Telomere replication, kinetochore organizers, and satellite DNA evolution (centromere/robertsonian rearrangement)

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1 Proc. Nti. Acd. Sci. USA Vol. 76, No. 9, pp , Septemer 1979 Genetics Telomere repliction, kinetochore orgnizers, nd stellite DNA evolution (centromere/roertsonin rerrngement) GERALD P. HOLMQUIST* AND BARRY DANCISt *Kleerg Genetics Center, Deprtment of Medicine, Bylor College of Medicine, 1200 Moursund Avenue, Houston, Texs 77030; nd tdeprtment of Biology, Temple University, Phildelphi, Pennsylvni Communicted y Ernest R. Sers, June 11, 1979 ABSTRACT Roertsonin rerrngements demonstrte onerek chromosome rerrngement nd the reversile ppernce nd disppernce of telomeres nd centromeres. Such events re quite discordnt with clssicl cytogenetic theories, which ssume ll chromosome rerrngements to require t lest two reks nd consider centromeres nd telomeres s immutle structures rther thn structures determined y mutle DNA sequences. Cytogenetic dt from spontneous nd induced telomeretelomere fusions in mmmls sup ort moleculr model of terminl DNA synthesis in which ll ttelomeres re similr nd recomine efore repliction nd susequent seprtion. This, long with evidence for hypotheticl DNA sequence, the kinetochore orgnizer, redily explins ltent telomeres, ltent centromeres, nd reversile (onerek) Roertsonin rerrngements. A second model, involving simply recomintion etween like stellite DNA sequences on different chromosomes, explins not only how one stellite cn simultneously evolve on different chromosomes, ut lso why stellite DNA is usully locted ner centromeres or telomeres nd why it mintins preferred orienttion with respect to the centromere. Roertsonin rerrngement, telomeres, nd centromeres The most esily oserved fetures of chromosome re its ends (telomeres) nd its primry constriction (centromere). Broken chromosome ends, s cused y xrys or stretching on the spindle, show cpcity for fusion; they ehve s if sticky (1) in tht they fuse with themselves ut not with nturl ends (telomeres). McClintock (2) showed tht fter chromosomes with two roken ends fused, resulting dicentrics were unstle; the two centromeres would often seprte to opposite spindle poles nd rek the chromtid. Muller (1) then formulted two rules of chromosome structure nd mechnics. (i) All vile chromosome rerrngements require t lest two reks with susequent rejoining of the roken ends. (ii) Rerrnged chromosomes must hve exctly two telomeres (orgnelles locted t the ends) nd one centromere (n internl orgnelle) to e mechniclly stle. Roertsonin rerrngements etween rod chromosomes (Fig. 1 upper) to produce metcentric irmed chromosomes (Fig. 1 lower) re common mechnism of kryotype evolution nd occur spontneously t n pprecile frequency in mmmlin tissue culture (5) or even in the somtic tissue of certin fish (6). Reciprocl trnsloctions (Fig. l) re consistent with Muller's rules. The reverse exchnges, Roertsonin fission of metcentric into two rod chromosomes, hve een oserved, nd some (Fig. 1 nd c) pper s onerek rerrngements which do not require centric frgment to supply new centromere nd telomeres to the new chromosomes (5, The puliction costs of this rticle were defryed in prt y pge chrge pyment. This rticle must therefore e herey mrked "dvertisement" in ccordnce with 18 U. S. C. 173 solely to indicte this fct , 8). In ddition, Roertsonin metcentrics generlly possess twice the centric structure of rod chromosomes (710). In the grsshopper Neopodismopsis, Moens' (11) electron microgrphs showed this douled "kno"like structure to e penetrted y twice s mny microtuules s the single centric kno of rod chromosomes. Thus, metcentric's centric region often ppers douled nd cple of splitting y fission, ech hlf ecoming functionl centromere (8, 10). Roertsonin rerrngements, especilly the fissions, revel the indequcy of Muller's rules, especilly his concept of centromeres nd telomeres s immutle structures (1), nd imply some or ll of the following: (i) dicentrics cn e stle; (ii) fissions cn result from onerek rerrngements; (iii) centromeres nd telomeres cn reversily pper nd dispper; nd (iv) centromeres nd telomeres cn e terminl coincident structures. Dicentrics cn e stle, showing prllel chromtid seprtion when the two centromeres re close together. Hir (12) oserved n isodicentric through mny vegettive genertions in the plnt Agropyron. The originl dicentric ws unstle t mitosis; crisscross nd interlocking seprtion produced rekgefusion cycle tht resulted in shorter intercentric distnces. Dicentrics with short intercentric regions, however, were mitoticlly stle, oth centromeres on one chromtid seprting to the sme pole. Dicentrics cn lso e stle when one centromere is ltent (see review, ref. 13). In humns, most Roertsonin metcentrics re dicentric (1) in tht they show pericentric heterochromtin from oth prentl chromosomes; however, one of the centromeres is often inctive in tht it does not produce secondry constriction nd region of tight sisterchromtid piring (15). In one t(7:15)(p21;p1l), the C nding pericentric heterochromtin of chromosome 15 identified the second centric region, ut this centromere exhiited neither primry constriction nor tight sisterchromtid piring nd did not Cd nd (16). One interprettion of these dt is tht fourrek rerrngement occurred, producing trnsloction with simultneous deletion of centromere. In ccord with Hsu et l. (13), we lterntively interpret this s tworek rerrngement involving trnsloction nd inctivtion of centromere with concomitnt loss of the ltent centromere's Cd ndedness. Telomeres cn lso e ltent (reviewed in ref. 13), s clssiclly demonstrted during chromtin diminution in Prscris (17). Here, few polycentric germline chromosomes rek down into multitude of smll monocentric chromosomes with the de novo cretion of mny telomeres, presumly y ctivtion of preexisting ltent telomeres. Activtion of ltent telomere ppers s onerek rerrngement tht genertes two nonsticky ends, telomeres. If ltent telomere were locted within centric region (Fig. l), ctivtion would lso generte second centromere. Arevition: KO, kinetochore orgnizer.

2 Genetics: Acrocentrics 0 +.I 0 C~ qc Holmquist nd Dncis Telocentrics c01 olv),7* D c Acrocentrics o o Ln Qn CZ? FIG. 1. One trnsloction nd two fissionfusion models of Roertsonin rerrngement re shown with Cd nds (3) produced y ctive centromeres. () Reciprocl trnsloction etween crocentrics produces metcentric nd smll centric frgment which, if susequently lost, mkes this process irreversile. Centromere nd telomere numer is conserved. () Reversile fusion of two telocentrics (chromosomes with terminl centromeres) to produce metcentric. The fusionfission rerrngement requires only one chromosome rek. Two telomeres nd centromere reversily pper or dispper. (c) Reversile fusion of two crocentrics to produce metdicentric with pir of Cd nds (). Two telomeres reversily pper or dispper. The centromere nd kinetochore orgnizer The ctive loclized centromere of mitotic irmed chromosomes ppers, y light microscopy, s negtively heteropycnotic constriction (primry constriction) tht moves first to the spindle poles during nphse nd is necessry for disjunction of sister chromtids. This region stins y the Cdnding technique (3) nd morphologiclly is DNAcontining, iprtite, reverserepet structure of severl chromomeres flnked y regions of tight sisterchromtid piring (18). In formlinfixed electron microscopic sections of oth plnts nd nimls, the centromere ppers not so much s constriction, ut s region of lightly stining, thin chromtin fiers (19) in which ech sister chromtid possesses kinetochore plte (20) [pir of kinetochore filments (19)]. Spindle microtuules penetrte the plte (20) nd the plte itself is cple of ctlyzing tuulin polymeriztion (21, 22). The kinetochore plte is the site of spindle ttchment nd microtuule polymeriztion nd reflects centromeric ctivity. It proly lso induces the ncillry ttriutes of n ctive centromere, including primry constriction, pericentric regions of tight sisterchromtid piring, nd Cd nding. Brinkley nd Stulefield (19) proposed the kinetochore to e specilized gene, much like the nucleolus orgnizer defined y McClintock (23), ecuse it contins DNA (18), segregtes with the chromtids, nd is ctivted t specific (mitosis) stge of the cell cycle. We shll cll this gene kinetochore orgnizer (KO). Just s the nucleolus orgnizer cn e seprted from the structurl 18S nd 25S rrna genes whose product it orgnizes (2), the KO need not contin the genes coding for kinetochoreplte proteins, ut the KO must orgnize plte proteins into functionl structure when it is ctivted. We propose only one dditionl property of the KO to mke it consistent with cytologicl dt (ref. 13; Fig. 1): the KO cn e permnently inctivted y rerrngement. Thus, visile centromere reflects n ctive KO nd ltent centromere, n inctive KO. Vile chromosomes must hve t lest one ctive KO for proper disjunction of sister chromtids. Telomere repliction The chromtid contins one doulestrnded DNA helix (25), the termini of which re proly included in its two telomeres. DNA termini cnnot e replicted y conventionl mens ecuse digestion of the terminl RNA primer leves 5' gp (Fig. 2) tht no known polymerse cn fill (26). Solutions to this *. 1 I 7Th )D )0 Proc. Ntl. Acd. Sci. USA 76 (1979) 567 c Z lid Hollidy Structure DDiDt H >>II>II.' smll Plindrome lrge tndem repet replicted 2ZZZZ intermedite i FIG. 2. Types of fused metphse chromosomes rising s repliction intermedites from vrious models of telomere repliction. () Repliction with digestion of RNA primers nd ligtion of Okski frgments leves terminl 5' gps (26). If telomeres re complementry s in T7 phge (26), complementry gps could se pir, forming conctmeric telomere fusions, which, eing endless, re le to e replicted. Alterntively, ligtion nd stggered nicking (not shown) of the fused junction could convert it into pir of replicle 3' gps (27). () Btemn (28) ssumed the telomere to e covlently closed hirpin, mking it different from "sticky" roken end. Repliction genertes plindrome tht is nicked y restriction endonuclese, refolded, nd ligted. (c) A fusioneforerepliction model. All telomeres re identicl, covlently closed, nd contin lrge, repeted sequence, the sic unit of which is represented y the letters A nd T. Two telomeres recomine, forming Hollidy (29) structure, which, fter ligtion, hs no ends nd is therefore le to e replicted. Newly replicted DNA is represented y the drker lines. Fission of the replicted intermedite proceeds s in. Okski terminl dilemm for eukryotic telomeres hve een proposed (2628, 30), nd representtives re shown (Fig. 2 nd ). To explin cytogenetic dt, we proposed tht the telomeres must fuse pirwise efore repliction (31, 32). The most resonle moleculr model (Fig. 2c) required ll telomeres in cell to recomine vi common sequences efore repliction nd is consistent with Ruin's (33) cloning of 12kilose DNA sequence, four tndem repets of 3kilose sequence, which hyridized in situ to ll Drosophil melnogster telomeres nd to the ectopic strnds tht connect them, nd with Forte's nd Fngmn's (3) discovery tht yest (Scchromyces cerevisie) telomeres re covlently closed hirpins. Eukryotic chromosomes, in some specil instnces, pper fused end to end, nd in rre extreme cses the entire genome ppers s gint ring of fused chromosomes (3537). White (38) inferred tht telomere sesequence homology cused occsionl endtoend ssocitions of meiotic chromosomes. We extended this concept of homology y interpreting mitotic ssocitions s repliction intermedites (31, 32). Senescing Xi

3 568 Genetics: Holmquist nd Dncis humn primry cell lines(39) nd lymphocytes from ptients with ThiergeWeissench syndrome (0) ehve s if the restriction endonuclese of Fig. 2c does not cut efficiently in tht mny chromosomes re seen permnently fused end to end t the first mitoses fter fusion. The fused configurtions predicted y other repliction models (Fig. 2 nd ) were not seen. Insted, sister telomeres were fused to nother pir of sister telomeres (Fig. 2c), s is consistent with telomere fusions occurring efore repliction. Rndom telomere pirs were fused; ll telomeres were similr in their fusion potentil (39, 0) nd not composed of complementry pirs s in conctmeric fusion process (26) (Fig. 2). BrdUrd pulse leling indicted tht the chromtin t the fusion junction of ThiergeWeissench lymphocyte chromosomes replicted very erly in the S phse preceding mitotic fusion (0). This suggests tht in norml cells, telomeretelomere fusion is temporry event occurring in erly S phse nd would not e detected in synchronous cultures y either viscometric DNA moleculr weight nlyses (25) or y psorlin crosslinking (1) to revel the cloverlefshped Hollidy intermedite of Fig. 2c. Fig. 3type telomeretelomere fusion figures were induced in the first metphse fter tretment of mouse cells with mitomycin C, DNA crosslinking gent (2). Such figures would, however, e expected from the repliction model in Fig. 2c. If one crosslink prevented terminl rnch migrtion of the Hollidy structure, one pir of chromtids would seprte fter repliction while the other pir would remin fused (32). In conclusion, we view telomeres s structures tht contin DNA termini nd solve the specil prolem of terminl repliction. Cytogenetic dt support model requiring telomeretelomere fusion efore repliction, nd moleculr dt re most consistent with recomintion (Fig. 2c) mechnism of fusion. The model lso explins ltent telomeres nd some spects of Roertsonin fusion. Telomeres (stle nonsticky chromosome ends) re creted de novo during the differentition of som from germ line in Prscris. This is esily explined if differentition promotes the seprtion of ltent telomeres (fused telomere pirs) or, more specificlly, ctivtes restriction endonuclese tht cleves those plindromes (Fig. 2c) tht re ltent telomeres (27). Activtion of ltent telomere would pper cytogeneticlly s onerek rerrngement, generting two telomeres, nd would violte Muller's (1) rules. Roertsonin fusion would e specil cse of telomeretelomere fusion, involving the centric ends of two rod chromosomes. In the moleculr model (Fig. 2c), muttion in terminl hirpin sequence could prevent recognition y the restriction endonuclese of the repliction intermedite's plindrome (Fig. 2c), resulting in fusion. A susequent ck muttion would pper s Roertsonin fission of ltent telomere. The fusionfirst repliction mechnism necessittes symmetry. Specificlly, if one telomere contined n inverted Ruin sequence, it could fuse with other telomeres ut the intermedite could not seprte (32). Thus, telomeres with common Ruinsequence orienttion would e selected for, s portryed in Fig. Sc y the letters A pointing towrd the terminl hirpin. The letters A re lso pointing wy from n internlly locted centromere; thus we cn define for ll such telomeres common centric orienttion. Centric orienttion nd stellite DNA evolution Mouse stellite DNA sequences ll hve the sme centric orienttion (Fig. 8). Cytologists found this y using romodeoxyuridine's quenching of fluorescent dyes. They followed the Trich stellite strnds during few genertions of BrdUrd incorportion nd determined their reltive orienttions y Tetocentrics Al T V T V Al TV TV AI TV TV 11 I11I 11 3' 5' 3'5' opposite Sme Proc. Ntl. Acd. Sci. USA 76 (1979) Sme Metcentrics some orienttion rms Roertsonion 3' I >5 Rerrngement 5' 3 or 3 >>> I>> 5'S11FE TrnslocOtion Opposite orienttion rms: not oserved ir_ ieon o5' F<< ~ ~ 3 Prcentric inversion not oserved Opposite He 7 F (~ ~~~ +, FIG. 3. Ce tric orienttion of mouse stellite DNA round the KO (dot). () the reltive centric orienttion of stellite locks is determined y the segregtion of Trich stellite strnds in metcentrics. () A sepiringdependent trnsloction (XX) etween terminl locks of either opposite or similrly oriented stellite DNA would produce either n centric chromosome nd dicentric frgment or terminl exchnge. (c) The product(s) of intrchromosoml, sepiringdependent exchnges etween similrly oriented stellites. Centric orienttion (rrows) of the included sequences remins unchnged. Becuse of the sepiring requirement, the prcentric exchnge is deletion nd not n inversion s in. their segregtion in Roertsonin metcentrics (3) nd x ryinduced dicentrics (). Even in mouse Lcell mrker chromosomes, which were generted y multirek rerrngement nd contin mny interclry locks of stellite, ll stellites' centric orienttions were invrily mintined the sme (5). A prcentric inversion is one of severl rerrngements tht cn reverse (Fig. S) nd thus rndomize centric orienttion, ut evidence for this ws not foundt (5). Selection must ct to preserve common centric orienttion, nd ny tenle theory of stellite DNA evolution must explin this fct. Computer simultions y Smith (7) hve shown tht DNA whose sequence is not mintined y selection cn theoreticlly develop hierrchy of tndem periodicities (i.e., form stellite When, s in humns, two or more different locks of A.Trich stellite DNA re present on the sme chromosome rm, switches in Trich strnds (6) my represent the junction of two different stellite locks nd not switch in ny one stellite's centric orienttion.

4 Holmquist nd Dncis Genetics: Holmquist nd Dncis ~~Proc. Nti. Acd. Sci. USA 76 (1979) 569 sequences) s result of se piring etween sister chromtids followed y unequl sisterchromtid exchnge t the piring site. The ptterns of restrictionendonuclesesensitive sites oserved in mouse (8), D. melnogster (9), nd clf (50) stellite DNA definitely support Smith's prediction of hierrchil repet pttern of longrnge periodicities. Two consequences of this sepiringdependent mechnism re tht stellites evolve in locks, contiguous stretches of like sequences s shown for Drosophil (51, 52), nd tht the sequences within ny one lock hve the sme orienttion, s shown for mouse stellite (5). Bov'ini nd Cprini, of the superfmily Bovide, diverged over io0 yers go from common ncestor nd yet they still shre wekly crosshyridizing stellite DNA (55). For ny one species in the superfmily, the pttern of restrictionendonuclesesensitive sites of the stellite is simple hierrchicl one (50) even though the stellite DNA comes from mny different chromosomes. The simple pttern mens tht within species, the stellite on mny different chromosomes is very similr in its longrnge periodicities. A recent slttory event with susequent dispersion to the different chromosomes cnnot explin this ecuse the sme stellite ws presumly present on different chromosomes io0 yers go. The stellite must hve coevolved on different chromosomes, with stellite muttions eing communicted from chromosome to chromosome y some interchromosoml interction. Bsepiringdependent interchromosoml exchnge etween stellite locks of like sequence would explin oth coevolution nd centric orienttion of stellites. Such n exchnge etween terminl stellite locks on different chromosomes would produce n centric chromosome if the locks were of opposite centric orienttion (Fig. S) nd result in selection ginst opposite orienttions in one species. Terminl stellites, s in the mouse, could thus coevolve y exchnge nd mintin prticulr orienttion reltive to the KG ecuse the structure the KG orgnizes is essentil to the existence of ech chromtid. Similr exchnges occurring etween stellites of the sme orienttion on the sme chromosomes would determine preferred distriution of stellite locks. Prcentric exchnges would delete intervening genes (Fig. Sc) so tht homologous stellite locks on the sme chromosome rm would e selected ginst. Similrly, pericentric exchnges could lter linkge groups (Fig. Sc), nd interstitil homologous stellites on opposite rms would e disfvored. However, if the locks were pericentric or terminl s in most eukryotes, such exchnges would not lter linkge groups. This postulted exchnge mechnism presents dynmic view of stellite evolution with locks of similr sequence locted usully penicentric or terminl nd showing their dynmic stte through popultion polymorphisms of lock sizes or pericentric inversions, s consistent with present cytogenetic dt. The whole chromosome Muller (1) considered centromeres nd telomeres, s immutle structures; y ssociting structure with the loction of the structurl determinnt, he thought telomere nd centromere could not shre the sme loction, s in telocentric, thus oscuring the mechnism of reversile Roertsonin rerrngements (Fig. 1). We define ctive telomeres s structures (the kinetochore plte nd ncillry centric chromomeres) expressing KG DNA sequences. Thus, telocentric chromosome could hve terminl centromere (kinetochore plte overlpping the telomere) nd telomere tht functions for terminl repliction without hving the KG s terminl sequence (Fig. ). Similrly, if fusion of telocentrics rought two KOs close together so tht the pltes they orgnized overlpped (Fig. ), Monocentric Telocertric Mo.nocentric C Acrocentric fusion Dicentric FIG.. () A monocentric chromtid hs one kinetochore plte (thick line) which is orgnized y the physiclly smller KO sequence (dot) nd contins pericentric stellite (lrge letters) nd telomeric Ruin sequences (smll letters). Telomere fusion cn e trnsient, s in repliction (Fig. 2c), or permnent, s in Roertsonin fissionfusion ( nd c). Stellite nd Ruin sequences mintin constnt reltive orienttion; thus, telomeretelomere fusions produce the contrlterl symmetry of pericentric stellite oserved in mouse metcentrics (Fig. 3). In c, the kinetochore pltes do not cover the ends of the crocentrics nd show douled nture in the fused metdicentric. the fusion product would hve one centromere, one centromere nd two telomeres hving disppered during the fusion. This would lso explin why the centromere of Roertsonin metcentrics often shows douled nture (711). Fig. lso indictes tht ltent telomeres should e common in centric regions nd is consistent with the oservtion of Kto et l. (5) tht 2.9% of the cells in their CHG line showed evidence of t lest one fission event, spontneous reking of the centromere with concomitnt "heling" of the roken ends. The moleculr models we present, while proly inccurte in detil, do unite disprte dt nd, more importntly, remove the mysteriously immutle or discrete structurl properties scried to telomeres nd centromeres, presenting them insted s the expressions of DNA sequences tht cn mutte, rerrnge, nd e regulted. We thnk Dvid Comings, T. C. Hsu, Sm Ltt, Gerld Ruin, John Wilson, Sheldon Wolff, nd E. R. Sers for dvice nd Dottie Holmquist for the rt work. This work ws supported y Ntionl institutes of Helth Grnts GM to B.D., GM23905 to G.P.H., nd GM to Thoms Cskey. 1. Muller, H. J. 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