binding protein: A circular dichroism study

Transcription

1 Proc. Ntl. Acd. Sci. USA Vol. 91, pp , August 1994 Biochemistry Promotion of prllel DNA qudruplexes y yest telomere inding protein: A circulr dichroism study RAFAL GIRALDO, MASASHI SUZUKI, LYNDA CHAPMAN, AND DANILA RHODS* Medicl Reserch Council Lortory of Moleculr Biology, Hills Rod, Cmridge CB2 2QH, United Kingdom Communicted y Aron Klug, April 15, 1994 (received for review Decemer 7, 1993) ABSTRACT Repressor-ctivtor protein 1 (RAP1) hs n essentil role in the mintennce of yest telomeres. Yest telomeric DNA consists of simple repeted G-rich sequences tht re ound y RAP1. We hve found tht RAP1, in ddition to its known inding ctivity for doule-strnded DNA, intercts with the G-rich strnd contining gunine se (G)-tetrds. We show here using circulr dichroism spectroscopy tht RAPi promotes the formtion of one prticulr type of DNA qudruplex, prllel G4-DNA. Furthermore, RAPi is le to ind to oth preformed prllel nd ntiprllel DNA qudruplexes. These results hve implictions for the possile use of DNA qudruplexes in telomere-telomere ssocition in vivo. The remrkle conservtion of telomeric DNA sequences suggests some importnt iologicl role. Telomeric DNA consists of simple repeted sequences in which one of the DNA strnds contins short trcts of gunines (such s T4G4 in Oxytrich, TG1_3 in Scchromyces, nd T2AG3 in vertertes) (for review, see ref. 1). These G-rich sequences cn self-ssocite in vitro to form different types of DNA qudruplexes, ll contining gunine se tetrds or qurtets (G-tetrds). A G-tetrd rises from the ssocition of four gunine ses into cyclic hydrogen onding rrngement. Ctions, such s K+ nd N+, stilize qudruplex structures y inding to the cges formed y the stcking of G-tetrds. DNA qudruplex formtion is very polymorphic nd is dependent on oth the concentrtion nd the numer of G-trcts in the sequence, nd the specific ction present (for review, see ref. 2). Different types of DNA qudruplexes, originlly proposed from iochemicl studies, hve more recently een oserved in numer of three-dimensionl structures determined using NMR nd x-ry crystllogrphic methods: The tetrmeriztion of four strnds gives rise to DNA qudruplex clled G4-DNA (3) in which the four strnds, in ll cses studied, re in prllel orienttion (4, 5); the dimeriztion of two hirpins, ech contining two G-trcts, gives rise to DNA qudruplex in which the strnds re necessrily in n ntiprllel orienttion (6-9); relted type rises from the intrmoleculr folding of n oligonucleotide contining four G-trcts (8). Although DNA qudruplex structures hve ttrcted much ttention nd role in telomere-telomere ssocition hs een proposed (6, 7, 10), the intermoleculr formtion of G4-tetrds in vitro requires high DNA concentrtions (10-6 to 10-3 M) nd is very slow, constituting kinetic rrier tht would preclude their formtion in vivo (2). Recently, however, it hs een reported tht the 3 suunit of the Oxytrich telomere inding protein is le to promote G-tetrd formtion (11, 12). We hve een studying the yest telomere inding protein repressor-ctivtor protein 1 (RAP1) nd hve found tht it lso is le to promote the formtion of G-tetrds nd ind to preformed DNA qudruplexes (13). RAP1, originlly identified s regultor of trnscription (14), is essentil for the mintennce of the telomeres of Scchromyces cerevisie (15) nd is loclized t the ends of chromosomes (16). In vitro inding studies show tht RAP1 is likely to e the mjor DNA inding protein of yest telomeres (17). On doule-strnded DNA, RAP1 inds to the consensus inding site 5'-GGTGTGTGGGTGT-3'. The inding ctivity of RAP1 for the telomeric GT-strnd is highly dependent on the sequence nd length of the GT-strnd nd requires the presence of t lest two RAP1 inding sites in tndem (13). From our previous study, using nd-shift nd footprinting nlyses, we could conclude tht G-tetrds were present, ut not wht type ofdna qudruplex ws promoted y RAP1. Knowledge of the type of DNA qudruplex involved, prllel or ntiprllel, would in turn provide informtion on the numer of strnds involved in G-tetrd formtion. From the comprison of iochemicl nd structurl nlyses with spectroscopic studies, it hs een possile to correlte prticulr circulr dichroism (CD) chrcteristics with prticulr type of DNA qudruplex (18-20). The prllel nd ntiprllel DNA qudruplexes give drmticlly different CD spectr: prllel qudruplexes hve positive ellipticity mximum t =260 nm nd negtive ellipticity minimum t 240 nm; ntiprllel qudruplexes hve mximum t etween 285 nd 295 nm nd minimum t 265 nm (21). The difference in CD chrcteristics correltes with different glycosidic torsion ngles tht re ll nti in prllel qudruplexes, ut lternte syn-nti long one strnd in ntiprllel qudruplexes, which in turn results in differences in the stcking of G-tetrds. We show here using CD spectroscopy tht RAPI is le to promote the formtion of the prllel type of DNA qudruplex. These results thus confirm nd extend our iochemicl nlysis (13) nd constitute spectroscopic evidence for the protein-promoted formtion of DNA qudruplexes. MATRIALS AND MTHODS Oligonucleotides. The oligonucleotide Ytelo (clled 4G3 in ref. 13) is derived from Scchromyces cerevisie telomeric DNA nd Oxytelo is from Oxytrich telomeric DNA. Sequences: Ytelo, 5'-AATTCTTGGTGTGTGQQTGTGTjQWTGTGTQGfTGTGG-3'; Oxytelo, 5'-jGCjTTTTQQ- WTTTTTTGGTTTTGGGG-3' (where G-trcts re underlined). Oligonucleotides were synthesized on n Applied Biosystems 380B DNA synthesizer nd purified using denturing polycrylmide gel electrophoresis s descried (13). Purifiction of RAPi nd Its DNA Binding Domin (DBD). RAP1 (n 827-mino cid residue protein) nd its DBD ( 247-residue protein) (22) were overproduced in scherichi coli nd purified to homogeneity (13). Protein preprtions were dilyzed extensively ginst 20 mm Tris HCI, ph 7.0/1 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 solely to indicte this fct Arevitions: RAP1, repressor-ctivtor protein 1; DBD, DNA inding domin. *To whom reprint requests should e ddressed.

2 Biochemistry: Girldo et l. mm DTA/5% (wt/vol) glycerol plus the pproprite slt for CD mesurements [250 mm (NH4)2SO4 or LiClJ. CD Spectroscopy. Oligonucleotide stocks were diluted to 0.8 A260 unit per ml (2,uM) in 20 mm Tris-HCl, ph 8.0/1 mm DTA (T uffer), heted t 90'C for 10 min, nd left to cool t room temperture for severl hours. Proteins nd the vrious slts were dded nd incutions were crried out t room temperture for 1 hr ( sufficient time to rech equilirium) prior to CD spectroscopy. CD spectr were recorded in Join Yvon CD6 spectropolrimeter using 1-cm pthlength cuvette set t 200C. The reported spectrum of ech smple represents the verge of five scns. The spectrl contriution of uffers nd proteins ws sutrcted s pproprite. Sutrctions nd spectr were clculted nd plotted using the softwre supplied with the spectrometer. Spectr re presented s plots of molr ellipticity Om expressed in degree cm2*dmol-1. Bnd-Shift Gel Anlysis. Binding rections were performed in T uffer supplemented with 100 mm KC1. The mount of protein required to sturte inding ws found y titrting the oligonucleotides t 0.8 A260 unit per ml, including 10 fmol of 5'-32P-leled oligonucleotide, with either RAP1 or DBD (13). Prior to the inding rections, the mixture ofleled nd unleled oligonucleotides ws nneled s descried ove. Binding rections were incuted for 1 hr t room temperture nd nlyzed y nondenturing gel electrophoresis in 1.0%6 grose gels in 45 mm Tris orte/l mm DTA (0.Sx TB). Gels were dried onto Whtmn D81 pper nd the results were visulized y utordiogrphy. RSULTS AND DISCUSSION The Yest Telomeric Oligonudeotide Ytelo Forms Prllel G4-DNA Qudruplexes in K+. The miniml length of the yest telomeric GT-strnd tht is recognized efficiently y RAP1 ws defined using nd-shift nlyses (13). The sme oligonucleotide, Ytelo, is used in the CD nlysis presented in this pper. First, we sked wht type of structure Ytelo forms in the sence of ny protein. Since the lkli metl ion cn determine the type of DNA qudruplex formed (3, 23), Ytelo ws incuted in vrious concentrtions of NCl nd KCl ( M). For comprison we lso nlyzed the Oxytrich telomeric oligonucleotide Oxytelo. Both ofthese oligonucleotides contin four G-trcts: G-trcts in the yest sequence contin three gunines, wheres G-trcts in the Oxytrich sequence contin four gunines; the linkers etween G-trcts differ in oth length nd sequence (TGTGT vs. TTTT). The CD mesurements presented were ll crried out t 2,uM oligonucleotide (0.8 A260 unit per ml), which is close to the lower limit for CD mesurements. For Oxytelo in KCl, we oserve mximum t 295 nm nd minimum t 265 nm (Fig. l). This is expected, s this type of CD spectrum hs een oserved for the oligonucleotide G4T4G4 nd correlted with ntiprllel DNA qudruplexes (18). Furthermore, the NMR nlysis of Oxytelo shows this oligonucleotide to e intrmoleculrly folded forming n ntiprllel DNA qudruplex (8). Spectr recorded in T uffer nd in NCl re identicl nd only slightly different from the one recorded in KCl, indicting tht under the experimentl conditions used, the folding of Oxytelo is essentilly independent of the ction used. The results for Ytelo re very different. Fig. l shows tht upon ddition of KCl, the spectrum chnges drmticlly from one contining two positive mxim of low ellipticity to one mximum of strong ellipticity t 264 nm nd minimum t 240 nm. These CD chrcteristics hve een identified with prllel G4-DNA: sequences of the Gn type, for which fier diffrction ptterns hve een interpreted in terms of prllel DNA qudrupled give similr CD spectrum (18); the sequence TG3T lso shows this type of CD spectrum, nd the Proc. NtL. Acd. Sci. USA 91 (1994) g 35 Io o.0-3 To I ' 40 g 20 r-r.o ~0 c -i N A Ṫo 0D no FIG. 1. CD spectr of the effects of N+ nd K+ on the formtion of DNA qudruplexes y Oxytelo nd Ytelo oligonucleotides. () Oxytelo ws mesured in the following uffers: T, -; T/0.1 M NCi, ; T/O.1 M KC1, -.-. () Ytelo/KCI titrtion: T, ; T/0.1 M KCI,-.-; T/0.5 M KCI, ; T/1.0M KCI, ---. (c) Ytelo/NCl titrtion: T, -; T/0.1 M NCi, --; T/0.5 M NCi, - ---; T/1.0 M NCi, -.-. Oligonucleotide concentrtions in this nd susequent experiments ws 2 pm. Spectr hve een plotted using different scles for the molr ellipticity. NMR structure shows it to e prllel G4-DNA qudruplex (19). The cquisition of the CD spectrum chrcteristic of G4-DNA is complete t 0.1 M KCl. The shoulder t 285 nm indictes tht the GT-strnd not only is in the pure G4-DNA form ut lso contins minor component of, proly, the

3 7660 Biochemistry: Girldo et l. intrmoleculrly folded conformtionl species. The effect of N+ ions is much less mrked (Fig. ic) nd gives rise to rod positive mximum centered on 275 nm, indicting the presence of mixture of conformers. The formtion, in the presence of K+ ions, of different types of qudruplexes y the Ytelo nd Oxytelo oligonucleotides, prllel nd ntiprllel, respectively, indictes tht oth the numer of gunines in the G-trcts nd the composition of the linker ply determinnt role. The formtion of G4-DNA y Ytelo could e due to the presence in the linkers of gunines tht cn form dditionl G-tetrds when the four strnds re in prllel orienttion. We do not oserve the expected "N+-K+ switch" (prllel to ntiprllel) reported y Sen nd coworkers (3, 23). This difference in ehvior proly rises from the very different oligonucleotide concentrtions used in the two studies, 2 pm in our study vs ,uM y Sen nd coworkers (3, 23), nd differences in the DNA sequences used. This in turn is likely to ffect the kinetic nd thermodynmic properties such tht t low concentrtions the Oxytrich sequence is trpped in the intrmoleculrly folded ntiprllel form, wheres the shorter G-trcts present in Ytelo llow rpid folding nd unfolding nd hence the formtion of G4-DNA. RAPi Binds to Both Prllel nd Antiprflel DNA Qudruplexes. Hving found tht in the presence of K+ ions, the oligonucleotides Ytelo nd Oxytelo form prllel nd ntiprllel DNA qudruplexes, respectively, we next sked whether RAPi is le to recognize these structures. As control we hve lso used the miniml DBD (17, 22). The DBD hs full DNA inding ctivity for doule-strnded telomeric sequences ut does not ind to the GT-strnd oligonucleotide Ytelo t nnomolr concentrtions (13). The two DNA qudruplexes were preformed in the presence of 50 f1 Proc. NtL. Acd. Sci. USA 91 (1994) KCl. Prior to the CD mesurements ech oligonucleotide (2 1LM) ws incuted with incresing concentrtions of RAP1 or DBD nd the nlysis ws crried out on nondenturing grose gels. Fig. 2 shows tht RAP1 inds to oth the folded Ytelo nd Oxytelo oligonucleotides wheres DBD, in greement with our previous results (13), does not. Comprison of the CD spectr otined (in KCl) in the sence nd in the presence of protein (t protein concentrtion tht gives full inding) shows tht there is no ltertion to the CD spectrum ofeither Ytelo or Oxytelo upon protein inding. These results indicte tht RAPi is le to recognize nd ind to oth prllel nd ntiprllel qudruplexes. vidence is lso forthcoming for other proteins tht recognize DNA qudruplexes, such s nuclese present in yest telomere extrcts (24). The fct tht RAP1 is le to ind with pproximtely the sme ffinity to oth prllel nd ntiprllel qudruplexes is surprising given tht the orienttion of the strnds, the width of the grooves, nd the glycosidic torsion ngles ll differ etween the two types of qudruplexes. However, the NMR structure (8) ofthe sme Oxytelo oligonucleotide (contining four G-trcts) s tht used in our studies my go some wy in reconciling this prolem of recognition. In this structure, djcent strnds re lterntely prllel nd ntiprllel, nd the groove etween the prllel strnds is of medium width, s found in prllel DNA qudruplexes (21). Thus, one of the fces tht this type of ntiprllel qudruplex presents to the protein hs fetures in common with those of the prllel DNA qudruplex. RAPi Promotes the Formtion of Prllel G4-DNA Qudruplexes. We next sked wht type of DNA qudruplex is promoted y RAP1. Oligonucleotide smples were prepred in the pproprite monovlent ction nd incuted for 1 hr in the presence nd sence of protein. To enle us to 70 / Xi P~~~ 30/3 1 ~~~~~~~~/1 535 / 0 7 R 0~~K1 I -60! i I L.L. / ; nm -70 protein - poino 0.5 1i[D DB I10C; S L.ZA 2.0 protein oligo RAP D 1,7S? C D05., -- O. 'r-.'.. IR _ et I R _--* F _-o " I _ OF -r F$I- ~~~... FIG. 2. Binding ofrapi nd its DBD to the Ytelo () nd the Oxytelo () oligonucleotides. (Upper) The CD nlysis ws crried out in T/O.1 M KCI s follows: free oligonucleotide, -; RAPl-oligonucleotide complex, -.-; DBD-oligonucleotide complex, -. (Lower) Bnd-shift gel nlysis of the sme oligonucleotides (2 pm, in T/0.1 M KCl) incuted with incresing concentrtions of RAPi or DBD (1-4 pm, s indicted). The protein/oligonucleotide molr rtios re indicted t the top of ech lne. The loctions of the free oligonucleotides (F) nd of RAPl-oligonucleotide complexes (1R) re indicted. The protein/oligonucleotide molr rtio used in the CD nlysis ws 1.75.

4 c I- Q I N. K Biochemistry: Girldo et L I? -04 " 0,I C *0 f- o7 K FIG. 3. CD spectr of the effects of RAPI, DBD, nd Hi on the structure of Ytelo nd Oxytelo oligonucleotides. () Ytelo (2 MM) with RAP1 or its DBD: T, -; T/50 mm (NH4)2SO4, -.-; T/50 mm (NH4)2SO4/3.5 pm RAP1, ---; T/50 mm (NH4)2SO4/3.5,M DBD, () Ytelo (2 P) with histone Hi in T/ 50 mm (NH4)2SO4: no Hi, -; 1 p.m Hi, -.-; 1.5 pm Hi, -; 2 um HI, (c) Oxytelo (2 M) with RAP1: T, -; T/0.1 M LiCI/3.5 pm RAP1, -.-; T/0.1 M NCI/3.5 pm RAP1, ----; T/o.1 M KCI/3.5 MM RAP1, oserve the effect ofthe protein on the structure of Ytelo, CD mesurements were crried out in the presence of NHW, n ion tht is considerly less efficient thn K+ in supporting qudruplex formtion (compre Fig. l with Fig. 3). Fig. 3 no Proc. NtL. Acd. Sci. USA 91 (1994) 7661 shows tht the ddition of 1.75 molr excess of RAP1 (3.5,uM) gives rise to n increse in ellipticity nd shifts the spectrum from one tht is predominntly ntiprllel in chrcter to one tht is chrcteristic of prllel G4-DNA (mximum t 264 nm). These spectroscopic results thus confirm the conclusion of our iochemicl study (13) on the role of RAP1 in the promotion of G-tetrds. Unexpectedly, we hve found tht DBD under the conditions of the CD nlysis lso promotes the formtion of G4-DNA (Fig. 3). This finding will e discussed elow. The effect of other ctions on the RAP1 promoted formtion ofg4-dna ws lso investigted. We find tht the height of the mximum t 264 nm decreses pproximtely with the size ofthe ionic rdius (NH' > N+ > Li+) (dt not shown), which is in greement with the different ility of these ctions to stilize DNA qudruplexes (25). In other words, the protein shifts the equilirium towrd the formtion of G4-DNA, ut the process lso requires the presence of n ion tht stilizes the DNA qudruplex. In the CD spectrum of the Ytelo-RAP1 complex (Fig. 3), there is positive mximum t 240 nm tht is reproducile for independent RAP1 preprtions nd is not due to some component of the uffer used. Furthermore, the mximum t 240 nm is not present in the CD spectrum of RAP1 ound to doule-strnded telomeric DNA (dt not shown). As RAP1 hs very high content of romtic residues (2 Trp, 25 Tyr, nd 33 Phe), the nomlous ellipticity is likely to e due to chnge in the environment of some of these residues upon GT-strnd inding. Since it hs een reported tht histone Hi ids the formtion of G-tetrds y n Oxytrich GT-strnd (11), we hve tested whether histone Hi promotes the formtion of DNA qudruplexes y the Ytelo oligonucleotide. The CD spectr recorded in the presence of NH4 nd incresing concentrtions (1-2 pm) of HI show tht Hi cuses the spectrum to shift towrd tht of G4-DNA. However, the two spectr do not coincide (Fig. 3) nd the increse in ellipticity is smller thn tht produced y RAP1. In ddition, t n Hi to Ytelo molr rtio of etween 0.5:1 nd 1:1, there is visile ggregtion nd t rtio of.1:1 the complex precipittes, wheres t this rtio RAPl-Ytelo complexes re solule. Thus Hi hs some effect on the structure of Ytelo, ut the effect is much less specific thn tht of RAP1 or its DBD. Since RAP1 inds to the ntiprllel Oxytelo qudruplex (Fig. 2), we investigted whether RAP1 lso promotes G4-DNA formtion y this oligonucleotide. Fig. 3c shows tht when RAP1 ws incuted with Oxytelo in the presence of Li+, N+, or K+, the CD spectrum remins chrcteristic of n ntiprllel qudruplex. This suggests tht the RAP1- dependent promotion of G4-DNA is specific for the yest telomeric sequence. As mentioned ove, the fct tht the DBD promotes the formtion ofg4-dna ws unexpected, since the iochemicl nlysis (crried out with Ytelo t nnomolr concentrtions) hd shown tht full-length RAP1 is required for the formtion of G-tetrds (13). Furthermore, the nd-shift nlysis (Fig. 2), crried out under the sme conditions s the CD spectroscopy, shows tht DBD does not form complex with preformed G4-DNA. It is therefore likely tht DBD inds very wekly to Ytelo in solution nd tht such complexes dissocite in the electric field nd thus cnnot e oserved in nd-shift gels. So wheres RAP1 promotes the formtion of G4-DNA nd inds reltively strongly to these structures, t the high concentrtions tht re necessry to crry out CD mesurement DBD seems to ct s "chperone," in the sme sense s reported for the /3 suunit of the Oxytrich protein. This protein ids the formtion of DNA qudruplexes ut does not pper to ind strongly to the structure it promotes, since no distinct protein-dna complex is oserved in nd-shift gels (11).

5 7662 Biochemistry: Girldo et l. Proc. Ntl. Acd. Sci. USA 91 (1994) Wht Is the Role of RAPi in DNA Qudruplex Formtion? The results from the spectroscopic nlysis presented in this pper show tht RAPi cn promote the formtion of prllel G4-DNA qudruplexes. These oservtions re consistent with our iochemicl dt from which we deduced tht RAPi promotes the formtion of G-tetrds (13). Furthermore, some of the results of the nd-shift nlyses hd indicted tht severl strnds re likely to e involved in G-tetrd formtion, consistent with the fct tht the formtion of G4-DNA necessrily involves four strnds. However, since CD spectroscopy nd the iochemicl nlysis were crried out t very different oligonucleotide concentrtions (micromolr vs. nnomolr, respectively), it remins to e estlished whether G-tetrd formtion t nnomolr oligonucleotide concentrtions involves G4-DNA or some other type ofdna qudruplex. Wht is the role of protein in DNA qudruplex formtion? For the 13 suunit of the Oxytrich protein, it hs een demonstrted tht it gretly increses the rte t which the two qudruplexes (dimers nd tetrmers) re formed (12) ut does not determine the specific fold of the GT-strnd oligonucleotide. The role of RAPi in qudruplex formtion ppers in some wys to e similr to tht of the Oxytrich protein in tht RAP1 promotes the formtion of the sme type of qudruplex tht is fvored y Ytelo itself in the presence of K+. A possile mechnism is tht the two telomeric proteins id qudruplex formtion y cting s surfces on which the DNA strnds re rought together, effectively incresing the locl concentrtion, nd in this wy llowing qudruplex formtion to tke plce t low oligonucleotide concentrtions. This might explin why H1, sic DNA inding protein, lso promotes the formtion DNA qudruplexes y Oxytrich telomeric DNA (11). Other spects of the role of RAPi pper to differ from those of the Oxytrich protein. Under conditions in which Ytelo hs mixture of structures (in NHZ), RAP1 drives the equilirium towrd one type of DNA qudruplex, the prllel form (Fig. 3). Also, contrry to wht hs een oserved for the Oxytrich protein (11), RAP1 forms distinct complex with the DNA qudruplex structure it promotes (Fig. 2). Additionl experimentl evidence will e required to permit direct comprison of the ction of the two telomeric proteins. Biologicl Implctons. Whether DNA qudruplexes re formed in vivo is not known. However, the demonstrtion tht oth the Oxytrich nd yest telomere inding proteins promote the formtion of DNA qudruplexes in vitro rgues for the presence of such structures in telomeres (11, 13). RAP1 could ct directly on the single-strnded GT-overhngs tht yest telomeres cquire in lte S phse (26). The formtion of DNA qudruplexes is consistent with the demonstrtion tht these GT-overhngs medite telomere-telomere piring in vivo (26). Another possiility is tht the GT-strnd of the long doule-strnded regions of telomeric DNA prticiptes in DNA qudruplex formtion (13). This possiility is suggested y the finding tht RAP1 inding results in distortion of the DNA doule helix so tht it cquires some single-strnded chrcter (17). The formtion ofdna qudruplexes in vivo would provide n ttrctive mechnism for telomere-telomere ssocition vi DNA-DNA interctions (6, 7, 10). The promotion of G4-DNA y RAP1 reported in this pper could e relted to the sitution when four chromtids of two homologous chromosomes ssocite during pchytene in meiosis (10). In ddition, DNA qudruplexes inhiit the ctivity of the telomere-specific polymerse (the telomerse) (27). The involvement of protein in promoting DNA qudruplex formtion provides the mens of regulting the folding nd unfolding of DNA qudruplexes, nd hence, ny iologicl role such structures my hve. Note Added in Proof. 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