-repeated sequence DNA [ 15,16*]. Clusters of tRNA genes are embedded within some of the repeated sequences [ 17*,18*]. At least seven tRNA genes are present in Cenl, 22 in Cen2 and nine in Cen3. Thus, over 10% of the S. ponzbe tRNA genes are clustered within a region that constitutes onl). 1.8% of the genome. Functional dissection of Cenl and Cen2 has been achieved by transforming S. pon&e with plasmid vectors containing defined regions of centromeric DNA [ I6*.19]. Subsequent measurement of the plasmid centromere activi~~ revealed that different aspects of centromere function are fulfilled by different regions of the centromere. Only a portion of the inverted repeat is required for normal mitotic chromosome segregation. For Cenl. the critical region spans 17 kb and includes the conserved core and some adjacent repeated sequences [ I6*]. In contrast, all the centromeric DNA is required for full meiotic centromere activiv and removal of even relatively small sections of Cenl or Cen2 causes precocious sister chromatid separation during meiosis 1 [16*,19]. In light of these findings it is interesting to note that in both CenI and Cen2 the central core and some of the adjacent repeated sequence DNA is not packaged into a standard nucleosome array [20]. The reduction or absence of nucleosomes in this region may reflect the binding of centromere-specific proteins that cause assembly of the chromatin into a kinetochore and mediate attachment of the chromosome to the spindle.
Mammalian
centromeres
In primates, the centromeric DNA consists primaril!, (if not solely) of 0.5-10 megabase blocks of a-satellite DNA. Each block of satellite DNA contains tandem repeats of a unique version of the basic 170-bp a-satellite sequence [21]. The centromeres of other mammals also contain satellite DNA, although the composition of the satellite is species-specific [3,22,23]. The abundance of repeated sequence DNA within mammalian centromeres has given rise to a model for centromere structure in which the centromere-kinetochore complex has a modular organization that mirrors the repeated arrangement of the DNA [ 3,24*,25**]. Specifically, proteins that make up the kinetochore (microtubule-binding proteins, motor proteins, etc.) are postulated to assemble on certain blocks of satellite DNA and hence form a series of functional kinetochore units along the DNA. Association of these functional units would then result in formation of the plate-like kinetochore complex.
One prediction of this model is that deletion of a substantial portion of the centromeric DNA would reduce the size of the centromere but would not destroy centromere function. In fact, this is what has been observed for human chromosome 17, when 2500 kb of the 3200 kb of a-satellite DNA are deleted [ 24.1. Visible etidence for a modular centromere-kinetochore structure has been obtained with caffeine-treated or physically stretched chromosomes. Following either treatment, the centromere appears to separate into a series of smaller units that lie along the chromatin fibre [25**]. Evidence that centromere assembly does occur in specific stages has been obtained from microinjection experiments using anti-centromere antibodies from CREST scleroderma patients [ 260*], Depending on what time in the cell cycle the antibodies are injected, both kinetochore assembly and centromere function are completely or partially inhibited. Identification of centromere proteins is progressing quite rapidly and 15-20 have now been identified ( [ 27e,28*,2y31,32*] and reviewed in [ 2,7] ). CENP-B and CENP-A are the best characterized structural proteins. CENP-A is a 17.kD centromere-specific histone that is homologous to histone H3 in select regions [27-l. As it is a component of nucleosonie core particles, it ma! pla!. a role in centrometic chromatin packaging. CENPB is a highly conserved helix-loop-helix protein that is present in the dense heterochromatin that underlies the kinetochore [ 21*,28*]. Human CENP-B binds specilically to a 17.bp sequence that is found in some, but not all, monomers of a-satellite DNA, as well as in the centromeric minor satellite DNA from mice. CENP-B may participate in centromere assembly either b!r mediating association between blocks of a-satellite DNA or 1~) promoting assembl!, of other centromere proteins into functional centromere-kirietochore units. The function of man!. of the other centromere proteins is still unclear. While some remain associated with the centromere throughout the cell cycle, others become associated with the centromere only during specific stages of mitosis [2931.32*]. For example, CENP-E appears at the centromere during prometaphase, remains there throughout metaphase, but then redistributes to the midplate at the onset of anaphase [32*]. Microinjection of antibodies against CENP-E into metaphase cells blocks progression into anaphase. suggesting that CENP-E function is required for the metaphase-anaphase transition. Telomeres Telomeric DNA from organisms as diverse as Gimdiu. ciliates, mammals and plants consists of a tandemly repeated &8-bp sequence [-i,5,33,34]. Although the toti length of the telomeric DNA varies dramatically from species to species (from 36 bp to more than 15 kbp) there are welt defined upper and lower limits for each species indicating that telomere length is quite closel). regulated. The telomeric repeated sequences are added to the 3’ end of the chromosome by the RNA-containing enzyme telomerase. The RNA component of telomerase is a structurally consenTed molecule that has a short region complimentary to the telomeric DNA; this region
Centromeres
acts as a template for synthesis of the telomeric repeats [4,5,35]. Although telomerase is processive in rlitro and adds hundreds of telomeric repeats to a primer, there is evidence that the telomerase activity is modulated in llizjo to make it distributive [ 36,37**]. This finding may explain why telomere length normally only increases by 3-10 bp per generation, and in some organisms the length is constant. Although identification of telomere-binding proteins has lagged behind characterization of the telomeric DNA, six or seven different telomere-binding proteins have now been isolated. These proteins seem to function in many different processes, including telomere length regulation, packaging of telomeric DNA and attachment of chromosomes to the nuclcdr matrix [-i,5,38*]. Teiomeric
DNA
sequence
and
structure
For many years, 1111the nuclear telomeric DNAs examined were found to have a very well defined organization with strict segregation of G and C residues to the 3’ and 5’ strands of the telomere and clustering of at least three GC base pairs per repeat eg.(CCCCAAM),.(GGGGTTTT),, or (AATCCCC),,.(TT’A GGGG), [4,5]. Because the G bias and G-rich nature of the 3’ strand appeared to be tightly conserved, these fe:ltures were thought to be somehow important for telomere function. This idea gained further support when an assortment of telomeric DNA sequences were found to form novel structures that contain non-Watsonxrick G.G base pairs [ 5,39,-iO]. The four-strdnded G-quartet is an example of such a structure. It was suggested that the G-containing structures might form at nahld telomeres and promote various aspects of telomere function, eg. regulation of telomerase or stabilization of chromosomes. A number of recent findings have brought into question the importance of the G-strand bias and the G-richness that is found in so many telomeric DNA sequences. First, strict segregation of the Gs and Cs to opposite strands is not found in the telomeric DNA from some organisms. eg. Ascal Iumbricoides (TTAGGC) [il.*] , Parascaris unirulens (ITGCA) [ 421, Plusmodium hergbei [ t3] and S.pombe (cited in [44] 1. Second, many telomeric repeats are not G-rich, eg. those of A Illmbricoides [+I**], P. unilulens [ 42], and CIJl~It?!))dot)1o11us reinbardtii (TTTTAGGG) [-45]. The CtX~~~~~~ionlorzm telomeric DNA cannot readily form a G-quartet structure under conditions that are likely to occur in llizjo [45]. Finally, neither the telomere-binding protein nor the telomerase that has been is&ted from Owyytricha tzolu recognize OSyfricha telomeric DNA when the DNA is folded into a G-quartet structure [46,47*3. As both the telomere-binding prctein and telomerase are known to interact with O.xyricha telomeres iu llizlo, these findings suggest that a G-quartet may not occur at native telomeres. De
nova
synthesis
of telomeres
H&ing of broken chromosomes by de noz’o telomere synthesis has been observed as a rare spontaneous event in cells that have suffered chromosome damage and as a routine event during the life cycle of cili-
and
telomeres
Price
ates [37*=,48*,49*]. Recent experiments have shown that routine chromosome healing also occurs in the nematode R Iumbricoides when the sequence (TTAGGC), is added to the ends of the somatic chromosomes following fragmentation and elimination of much of the germlinespecific chromatin [41**]. In all three cases, telomeric repeats are added to non-telomeric sequences present at the end of a broken chromosome. While it has been suspected that telomerase is responsible for addition of the telomeric sequences, the mechanism of addition posed a dilemma because early experiments had suggested that telomerase uses only telomeric DNA sequences as a primer. Two recent papers have demonstrated that the primer requirements for telomerase recognition are quite relaxed [ 48*,49*]. Telomerase from humans ( HeLa cells) and Tefral$anena cdn add telomeric repeats to primers that have very little homology to the telomerase template. In fact, human telomerase can add telomeric repeats to a sequence that corresponds to a naturally healed break in human chromosome 16 (49*]. Only three base pairs can fomi between the 3’ terminus of this sequence and the proposed telomerase RNA template. It seems that telomerase interacts both with sequences at the 3’ end of the primer and sequences that are internal to the primer [ 48*49*], Strong evidence that telomerase adds telomeric repeats directly onto the ends of broken chromosomes in rlitlo has been obtained from studies of developmentally programmed de t?ozlo telomere synthesis in Tenzlh?,mena [37**]. TetraLynena were transformed with telomerase RNA genes encoding an altered template sequence and the cells were then mated so that chromosome fragmentation and de tzoz’o telomere sythesis would occur. When the newly synthesized telomeres were sequenced, many of them were found to have the sequence corresponding to the mutant telomerase template added directlv to the end of the fragmented chromosome. As both itz rlillo and ill ritro experiments indicate that telomerase can use a non-telomeric sequence to prime addition of telomeric repeats, it now seems almost certain that telomerase is generally responsible for the healing of broken chromosomes. Telomere-binding
proteins
Two groups of telomere-binding proteins have so far been isolated. These include proteins that bind only to the extreme end of the telomeric DNA, and proteins that bind along the length of the telomeric DNA. Proteins that bind specifically to the end of the DNA have been isolated from the ciliates Owyyhichn nova and ffuplotes cru;cu.s [ 50*,51]. The proteins from both species bind the T,G,-containing extension on the 3’ strand. The O.xytricba protein is composed of two subunits (CLand p). Although contact with the DNA is predominantly \ia the a-subunit, both subunits are required to form the full DNA-binding site. End-binding proteins such s the Oqftricha and Euplotes telomere proteins are thought to promote chromosome stability by forming a protective cap over the end of the chromosome. However, they may also regulate telomere length either by directly competing with telomerase for the end of the chromosome, or by modulating telomerase activity.
381
382
Nucleus
and gene expression
Proteins that bind internal stretches of telomeric DNA have been isolated from Pkysurum (PPT) [52] and yeast (TEiFa and RAP1 )[5355,56*]. PPT is a small (10 kD) heat-stable protein that is thought to coat the length of the double-stranded (T2AG& telomeric DNA (521. TE%Fa appears to bind at the junction between the GImjT telomeric repeats and the subtelomeric X sequence 1531. RAP1 is a multifunctional protein that binds a consensus sequence which occurs within the telomeric Gl.jT repeats as well as in the upstream activating site of many genes and the silencer elements flanking the silent matingtype loci HMR and HML. The role played by RAP1 in telomere length regulation appears to be complex as mutation or over-expression of RAP1 can make telomeres grow either longer or shorter [ 54,55,56**]. Various experiments suggest that mutations in RAP1 affect both telomere length and silencing by altering chromatin structure. RAP1 appears to interact with various proteins via the carboxyi-terminus, thus mutations that affect this terminus may alter the interactions between RAP1 and other chromosomal proteins [ 56**]. This in turn could alter the telomeric chromatin structure so that the telomeric DNA becomes more or less accessible to telomerase or nucleases. Telomeric chromatin structure appears to influence not only telomere length but also expression of telomere proximal genes. When genes are placed near a telomere their transcription is often repressed [ 57,581. However, in yeast this telomere position effect is relieved by mutations in a variety of proteins that are known to influence chromatin structure [ 59~01. It seems likely that derepression of transcription arises because the mutant proteins alter the chromatin structure in the telomere-proximal region of the chromosome.
plex than was previously thought. It is becoming apparent that the extent to which telomeric DNA is elongated by telomerase is influenced by telomere-binding proteins. Recent experiments with yeast suggest that this regulation of telomerase activity may occur via alteration of the telomeric chromatin structure. However, direct interactions between telomerase and telomere-binding proteins may also occur. Acknowledgements I wish to thank this manuscript
References Papers of view. have . of .. of
Research on telomeres published over the past year has demonstrated that we need to reconsider some ideas about the type of sequence that constitutes telomeric DNA and about how this sequence is recognized by telomerase. As not all telomeric DNAs exhibit G-richness or segregation of the Gs to one strand, these features are unlikely to be important. Moreover, it has been demonstrated that telomerase can add telomeric repeats directly to the ends of broken chromosomes that lack pre-existing telomeric DNA. This finding indicates that primer recognition by telomerase is clearly more com-
and recommended
particular interest, published been highlighted as: special interest outstanding interest
for reading
reading within
of Budding
the annual
and Fission
period
Yeast.
of re.
1.
CLARU L Centromeres Gener 1990, 6:15&154.
2.
PLUTA AF, CCXXE man Centromere 15:1Rl-185.
3.
WILLARD H: Centromeres of Trends Gener 1990, 6:4l(kl5.
4.
ZAUU V. A: Structure and Ret, Genet 1989, 23:57%64X.
Function
5.
BIACICBII~ E: Structure 1991, 350:56%5?3.
Function
6
BOLIRGAIN FM, KA~NKA MD: Telomeres Inhibit End to End Fusion and Enhance Maintenance of Linear DNA Molecules Injected into the Paramecium prfmaureliu Macronucleus. n~~dc~ic AcidsRes 1991, 19:15.i&l5+7.
7.
EARNSIC\W’ W When Cell Sci 1991, 99:1-+.
8.
DENS~~ORE L, PAYXE W. FITSCER~LD-HAYES M: In nomic Footprint of a Yeast Centromere. hJoJ Cd 11:15-t-165.
9.
IMEUOR J, JMNG W, FUNK IM, RXHJEN J. BARNES C. MINZ T, HEGEMANN J, PHIUPSEN P: CPFI, a Yeast Protein which Functions in Centromeres and Promoters. EZU30 J 1990, 940171026.
10.
NIEDE~IHAL R. STOIL R, HEGEI~ANN J: In Viva Characterization of the Succhuronzyces cerevisiue Centromere DNA Element I, a Binding Site for the Helix-Loop-Helix Protein CPFI. nlol Cell Biol 1991. 11:354!%3553.
11.
MUOR J. RATHJEN J, J~ANG W, DOWELL S: DNA-binding of CPFI is Required for Optimal Centromere Function but not for Maintaining Methionine Prototropy in Yeast. Nrrcleic Acids Res 1991, 19:2961-2969.
Conclusions Many of the past studies of centromere structure and function have, of necessity, concentrated on iden@ ing the DNA sequences and the proteins that make up a functional centromere. However, now that many of the essential components have been identified, it has become possible to move on and ask what role these proteins and DNA sequences play in centromere function. The in rlitro assay for centromere activity that has been developed b) Kingsbury and Koshland [14-l should greatly facilitate such studies in the S. cmez&zze system. While microinjection of antibodies to centromere proteins is proving a useful tool in the mammalian system, in t&r0 assays for centromere function still need to be developed.
J. Berman, S. Darr and D. Shippen.Lentz and making helpful comments.
Trends
CA. EARNSHAW’ WC: Structure of the Huat Metaphase. Trenuk Rio&em Sci 1990,
and
Mammalian
is a Centromere
Chromosomes.
of Telomeres. of Telomeres.
not
Annzr Nurlcre
a Kinetochore?J Viuo GeBid 1991,
12. .
LECHNER J, CARBON J: A 240 kd Multisubunit Protein Complex, CBF3 is a Major Component of the Budding Yeast Centromere. Cell 1991, 641717.725. Reports the purification and characterization of a novel centromere. binding protein CBF3. This protein binds to the essential element CDE III and hence is probably an important determinant of centromere function. 13. .
SHERO J, HIETER PA: Suppressor of a Centromere DNA Mutation Encodes a Putative Protein Kinase (MCKI). Ce;erzes Dev 1991, 5:54+560.
Centromeres Links protein phosphorylation to regulation of centromere activity. In. creased dosage of MCKl specifically suppresses mutations in CDE II! but not mutations in CDE I and CDL; II. Disruption of MCKl results in decreased growth and increased chromosome instability when cells are grown in conditions that destabilize microtubules. MCKl shows homol0~ to multiple protein kinases and appears to be a member of the .serine/threonine kinase group. 14. ..
KINGSBURY J, KOSHLWD D: Centromere-dependent Binding of Yeast Minichromosomes to Microtubules in Vitro. Cell 1991, rX483-495. Reports the development of an in (vitro assay for the binding of centromeres to microtubules, one aspect of centromere function. The data clearly show that the assay can discriminate between active and inactive centromeres. The great utility of the arsay is demonstrated in studies of cell cycle related variation in centromere activity. 15.
CHIKA‘~IIIGE Y, KINOSHITA N. NAKASEKO Y, Mmwh~o’ro MLIKAKAMI T, NIWA S, YANACII~A M: Composite Motifs Repeat Symmetry in S. Porn&e Centromeres: Direct ysis by Integration of Not1 Restriction Sites. Cell 57:73%751.
T, and Anal1989,
16. .
HAHNENHERGER K, CARHON J, CIARKE L: Identification of DNA Regions Required for Mitotic and Meiotic Functions Within the Centromere of Schizosaccharomyces porn&e Chromosome I. Afol &II Rio/ 1991, 11:22062215. Describes the structural organization of the different classes of repe;lted DNA sequences within Crnl. Maps the regions of Cerzl that are re quired for full centromere activity during mitosis and meiosis. Shows that different aspects of centromere function are fulfilled by different regions of the centromere. 17. .
KLIH~’ R, CIARKE I, CARBON J: Clustered tRNA Genes in Schizosaccharomyces pombe Centromeric DNA Sequence Repeats. Proc Nail Acad Sci (ISA 1991. 88:13061310. Identities 22 tRNA genes that are clustered within the B’ sequence of Cen2 These genes are structurally normal and may be transcribed 18. .
TAKAHASHI K, MAUAKANI S C~~IKAS~IICX Y, N~A 0. YANAGIIIA M: A Large Number of tRNA Genes are Symmetrically Located in Fission Yeast Centromeres. J rllol Rio/ 1991. 218:13-17. Identities 36 tRNA genes within the three ?; pot&e centromeres; seven genes from Cenl. 20 genes from Cefr-7 and 9 genes from Cen.3. These genes encode 11 different tRNAs and they all appear to be structurally normal. 19.
CIAIUZ 1 BAO~~ M: Functional Analysis of a Centromere t?om Fission Yeast: a Role for Centromere-specific Repeated DNA Sequences. 11101 Cell Hiol 1990, 103186%3872.
20.
POLIZI C, Cm L: The Chromatin Structure of tromeres from Fission Yeast: Differentiation of the tral Core that Correlates with Function. / CeN Rio1 112:191-201.
21.
WE~RICK R, WIIL.\RI) I-1: Physical Map of the Centromeric Region of Human Chromosome 7: Relationship between Two Distinct Alpha Satellite Arrays. Nlicleic Acid Res 1991. 19:2295-2301.
22.
V~Y J, MACC~GOR H. BARXI.X-~ L Characterization Short, Highly Repeated and CentromericaUy Localized Sequence in Crested and Marbled Newts of the Genus urus. chonracoff?ci 1990. lot: 15-3 1.
23.
RICHARDS E. CkXIDhLW H. A~IXI~I:I. F: The CentrOmere Of Arubidopsis thafiana Chromosome I Contains Telomeresimilar Sequences. Ntrciek Acid Rev 1991, 19:3351-3357.
24. .
WEVKlCK R, EARNSHAW’ W. HuwARI~-PI:EH~S P, WIU.4RD H: Partial Deletion of Alpha Satellite DNA Associated with Reduced Amounts of the Centromere Protein CENP-B in a MitoticaUy Stable Human Chromosome Rearrangement. Mel Cell Biol 1990, 10:6374380. A rearrangement Ltithin chromosome 17 result4 in deletion of 20.30% of the a-satellite DNA This rearranged chromosome was inherited b! two offspring and appeared to retain full mitotic centromere activity. The data provide tlidence that CENP-B is associated with a.satellitc DNA in ttrlo and that human centromeres are structurally and functionally repetitive.
Price
25. . .
ZINKOW%I R. ME~ENE J, BRINKLEY BR: The CentromereXinetochore Complex: a Repeat Subunit Model. / Cell Biol1991. 113:1091-l 110. Examination of the 3-dimensional structure of the kinetochore. The kinetochore was separated into small subdomains following detachment of the kinetochore from the chromosome with caffeine, or physi. cal stretching of the chromosome by hypotonic or shear forces. Pro. vides visual evidence that the kinetochore is assembled from repeating DNA-protein complexes that are tandemly arranged along the length of the centromeric DNA 26. . .
BERNAT R, DE~ANNOY M, ROTHFIEID N, EARNSHAW W: Dirup tion of Centromere Assembly During Interphase Inhibits Kinetochore Motphogenesis and Function in Mitosis. Cell 1991, 66:122’+1238. A successful attempt to identify some of the steps in klnetochore as sembly. Microinjection of anti-centromere antibodies (predominandy anti-CENP.A and anti-CENP.B) into G1- or S-phase cells prevents assem. bly of any visible kinetochore structure and results in cell cycle arrest at prometaphase. Injection of the antibodies during Gz prevents assembly of the inner plate of the kinetochore and compaction of the heterochromatin directly beneath the kinetochore, and results in inhibition of the metaphaseanaphase transition. 27. .
PAIMER D, O’DAY K. TRONC H, CHAR~ONNEALI H: Purification of the Centromere-specific Protein CENP-A and Demonstration that it is a Distinctive Histone. Proc Null Acud Sci USA 1991, 88:3734-3738. Purificadon and amino acid sequence analysis of CENP-A Demonstration that segments of CENP-A are very similar in sequence to histone H3 although other segmenLs show no such sequence conservation. 28. .
SLIIUVAN K, GLA? C: CENP-B is a Highly Conserved Mammalian Centromere Protein with Homology to the Helix-Loop-Helix Family of Proteins. aromasomu 1991, 100:36G370. Cloning and sequencing of CENF-B gene from mouse cells. Demonstration that CENP.B is highly conserved in mammalian cells and thus is likely to be important for centromere function. 29.
COM~TON D. YEN T. CLEVELAND D: Identification of Novel Centromere/Kinetochore-associated Proteins Using Monoclonal Antibodies Generated Against Human Mitotic Cluomosome Sctiolds. / Cell Biol 1991, 112:1083-1097.
30.
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31.
EARNSHAW W, COOKE C: Analysis of the Distribution of the INCENP’s throughout Mitosis Reveals the Existence of a Pathway of Structural Changes in the Chromosomes during Metaphase and Early Events in Cleavage Furrow Formation. J Cell Sci 1991, 98:4+461.
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YEN T. COLOPHON D, WISE D, ZINKOWSKI R, Bw B, E,~~NSHAW’ W, CLEVEIAND D: CENP-E, a Novel Human Centromere-associated Protein Required for Progression from Metaphase to Anaphase. EMBO J 1991, 10:124%1254. - . Monoclonal antibodies made against human chromosome scattold proteins recognize a new centromere protein (CENP-E) of 250-300kD. CENP-E appears at the centromeres at prometaphase, remains there during metaphase, and dissociates to the midplate at anaphase. Micro. injection of anti-CENP-E antibodies inhibits the metaphase-anaphase transition. 32.
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ACEVED~ 0. herence of 19:34w3419.
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T. THO~W C: The CoNfdcleic Acid< Kes 1991.
Shows that a sequence corresponding to a naturally hcdlrd break in human chromosome 16 senres as a primer for human telomerace. This sequence hns little homoloR to the putative telomerase RNA template. The dara suggest that telomerase has the capacit) to heal chromosomes by the de ~01’0 addition of telomeric DNA to broken ends that have no preexisting telomeric sequences. 50. .
GRAS JT. CEL~~DER DW, PRICE CM, CECH TR: Cloning and Expression of Genes for the O.\ytricba Telomere-binding Protein: Specific Subunit Interactions in the Telomeric Complex. Cc,// 199 I. 67,H07-8 11. Examines the relative conrributions of the (1. and P-subunits of the O.yVricha t&mere protein 10 DNA-binding.
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42.
TISCHW C. tilJIDt% G. MOIUT?! K: The Highly Variable Pentameric Repeats of the AT-rich Germline Limited DNA in Parascarik univalens are the Telomeric Repeats of Somatic Chromosomes. Nucleic .&xi.% Res 1991, 19:2677-26%
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RACHllRAh!.&~ MK, CECH TR: Effect of Monovalent Cationinduced Telomeric DNA Structure on the Binding of Oxytricha Telomeric Protein. Nlrcleic .4cids f&.v 1990. 18454h551.
ZAHER A, WIIUAAISON J, CECH T, PRESCOTT D: Inhibition of Telomerase by G-quartet DNA Structures. Narrrre 1991. 350:718-720. Demonstrates that oligonucleotides with the same sequence ;LS Oqlricba telomeres (T,t&), only act as primers for Oq~rid~u relomeraSe when the terminal T,G, repeat is not part of a Gquartet structure. 48. HARRINCTON L, GREIDER C: Telomerase Primer Specificity and . Chromosome Healing. Nature 1991. 353:451&453. Demonstrates that Tetrabymena telomerase can add TTGGGG repeats to the end of primers that are not homologous at the 3’ end to the telomerase template RNA However, an internal region that is homologous to the template is required. The data suggest that telomemse has the capacity to heal chromosomes by the de ?ror’o addition of telomeric DNA to broken ends that have no preexisting telomeric sequences.
49.
111’ %. RI! 13: A Yeast Telomeres and Conserved l?,cV~ 1991. 5:4%59.
54.
Lr’srlc; .\I. KlXlX s. SHAKY D: Involvement of the Silencer and llAS Binding Protein RAPI in Regulation of Telomere Length. Scie~cr 1990. 750:5-19-ii?.
55.
CONIUI) MN. WKICHT JI 1. w’0lP Al, T>KuI\: Interacts with Yeast Telomeres itz lttla Alters T&mere Structure and Decreases bility. Ccl/ 1990. 63:‘jSr-50.
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Protein Yeast
that Binds to Vertebrate Telomeric Junctions. &f/e.<
\‘A: RAP1 Protein Over Production Chromosome Sta-
56 ..
SLhSlil. I.. SHOKI; D: Separation of Transcriptional Activation and Silencing Functions of the RAPl-encoded Repressor/Activator Protein 1: Isolation of Viable Mutants Affecting Both Silencing and Telomere Length. Pra,Vcrfl Acad Sci 1 ‘SA 1991. 88:T4~+753. Shows that mutlitions at specitic amino ac~cls in the carbo~~l-trrrninlls of RAP1 holh suppress silencing of rhr silent mating npr Ioci and illcrease telomcrr Irngdi. Sugg:r.srs that mutations in /2-11’1 m&c t&m eric chromatin structure and hence the accesslbili~ of telomeric DNA IO t&nl~ns~. 57.
GOTTSCHIJN~; D. AI’.AtuCIO 0. MIJJNWON B. %WAN \‘: Position Effect at S. ceretpisiae Telomeres: Reversible Repression of Pol 111 Transcription. Cell 1990. 63:751-762
5X.
7x1.~ Tretrth
VA. SAVDEIJ. L: Telomeric Cell Hiol 1992. 210-14.
Position
Effect
in Yeast.
59. ..
APARICIO 0. HIUJNGTON H. G~ITSCHUNG D: Modifiers of Position Effects are Shared between Telomeric and Silent Mating-type Loci in S. cerec:isiae. Cell 1991, i&1273-1288 Shows that transcriptional repression of tekmlere pr0xinlal genvs is derepressed by mutations in S/K-, Sl&. SIR4, X’TI. Ah’DI wd HI~IFZ. AIl the gene prtducts are required for silencing of the mating ripe Ioci and they are all bt+rved to modify chromatin structure. Suggests that the telomere position effect is caused by the telomeric chromatin strucfure.
CM Price, Nebraska,
Department of Chemistr), Lincoln, Nebraska 68588,
610 Hamilton LISA
Hall.
liniversity
of