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Structural properties of Friedreich’s ataxia d(GAA) repeats
Biochimica et Biophysica Acta 1444 (1999) 14^24
Structural properties of Friedreich's ataxia d(GAA) repeats Iang-Shan Suen a , Jamie N. Rhodes b , Mellisa Christy a , Brian McEwen a , Donald M. Gray b , Michael Mitas a; * a
b
Department of Biochemistry and Molecular Biology, Oklahoma State University, 246 Noble Research Center, Stillwater, OK 74078-3035 USA University of Texas at Dallas, Department of Molecular and Cell Biology, Box 830688, Richardson, TX 75083-0688, USA Received 16 September 1998; received in revised form 23 November 1998; accepted 26 November 1998
Abstract The expansion of trinucleotide repeat sequences is the underlying cause of a growing number of inherited human disorders. To provide correlations between DNA structure and mechanisms of trinucleotide repeat expansion, we investigated potential secondary structures formed from the complementary strands of d(GAAWTTC)n , a sequence whose expansion is associated with Friedreich's ataxia. In 50 mM NaCl, pH 7.5, d(GAA)15 exhibited a cooperative and reversible decrease in large circular dichroism bands at 248 and 272^274 nm over the temperature range of 5^50³C, providing evidence for a base-paired structure at reduced temperatures. Ultraviolet absorbance melting profiles indicated that the melting temperature (Tm ) of d(GAA)15 was 40³C. At 5³C, the central portion of d(GAA)15 was hypersensitive to single-strand-specific P1 nuclease degradation and diethyl pyrocarbonate modification, providing evidence for a hairpin conformation. At temperatures between 25 and 35³C in 50 mM NaCl, the triplet repeat region of d(GAA)15 was uniformly resistant to degradation by P1 nuclease, including the central portion of the sequence. Our results indicate that the structure of d(GAA)15 is a hairpin at 5³C, unknown but partially base-paired at 37³C, and an approximately random coil above 65³C. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Hairpin structure; Triplet repeat expansion disease; Circular dichroism; Ultraviolet absorbance; Chemical modi¢cation; Electrophoretic mobility; P1 nuclease
1. Introduction Triplet repeat expansion diseases (TREDs) are a unique family of inherited human disorders [1]. They are typically characterized by expansion of d(CTGWCAG) or d(CCGWCGG) triplet repeat sequences located within speci¢c genes. Expansions * Corresponding author. Present address: Hollings Cancer Center, Room 313, 86 Jonathan Lucas St., Medical University of South Carolina, SC 29425, USA. Fax: +1 (843) 7923940; E-mail: [email protected]
generally continue in o¡spring of a¡ected individuals, resulting in progressively increased severity of the disease and/or an earlier age of onset, phenomena clinically referred to as `anticipation'. Members of the TRED family include fragile X syndrome, Huntington's disease, and Friedreich's ataxia (for a review of the genetic and clinical features of TREDs, see [1^3]). Biophysical and biochemical studies have shown that the GC-rich triplet repeat sequences (d(CTG)n , d(CGG)n , d(CAG)n , d(CCG)n , d(GTC)n , and d(GAC)n ), which undergo expansion in human genes
0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 2 6 7 - X
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(reviewed in [4]) or in yeast [5^7] and bacterial systems [8^11] form hairpin structures. Each hairpin is endowed with a unique structural feature that is dictated by mismatched bases surrounded by GC or CG base pair steps. For example, in the d(CCG)15 hairpin, the mismatched cytosines are protonated and interact with one another in an unknown manner, which results in severe distortion of the DNA helix [12]. In addition to being able to form a hairpin [13], the d(CGG)n sequence has the capability of folding into a tetraplex structure [14] which most likely contains G4 and C4 quartets in a 2:1 ratio (K. Usdin, personal communication, but see also [15,16]). The observation that a stable secondary structure is adopted by each of the complementary strands associated with TREDs has led to the conclusion that hairpin (or hairpin-like) structures form at the replication fork [17^22] (presumably at the lagging daughter strand [23,24]) and play a major role in expansion events. However, the validity of this conclusion has come into question with the recent discovery that Friedreich's ataxia (FA) is associated with expansion of d(GAAWTTC) repeat sequences [25]. FA is the only TRED in which (i) the inheritance pattern is recessive, (ii) the expanded triplet repeat is within an intron, and (iii), the expanded repeat is not GC-rich [25]. Since GAA repeats cannot form hairpins similar to those containing GC-rich sequences, some have argued that all triplet repeat sequences might undergo expansion [26], including those sequences that lack secondary structure. Others have argued that there might be two separate mechanisms of expansion [2], the ¢rst of which involves hairpin structures. The second mechanism might involve triplex structures of the YWRY type, such as those formed with the FA GAA repeats [27]. However, in contrast to the above possibilities, a more parsimonious explanation is that expansion of triplet repeat sequences requires the formation of a unimolecular structure, and the one adopted by Friedreich's ataxia d(GAA)n is unique compared to the GC-rich triplet repeats. To resolve the issue of whether Friedreich's ataxia d(GAA) repeats adopt a stable intramolecular conformation, we investigated the structure of d(GAA)15 by circular dichroism (CD), UV absorbance, electrophoretic, P1 nuclease digestion, and chemical modi¢cation techniques. We ¢nd that at or below 37³C,
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d(GAA)15 shows a clear propensity to adopt a stable unimolecular structure. 2. Materials and methods 2.1. Circular dichroism and UV absorbance The 54-mer oligomer sequence d[AACC(GAA)15 GGATC] was purchased from Midland Certi¢ed Reagent Company (Midland, TX), puri¢ed by reverse phase HPCL and provided as the ammonium salt. It was dissolved in distilled, deionized water and then diluted with 2Ubu¡er to give samples in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , and 50 mM NaCl. For samples at pH 6.5 and 8.5, the pH was adjusted with small aliquots of 1.2 M HCl and 1 M NaOH, respectively. The extinction coe¤cient at 260 nm for the 54-mer oligomer at 90³C was taken to be the average of the monomer values [28], which yielded a value of the extinction coe¤cient corrected to 20³C of 1.06U104 M31 cm31 , per mole of nucleotide. Based on this value and absorption measurements, typical concentrations of the 54-mer were 5.8^6.6U1035 M in nucleotides for the CD experiments. Absorbance readings were taken with an Olismodi¢ed Cary 14, and CD measurements were taken with a Jasco Model J710 spectropolarimeter. Acquisition of CD spectra and melting pro¢les was essentially as described in previous work [29], except that smoothing of the CD data was by the Savitzky-Golay algorithm [30] provided by Jasco. CD data are plotted as OL 3OR in units of M31 cm31 , per mole of nucleotide. 2.2. Plasmid DNA preparation Oligonucleotides used for plasmid puri¢cation were synthesized by the OSU Recombinant DNA/ Protein Resource Facility on an Applied Biosystems 381A oligonucleotide synthesizer (Foster City, CA) with the trityl group on and puri¢ed with oligonucleotide puri¢cation cartridges (Cruachem, Glasgow, UK). Sequences of oligonucleotides were AGCTTGTTAACC(GAA)15 G and GATCC(TTC)15 GGTTAACA. Five Wg of each synthetic oligonucleotide was phosphorylated with ATP and T4 polynucleotide
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kinase (Boehringer Mannheim, Indianapolis, IN) in bu¡er containing 1 mM DTT, 1 mM ATP, 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2 , 0.1 mM spermidine, 0.1 mM EDTA and 20 units of T4 polynucleotide kinase (New England Biolabs, Beverly, MA) in a ¢nal volume of 100 Wl. Complementary pairs were combined, heated to 100³C, and allowed to cool to room temperature over a period of at least 4 h and ligated into the BamHI and HindIII sites of pBluescript SK (Stratagene, La Jolla, CA) as previously described [17]. Plasmid DNA was transformed into competent XL-1Blue cells (Stratagene) and puri¢ed by cesium chloride centrifugation [31]. DNA sequence analysis performed by the OSU recombinant DNA/protein resource facility on an ABI 373 automated sequencer with the use of the M13 universal primer revealed no mutations in the insert region. The sequenced plasmid was named pGAA15. 2.3. Plasmid labeling Fifteen Wg of pGAA15 was ¢rst cleaved with HpaI. The GAA-containing strand was then labeled at the 3P-end with the use of [K-32 P]dCTP, [K-32 P]dATP (ICN, Irvine, CA) and Klenow enzyme (New England Biolabs, Beverly, MA) as previously described [32]. The double-stranded DNA fragment containing the repeat was then liberated from plasmid by cleavage with the restriction enzyme HindIII. For labeling of the TTC-containing strand, the order of enzyme additions was reversed. 2.4. Electrophoretic analysis Temperature-dependent electrophoretic analyses of the d(GAA)15 and d(TCC)15 were performed as described previously (Yu, 1995). Brie£y, to obtain a homogeneous population of labeled d(GAA)15 derived from plasmid, 1.4 pmol of unlabeled synthetic oligonucleotide of the corresponding sequence as the labeled strand (approx. 0.7 fmol) was added to the labeled strand, placed in a 90³C H2 O bath for 5 min, and placed in ice for 5 min. Labeled DNAs (4U104 dpm) were diluted to 10 Wl in bu¡er containing 8% glycerol, 10 mM HEPES, pH 7.5^8.5, and 1 mM EDTA. One Wl of loading dye (50% glycerol, 0.4% bromophenol blue) was added to the DNA samples prior to gel electrophoresis. Electrophoresis was per-
formed in a Hoe¡er (San Francisco, CA) SE600 series unit at various temperatures at 25 mA/gel in 45 mM Tris-borate (pH 8.5) and 1 mM EDTA (TBE). Gel plates were 14 cm (length)U16 cm (width)U1.5 mm (thickness). Electrophoresis was stopped when the bromophenol blue marker migrated approx. 10 cm. Dried gels were placed between two intensifying screens (Dupont) and exposed to Fuji RX ¢lm for 2^5 h. Temperature of the polyacrylamide gels was controlled as previously described [32]. 2.5. P1 nuclease digestion d(GAA)15 was labeled as described for the electrophoretic studies. The labeled oligonucleotide was separated from labeled vector by electrophoresis in a 2% agarose gel. Fragments were excised from gels and puri¢ed with glass beads (Mermaid Kit, Bio101, La Jolla, CA). Labeled oligonucleotide (4U103 dpm; 0.7 fmoles) was added to a synthetic oligonucleotide (1 WM) of the same sequence and placed in a 70³C bath for 3 min and placed on ice for 5 min. P1 nuclease digestions were performed in 50 mM TrisHCl (pH 7.5), 10 mM MgCl2 , and 50 mM NaCl at various temperatures essentially according to the method of Wohlrab [33] as previously described [32]. 2.6. Chemical modi¢cations 2.6.1. Diethylpyrocarbonate The GAA-containing strand was labeled as described above for the P1 nuclease analysis. Diethyl pyrocarbonate reactions were performed essentially according to the method of McCarthy et al. [34]. Modi¢cation reactions were performed at various times in bu¡er containing 50 mM Na cacodylate (pH 7.5), 2 mM EDTA, and 1 WM of synthetic oligonucleotide of the same sequence. The reaction was stopped by the addition of a 0.25 ml stop solution (0.3 M sodium acetate (pH 5.2), 0.1 mM EDTA, 25 Wg/ml tRNA) and 0.75 ml cold ethanol. The precipitated DNA was resuspended in 100 Wl 1 M piperidine and heated for 30 min at 92³C to generate strand breaks. After removal of piperidine in vacuo (U2), the DNA was resuspended in formamide loading bu¡er, placed in a boiling water bath for 2 min, immediately chilled on ice, and loaded on a DNA
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sequencing gel containing 20% polyacrylamide and 8 M urea. 2.6.2. Dimethyl sulfate The GAA-containing strand was labeled as described above for the P1 nuclease analysis. Dimethyl sulfate (DMS) reactions were performed at 21 mM with the DNAs essentially according to the method of Maxam and Gilbert [35]. 3. Results The CD spectrum of a synthetic 54-mer oligomer sequence d[AACC(GAA)15 GGATC] (referred to as d(GAA)15 ) in 50 mM NaCl was temperature-dependent, as shown in Fig. 1. The decrease in large CD bands at 248 and 272^274 nm provided evidence for the disruption of a base-paired structure as the tem-
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perature increased. A sample immersed in boiling water and then quick-chilled in an ice bath also had a CD spectrum at 5³C (not shown) that was essentially identical to the spectrum in Fig. 1 at 6³C. The later result provided evidence that the putative base-paired structure of d(GAA)15 was unimolecular. Further, an isodichroic point at 257 nm indicated that the CD transition was two-state. The structural change was also cooperative and reversible, as shown by the CD melting and annealing data and the absorbance melting data of Fig. 2. The percent hyperchromicity at 260 nm was 15% from 25 to 50³C, which included the cooperative portion of the transition. The Tm was 40³C ( þ 1³C range) from the derivatives of three melting pro¢les. CD data for samples at di¡erent starting pH values were very similar (Fig. 2), which showed that spectral changes were not the result of temperature-dependent changes in the Tris bu¡er pH.
Fig. 1. CD spectra as a function of temperature for d(GAA)15 . Values were obtained at pH 7.5 and 6³C (999), 15.5³C (a), 25³C (8), 34³C (b), 43.5³C (R), 53³C (O), and 62³C (- - -). See Section 2 for other information.
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Fig. 2. CD values at 270 nm and absorbance values at 260 nm as a function of temperature for d(GAA)15 . Di¡erent symbols represent data from independent experiments. CD(270) values at pH 7.5 and increasing temperatures (8,R), at pH 7.5 and decreasing temperatures (b), at pH 6.5 and increasing temperatures (a); and at pH 8.5 and increasing temperatures (O). Absorbance values at pH 7.5 and increasing temperatures (-b-b-b-).
The reversible nature of the CD measurements shown in Fig. 2 suggested that d(GAA)15 formed a unimolecular structure. To further investigate this possibility, the molecular composition of d(GAA)15 , as well as its control complementary d(TTC)15 sequence was determined by assessing its electrophoretic mobility as a function of DNA concentration. To perform this and subsequent experiments, we cloned a double-stranded (ds) oligonucleotide containing d(GAAWTTC)15 into plasmid as described in Section 2. Cleavage of plasmid DNA by the appropriate enzymes results in the liberation of an unequivocally full-length sequence consisting of d[AACC(GAA)15 GGATC] (i.e., the same sequence as the synthetic oligonucleotide used for CD and UV studies). The Watson-Crick (W-C) oligonucleotide pair d(TTCWGAA)15 was ¢rst prepared such that
the 32 P label was on the TTC-containing strand only. The oligonucleotide pair was then subjected to electrophoresis before and after heating (to 70³C), thus revealing the electrophoretic positions of d(TTCWGAA)15 and d(TTC)15 (Fig. 3A). The electrophoretic mobility of d(TTC)15 , relative to its W-C oligonucleotide pair d(TTCWGAA)15 , was 0.88, a value (referred to as Mrel ) corresponding to DNA that contains no preferred secondary structure [12,13,32,36]. Addition of a 105 -fold molar excess (¢nal DNA concentration = 7 WM) of unlabeled synthetic d(TTC)15 did not result in the formation of a slower migrating complex, indicating that d(TTC)15 did not form a stable intermolecular structure. We conclude that the electrophoretic properties of d(TTC)15 are consistent with a DNA molecule that contains no preferred secondary structure. This con-
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rich triplet repeats (Mrel values at pH 8.5, 28³C range from 1.02 for d(CCG)15 [12] to 1.17 for d(CGG)15 [13]), it is higher than that of random coil singlestranded DNA (ssDNA). These results, which are consistent with the CD and UV studies described above, indicate that d(GAA)15 forms a stable intramolecular structure at or below 28³C. We note that at 6³C (data not shown) and 28³C, d(GAA)15 migrated as a single molecular species by electrophoretic analysis. This result provides evidence that at 6 or 28³C (and low strand concentration), d(GAA)15 does not £uctuate signi¢cantly from one structural conformer to another. 3.1. d(GAA)15 is refractory to digestion by P1 nuclease
Fig. 3. Structure of d(GAA)15 is concentration-independent. A ds oligonucleotide containing the Watson-Crick (W-C) oligonucleotide pair d(GAAWTTC)15 was excised from plasmid pGAA as described in Section 2. Strands end-labeled with the use of Klenow enzyme were (A) d(TTC)15 , and (B) d(GAA)15 . Prior to gel electrophoresis, the indicated amounts of unlabeled synthetic oligonucleotides d(TTC)15 or (GAA)15 were added to labeled oligonucleotides d(TTC)15 or d(GAA)15 , respectively, incubated at 70³C for 5 min, and placed on ice for 5 min. Electrophoresis was performed at 28³C in gels containing 8% polyacrylamide.
clusion was further supported by P1 nuclease studies, which revealed substantial cleavage of all phosphodiesters in d(TTC)15 (data not shown). Electrophoretic mobility experiments were performed with labeled d(GAA)15 mixed with various amounts of unlabeled synthetic oligonucleotide of the same sequence (Fig. 3B). The results were similar to those described above (Fig. 3A), with the exception that the Mrel of d(GAA)15 was 0.94 at 28³C (Fig. 3B) or 6³C (data not shown). Although this value is lower than that of hairpins containing GC-
To further investigate the possibility that the structure of d(GAA)15 contained base pairs, it was incubated with single-strand-speci¢c P1 nuclease in 50 mM NaCl at 5^65³C, pH 7.5. At 25³C, P1 nuclease failed to cleave the phosphodiester linkages in the triplet repeat region of d(GAA)15 (Fig. 4a; with the exception of moderate cleavage in triplet XV), providing evidence for formation of a secondary structure containing base pairs. Two major sites of P1 nuclease digestion were detected 3P to the triplet repeat region, both of which involved purine dinucleotides (G50pG51 and G51pA52). The observation that P1 nuclease was capable of cleaving the purine dinucleotides G50pG51 and G51pA52 provides an internal positive control (for P1 nuclease reactivity) and rules out the possibility that lack of cleavage observed in the triplet repeat region was due to the purine-rich nature of the sequence. At 5³C (Fig. 4a) and 15³C (data not shown), P1 nuclease cleavage sites were observed in the middle of the triplet repeat region (at the G26pA27, A27pA28 and A28pG29 phosphodiesters), indicating that d(GAA)15 folded into a hairpin structure at these temperatures. The amounts of cleavages of the three phosphodiesters appeared minor compared to the 3P-end of the sequence (i.e., at G50pG51 and G51pA52), providing evidence that the nucleotides in the loop were involved in extensive stacking interactions. Another interesting but subtle di¡erence was observed in the P1 nuclease cleavage pattern at the lower temperature: in contrast to results obtained at
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Fig. 4. P1 nuclease digestion of d(GAA)15 . d(GAA)15 , the complete sequence of which is shown on the left, was isolated from plasmid pGAA15 and incubated with P1 nuclease as described in Section 2 at pH 7.5. Samples were applied to a DNA sequencing gel containing 8 M urea and 20% polyacrylamide. (A) P1 nuclease analysis in 50 mM NaCl. From left to right, the amount of P1 nuclease (and time of incubation) added in each reaction were 9U1032 U (10 min), 2.2U1032 U (5 min), 2.8U1033 U (5 min), and 2.8U1033 U (5 min). Dimethyl sulfate (21 mM) modi¢cation was performed at 25³C in 50 mM NaCl as described in Section 2. A hairpin structure that is consistent with the P1 nuclease data at 5³C is shown to the left. Arrows indicate phosphodiester bonds cleaved by P1 nuclease. Lines between bases indicate formation of presumed H-bonds. Roman numerals correspond to triplet repeats. Conventional Arabic numerals indicate the position of a nucleotide with respect to the 5P-end. (B) P1 nuclease analysis in 150 mM NaCl. Where indicated, 9U1032 U of P1 nuclease was incubated with d(GAA)15 for 10 min at 5³C. Control lane contained no P1 nuclease. Dimethyl sulfate (21 mM) modi¢cation was performed at 5³C in 150 mM NaCl. Amount of P1 nuclease = 0.09 U, 10 min.
25³C, the A49pG50 phosphodiester bond was completely protected from digestion. The signi¢cance of this result is discussed below. Cleavages of the three central phosphodiesters
were also observed at 5³C at pH 6.5 or 8.5 (data not shown). The result at the higher pH provides evidence that the putative hairpin structure of d(GAA)15 does not require protonation of the adenine residues, which is necessary for a hairpin containing Aanti W+Gsyn base pairs [37] or for a helical single-stranded structure containing alternating As and Gs [38^40]. At 5³C, 150 mM Na , pH 7.5, the sites of P1 nuclease cleavage were similar to those generated in 50 mM Na , with the exception that the cleavage at the A28pG29 phosphodiester was more pronounced (Fig. 4b). At 45 and 65³C, minor and uniform cleavage of the triplet repeat region of d(GAA)15 was observed (Fig. 4a). However, the relative amount of cleavage was reduced with respect to the phosphodiesters at the 3P-end of the sequence. These results at 45 and 65³C are consistent with a DNA structure in which (i) no loop is present, and (ii) the bases are partially stacked. Incubation of d(GAA)15 with DMS in 50 mM Na at 25³C (Fig. 4a) or 5³C (Fig. 4b) resulted in uniform modi¢cation of all guanines. This result provides evidence that the guanine N7s of the d(GAA)15 structure are not involved in H-bond formation. 3.2. The central adenines of d(GAA)15 are hypersensitive to modi¢cation by diethylpyrocarbonate at 5³C To further investigate the possibility that d(GAA)15 contained secondary structure, it was incubated with diethylpyrocarbonate (DEPC). This compound preferentially alkylates the N7 of adenine residues that are not involved in stacking interactions [41]. At 5³C, the adenines in triplet VIII were extensively modi¢ed by DEPC (Fig. 5), providing evidence that a loop was contained in the central region of d(GAA)15 (i.e., the sequence formed a hairpin). The adenines in triplet VII, as well as the adenines at nucleotide positions 6 and 9 were also highly susceptible to modi¢cation (Fig. 5). Of the remaining adenines, those that were immediately 3P to the nearest guanine appeared to be more reactive compared to those that were 5P (Fig. 5). This result provides evidence that at least some of the 5P adenines were well stacked in a DNA structure. At 15^45³C, the reactivity of the adenines in the central portion of the
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Fig. 5. Diethylpyrocarbonate modi¢cation of d(GAA)15 . d(GAA)15 , the complete sequence of which is shown on the left, was isolated from plasmid pGAA15 and incubated with 0.46 M diethylpyrocarbonate at the indicated temperatures as described in Section 2. Times of incubation were 60 min (5 and 15³C), 15 min (25 and 35³C), 4 min (45³C), and 2 min (55³C). Arrows point to adenines that were extensively modi¢ed by DEPC at 5³C. A hairpin structure that is consistent with the modi¢cation data is shown to the left.
triplet repeat decreased (Fig. 5), providing evidence that either the structure of d(GAA)15 did not contain a loop, or alternatively, that the adenines in the loop were not hypersensitive to DEPC. 4. Discussion CD and absorption data gave one cooperative transition of d(GAA)15 from an ordered state below 25³C having large CD bands to a hyperchromic state having small CD bands above 50³C. The latter state
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is probably a stacked single-stranded state since there was a further gradual increase in hyperchromicity as the temperature increased above 50³C. Our data (discussed below) suggest that the structure of the ordered (`self-complexed') form of the d(GAA)15 is a hairpin. Self-complexes with similarly large CD bands have been observed for other A- and G-containing polymers and oligomers. Lee et al. [42] reported CD spectra for the RNA polymer poly[r(GAA)] in 10 mM Tris-HCl, pH 8, 0.1 M EDTA, and 0.25 M NaCl. At 15³C, the CD spectrum of this RNA analogue, with a large positive CD band (OL 3OR W9 M31 cm31 ) at 280 nm, is similar to the spectra of d(GAA)15 at low temperatures shown in Fig. 1. Self-complexes of poly[d(GA)] and oligo[d(AG)n ] have been studied by Lee et al. [43] and Antao et al. [44]. Oligo[d(AG)n ] (10 6 n 6 30) was shown to form a self-complex with a large positive CD band (OL 3OR W8 M31 cm31 ) at 260 nm) at neutral pH and a Na concentration above 0.2 M [44]. An ordered structure of oligo[d(AG)30 ] could also be induced by lowering the pH to 4 at a lower Na concentration of 0.01 M [44]. The self-complex of poly[d(GA)] has a Tm of about 38³C over the pH range of 5^8 in a bu¡er of 10 mM sodium acetate, 0.1 mM EDTA, and the Tm of the polymer increases with increasing ionic strength [43]. At low strand concentration (6U1037 M), neutral pH, low temperature (5^15³C) and physiologic salt concentration (150 mM Na ), Fresco and colleagues observed that d(GA)10 adopted a hairpin conformation as evidenced by relative rapid electrophoretic mobility and cleavage in the central portion of the sequence by S1 nuclease [45]. Similar to the results obtained by Fresco and colleagues [45], we observed that at 5³C, pH 7.5, 50 or 150 mM NaCl, d(GAA)15 adopted a hairpin conformation. Data supporting this conclusion were P1 nuclease digestion of three phosphodiester linkages in the central portion of the triplet repeat sequence (Fig. 4), and hypersensitivity of the adenines in triplets VII and VIII to modi¢cation by DEPC (Fig. 5). Based primarily on the P1 nuclease cleavage pattern at the 3P-end of the d(GAA)15 sequence (Fig. 4), and to a lesser extent on the modi¢cation pattern by DMS (Fig. 4) and DEPC (Fig. 5), we have tentatively assigned a base-pairing arrangement of the antiparallel strands in the d(GAA)15 hairpin such that a 5P-GAA-3P re-
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Fig. 6. Potential conformers of d(GAA)15 . The schema depicts potential structures formed by the d(GAA)15 sequence as a function of temperature. The hairpin structure on the left represents the one shown in Fig. 4. The hairpin at the top portion of the ¢gure is speculative. The di¡erence in asymmetries of the two hairpins implies di¡erences in base-pairing arrangements, as discussed in the text.
peat on the 5P-side is paired to a 5P-AGA-3P repeat on the 3P side (i.e., a hairpin containing AWG and AWA pairs in a 2:1 ratio; Figs. 4 and 5). In an interesting study on a 20-mer, d(GATA)5 , Vorlickova¨ et al. [46] found that there were two types of self-complexes. One type, in a bu¡er of 10 mM sodium phosphate, 0.3 mM EDTA, pH 7, plus 0.7 M NaCl, had a CD spectrum characteristic of alternating AT base pairs, as if the G were taking the place of T. At a higher salt concentration of 5 M NaCl, another form of the 20-mer, called the `GA duplex', had a CD spectrum very much like that of d(GAA)15 shown in Fig. 1. d(GATA)5 also melted in a twostate fashion with an isodichroic point at 255.5 nm [46]. While the base pairing is not known for the GA form of the 20-mer, the pairing appears to be between two strands (from concentration dependence and gel mobility shifts) and to be of a type such that T is not involved or it takes the place of G. The d(GATA)5 could have the same base pairing as our d(GAA)15 if the T looped out of the former sequence at high salt concentrations. Results from three independent studies (CD (Figs. 1 and 2), UV absorbance (Fig. 2), and electrophoretic mobility (data not shown)) suggested a single structural transition of d(GAA)15 between the temperatures of 5 and 45³C. Our P1 nuclease data suggested that the transition at these temperatures was from a hairpin in which the G50pG51 and G51pA52 phosphodiesters were extensively cleaved (Fig. 4), to an unknown structure that retained a similar cleavage pattern at the 3P-end. However, at 25³C (Fig. 4)
or 37³C (data not shown), P1 nuclease was not able to cleave the phosphodiesters located in the central portion of the helix. Although the structure of d(GAA)15 at 25 or 37³C is unknown, a close analysis of the P1 nuclease pattern at the 3P-end perhaps provides some clues to the identity of the structure. As described above, cleavage of the A49G50 phosphodiester was observed at 25 or 37³C, but not at 5³C. Possible explanations for these somewhat paradoxical results are: (1) Although the base-pairing arrangements of the hairpins formed at 5 and 37³C are the same, the loop region is better stacked at the higher temperature. (2) At the higher (25^37³C) temperatures, a hairpin is formed in which the basepairing arrangement is di¡erent compared to 5³C; stacking interactions of the loop increase, while those at the 3P-end decrease (Fig. 6). (3) At the higher temperatures, a well-de¢ned stacked structure is formed that lacks a loop region. We favor possibility 1, since the conformational change of the loop region must be minor enough so that it is not detected as a separate transition by CD. In support of possibility 1, studies have shown that when GdAGA [47], d(GAAA) [48] or d(GNA) [49] sequences are present within the loops of hairpin structures, they are stabilized by extensive stacking interactions. In summary, the d(GAA)15 can form a self-complex that has a CD spectrum that is most like that of the self-complex of poly[r(GAA)] and the `GA form' of d(GATA)5 . It melts in a two-state fashion, so that any intermediate form that is resistant to P1 digestion likely re£ects a minor perturbation on the base
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pairing or loop structure. The type of base pairing is not known, but from the work by Vorlickova¨ et al. [47] it would appear not to involve GWA pairs of a type that could stack and coexist with AWT pairs. Acknowledgements We thank Drs. X. Gao and G. Gupta for helpful discussions, U. Melcher for critical review of this manuscript. P1 nuclease, electrophoretic mobility, and chemical modi¢cation studies were supported by the Oklahoma Center for the Advancement of Science and Technology (Project HN6-019), by the Oklahoma Agricultural Experiment Station at OSU, and from an OSU Wentz Project awarded to M.C. CD and UV absorbtion studies were supported by Grant AT-503 from the Robert A Welch Foundation and funds from Cytoclonal Pharmaceutics, Inc., of Dallas, TX. J.N.R. was a Clark Undergraduate Scholar at UT-Dallas. References [1] J.C.T. Ashley, S.T. Warren, Annu. Rev. Genet. 29 (1995) 703^728. [2] L.T. Timchenko, C.T. Caskey, FASEB J. 10 (1996) 1589^ 1597. [3] A.R. La Spada, Brain Pathol. 7 (1997) 943^963. [4] M. Mitas, Nucleic Acids Res. 25 (1997) 2245^2253. [5] J.J. Miret, L. Pessoa-Brandao, R.S. Lahue, Mol. Cell. Biol. 17 (1997) 3382^3387. [6] J.K. Schweitzer, D.M. Livingston, Hum. Mol. Genet. 7 (1998) 69^74. [7] C.H. Freudenreich, S.M. Kantrow, V.A. Zakian, Science 279 (1998) 853^856. [8] R.D. Wells, J. Biol. Chem. 271 (1996) 2875^2878. [9] G.M. Samadashwily, G. Raca, S.M. Mirkin, Nat. Genet. 17 (1997) 298^304. [10] S. Schumacher, R.P. Fuchs, M. Bichara, J. Mol. Biol. 279 (1998) 1101^1110. [11] R.D. Wells, P. Parniewski, A. Pluciennik, A. Bacolla, R. Gellibolian, A. Jaworski, J. Biol. Chem. 273 (1998) 19532^ 19541. [12] A. Yu, M.D. Barron, R.M. Romero, M. Christy, B. Gold, J. Dai, D.M. Gray, I.S. Haworth, M. Mitas, Biochemistry 36 (1997) 3687^3699. [13] M. Mitas, A. Yu, J. Dill, I.S. Haworth, Biochemistry 34 (1995) 12803^12811. [14] K. Usdin, K.J. Woodford, Nucleic Acids Res. 23 (1995) 4202^4209.
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[15] A. Kettani, R.A. Kumar, D.J. Patel, J. Mol. Biol. 254 (1995) 638^656. [16] J.M. Darlow, D.R.F. Leach, J. Mol. Biol. 275 (1998) 3^ 16. [17] M. Mitas, A. Yu, J. Dill, T.J. Kamp, E.J. Chambers, I.S. Haworth, Nucleic Acids Res. 23 (1995) 1050^1059. [18] A.M. Gacy, G. Goeliner, N. Juranic, S. Macura, C.T. McMurray, Cell 81 (1995) 533^540. [19] X. Chen, S.V.S. Mariappan, P. Catasti, R. Ratli¡, R.K. Moyzis, A. Laayoun, S.S. Smith, E.M. Bradbury, G. Gupta, Proc. Natl. Acad. Sci. USA 92 (1995) 5199^5203. [20] S.V.S. Mariappan, P. Catasti, X. Chen, R. Ratcli¡, R.K. Moyzis, E.M. Bradbury, G. Gupta, Nucleic Acids Res. 24 (1996) 784^792. [21] C.E. Pearson, R.R. Sinden, Biochemistry 35 (1996) 5041^ 5053. [22] S. Kang, K. Ohshima, A. Jaworski, R.D. Wells, J. Mol. Biol. 258 (1996) 543^547. [23] S. Kang, A. Jaworski, K. Ohshima, R.D. Wells, Nat. Genet. 10 (1995) 213^218. [24] D.A. Gordenin, T.A. Kunkel, M.A. Resnick, Nat. Genet. 16 (1997) 116^118. [25] V. Campuzano, L. Montermini, M.D. Molto©, L. Pianese, M. Cosse¨e, F. Cavalcanti, E. Monros, F. Rodius, F. Duclos, A. Monticelli, F. Zara, J. Can¬izares, H. Koutnikova, S.I. Bidichandani, C. Gellera, A. Brice, P. Troullas, G. De Michele, A. Filla, R. De Frutos, F. Palau, P.I. Patel, S. Di Donato, J.-L. Mandel, S. Cocozza, M. Koenig, M. Pandolfo, Science 271 (1996) 1423^1427. [26] S.T. Warren, Science 271 (1996) 1374^1375. [27] A.M. Gacy, G.M. Goeliner, C. Spiro, X. Chen, G. Gupta, E.M. Bradbury, R.B. Dyer, M.J. Mikesell, J.Z. Yao, A.J. Johnson, A. Richter, S.B. Melancon, C.T. McMurray, Mol. Cell 1 (1998) 583^593. [28] D.M. Gray, S.-H. Hung, K.H. Johnson, Methods Enzymol. 246 (1995) 19^34. [29] G.M. Hashem, L. Pham, M.R. Vaughan, D.M. Gray, Biochemistry 37 (1998) 61^72. [30] A. Savitzky, M.J.E. Golay, Anal. Chem. 36 (1964) 1627^ 1639. [31] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning, 2nd edn., Cold Spring Harbor Laboratory Press, Plainview, NY, 1989. [32] A. Yu, J. Dill, S.S. Wirth, G. Huang, V.H. Lee, I.S. Haworth, M. Mitas, Nucleic Acids Res. 23 (1995) 2706^2714. [33] F. Wohlrab, Methods Enzymol. 212B (1992) 294^301. [34] J.G. McCarthy, L.D. Williams, A. Rich, Biochemistry 29 (1990) 6071^6081. [35] A.M. Maxam, W. Gilbert, Methods Enzymol. 65 (1980) 499^560. [36] A. Yu, J. Dill, M. Mitas, Nucleic Acids Res. 23 (1995) 4055^ 4057. [37] G.G. Prive, U. Heinemann, S. Chandrasegaran, L.S. Kan, M.L. Kopka, R.E. Dickerson, Science 238 (1987) 498^504. [38] I. Mukerji, M.C. Shiber, T.G. Spiro, J.R. Fresco, Biochemistry 34 (1995) 14300^14303.
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[39] N.G. Dolinnaya, J.R. Fresco, Proc. Natl. Acad. Sci. USA 89 (1992) 9242^9246. [40] K. Rippe, V. Fritsch, E. Westhof, T.M. Jovin, EMBO J. 11 (1992) 3777^3786. [41] A. Krol, P. Carbon, Methods Enzymol. 180 (1989) 212^227. [42] J.S. Lee, D.H. Evans, A.R. Morgan, Nucleic Acids Res. 8 (1980) 4305^4320. [43] J.S. Lee, D.A. Johnson, A.R. Morgan, Nucleic Acids Res. 6 (1979) 3073^3091. [44] V.P. Antao, D.M. Gray, R.L. Ratli¡, Nucleic Acids Res. 16 (1988) 719^738.
[45] M.C. Shiber, E.H. Braswell, H. Klump, J.R. Fresco, Nucleic Acids Res. 24 (1996) 5004^5012. [46] M. Vorlickova, I. Kejnovska, J. Kovanda, J. Kypr, Nucleic Acids Res. 26 (1998) 1509^1514. [47] M. Orita, F. Nishikawa, T. Kohno, T. Senda, Y. Mitsui, Y. Endo, K. Taira, S. Nishikawa, Nucleic Acids Res. 24 (1996) 611^618. [48] H.A. Heus, A. Pardi, Science 253 (1991) 191^194. [49] S. Yoshizawa, G. Kawai, K. Watanabe, K. Miura, I. Hirao, Biochemistry 36 (1997) 4761^4767.
BBAEXP 93233 20-1-99
Structural properties of Friedreich's ataxia d(GAA) repeats Iang-Shan Suen a , Jamie N. Rhodes b , Mellisa Christy a , Brian McEwen a , Donald M. Gray b , Michael Mitas a; * a
b
Department of Biochemistry and Molecular Biology, Oklahoma State University, 246 Noble Research Center, Stillwater, OK 74078-3035 USA University of Texas at Dallas, Department of Molecular and Cell Biology, Box 830688, Richardson, TX 75083-0688, USA Received 16 September 1998; received in revised form 23 November 1998; accepted 26 November 1998
Abstract The expansion of trinucleotide repeat sequences is the underlying cause of a growing number of inherited human disorders. To provide correlations between DNA structure and mechanisms of trinucleotide repeat expansion, we investigated potential secondary structures formed from the complementary strands of d(GAAWTTC)n , a sequence whose expansion is associated with Friedreich's ataxia. In 50 mM NaCl, pH 7.5, d(GAA)15 exhibited a cooperative and reversible decrease in large circular dichroism bands at 248 and 272^274 nm over the temperature range of 5^50³C, providing evidence for a base-paired structure at reduced temperatures. Ultraviolet absorbance melting profiles indicated that the melting temperature (Tm ) of d(GAA)15 was 40³C. At 5³C, the central portion of d(GAA)15 was hypersensitive to single-strand-specific P1 nuclease degradation and diethyl pyrocarbonate modification, providing evidence for a hairpin conformation. At temperatures between 25 and 35³C in 50 mM NaCl, the triplet repeat region of d(GAA)15 was uniformly resistant to degradation by P1 nuclease, including the central portion of the sequence. Our results indicate that the structure of d(GAA)15 is a hairpin at 5³C, unknown but partially base-paired at 37³C, and an approximately random coil above 65³C. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Hairpin structure; Triplet repeat expansion disease; Circular dichroism; Ultraviolet absorbance; Chemical modi¢cation; Electrophoretic mobility; P1 nuclease
1. Introduction Triplet repeat expansion diseases (TREDs) are a unique family of inherited human disorders [1]. They are typically characterized by expansion of d(CTGWCAG) or d(CCGWCGG) triplet repeat sequences located within speci¢c genes. Expansions * Corresponding author. Present address: Hollings Cancer Center, Room 313, 86 Jonathan Lucas St., Medical University of South Carolina, SC 29425, USA. Fax: +1 (843) 7923940; E-mail: [email protected]
generally continue in o¡spring of a¡ected individuals, resulting in progressively increased severity of the disease and/or an earlier age of onset, phenomena clinically referred to as `anticipation'. Members of the TRED family include fragile X syndrome, Huntington's disease, and Friedreich's ataxia (for a review of the genetic and clinical features of TREDs, see [1^3]). Biophysical and biochemical studies have shown that the GC-rich triplet repeat sequences (d(CTG)n , d(CGG)n , d(CAG)n , d(CCG)n , d(GTC)n , and d(GAC)n ), which undergo expansion in human genes
0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 2 6 7 - X
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(reviewed in [4]) or in yeast [5^7] and bacterial systems [8^11] form hairpin structures. Each hairpin is endowed with a unique structural feature that is dictated by mismatched bases surrounded by GC or CG base pair steps. For example, in the d(CCG)15 hairpin, the mismatched cytosines are protonated and interact with one another in an unknown manner, which results in severe distortion of the DNA helix [12]. In addition to being able to form a hairpin [13], the d(CGG)n sequence has the capability of folding into a tetraplex structure [14] which most likely contains G4 and C4 quartets in a 2:1 ratio (K. Usdin, personal communication, but see also [15,16]). The observation that a stable secondary structure is adopted by each of the complementary strands associated with TREDs has led to the conclusion that hairpin (or hairpin-like) structures form at the replication fork [17^22] (presumably at the lagging daughter strand [23,24]) and play a major role in expansion events. However, the validity of this conclusion has come into question with the recent discovery that Friedreich's ataxia (FA) is associated with expansion of d(GAAWTTC) repeat sequences [25]. FA is the only TRED in which (i) the inheritance pattern is recessive, (ii) the expanded triplet repeat is within an intron, and (iii), the expanded repeat is not GC-rich [25]. Since GAA repeats cannot form hairpins similar to those containing GC-rich sequences, some have argued that all triplet repeat sequences might undergo expansion [26], including those sequences that lack secondary structure. Others have argued that there might be two separate mechanisms of expansion [2], the ¢rst of which involves hairpin structures. The second mechanism might involve triplex structures of the YWRY type, such as those formed with the FA GAA repeats [27]. However, in contrast to the above possibilities, a more parsimonious explanation is that expansion of triplet repeat sequences requires the formation of a unimolecular structure, and the one adopted by Friedreich's ataxia d(GAA)n is unique compared to the GC-rich triplet repeats. To resolve the issue of whether Friedreich's ataxia d(GAA) repeats adopt a stable intramolecular conformation, we investigated the structure of d(GAA)15 by circular dichroism (CD), UV absorbance, electrophoretic, P1 nuclease digestion, and chemical modi¢cation techniques. We ¢nd that at or below 37³C,
15
d(GAA)15 shows a clear propensity to adopt a stable unimolecular structure. 2. Materials and methods 2.1. Circular dichroism and UV absorbance The 54-mer oligomer sequence d[AACC(GAA)15 GGATC] was purchased from Midland Certi¢ed Reagent Company (Midland, TX), puri¢ed by reverse phase HPCL and provided as the ammonium salt. It was dissolved in distilled, deionized water and then diluted with 2Ubu¡er to give samples in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , and 50 mM NaCl. For samples at pH 6.5 and 8.5, the pH was adjusted with small aliquots of 1.2 M HCl and 1 M NaOH, respectively. The extinction coe¤cient at 260 nm for the 54-mer oligomer at 90³C was taken to be the average of the monomer values [28], which yielded a value of the extinction coe¤cient corrected to 20³C of 1.06U104 M31 cm31 , per mole of nucleotide. Based on this value and absorption measurements, typical concentrations of the 54-mer were 5.8^6.6U1035 M in nucleotides for the CD experiments. Absorbance readings were taken with an Olismodi¢ed Cary 14, and CD measurements were taken with a Jasco Model J710 spectropolarimeter. Acquisition of CD spectra and melting pro¢les was essentially as described in previous work [29], except that smoothing of the CD data was by the Savitzky-Golay algorithm [30] provided by Jasco. CD data are plotted as OL 3OR in units of M31 cm31 , per mole of nucleotide. 2.2. Plasmid DNA preparation Oligonucleotides used for plasmid puri¢cation were synthesized by the OSU Recombinant DNA/ Protein Resource Facility on an Applied Biosystems 381A oligonucleotide synthesizer (Foster City, CA) with the trityl group on and puri¢ed with oligonucleotide puri¢cation cartridges (Cruachem, Glasgow, UK). Sequences of oligonucleotides were AGCTTGTTAACC(GAA)15 G and GATCC(TTC)15 GGTTAACA. Five Wg of each synthetic oligonucleotide was phosphorylated with ATP and T4 polynucleotide
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kinase (Boehringer Mannheim, Indianapolis, IN) in bu¡er containing 1 mM DTT, 1 mM ATP, 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2 , 0.1 mM spermidine, 0.1 mM EDTA and 20 units of T4 polynucleotide kinase (New England Biolabs, Beverly, MA) in a ¢nal volume of 100 Wl. Complementary pairs were combined, heated to 100³C, and allowed to cool to room temperature over a period of at least 4 h and ligated into the BamHI and HindIII sites of pBluescript SK (Stratagene, La Jolla, CA) as previously described [17]. Plasmid DNA was transformed into competent XL-1Blue cells (Stratagene) and puri¢ed by cesium chloride centrifugation [31]. DNA sequence analysis performed by the OSU recombinant DNA/protein resource facility on an ABI 373 automated sequencer with the use of the M13 universal primer revealed no mutations in the insert region. The sequenced plasmid was named pGAA15. 2.3. Plasmid labeling Fifteen Wg of pGAA15 was ¢rst cleaved with HpaI. The GAA-containing strand was then labeled at the 3P-end with the use of [K-32 P]dCTP, [K-32 P]dATP (ICN, Irvine, CA) and Klenow enzyme (New England Biolabs, Beverly, MA) as previously described [32]. The double-stranded DNA fragment containing the repeat was then liberated from plasmid by cleavage with the restriction enzyme HindIII. For labeling of the TTC-containing strand, the order of enzyme additions was reversed. 2.4. Electrophoretic analysis Temperature-dependent electrophoretic analyses of the d(GAA)15 and d(TCC)15 were performed as described previously (Yu, 1995). Brie£y, to obtain a homogeneous population of labeled d(GAA)15 derived from plasmid, 1.4 pmol of unlabeled synthetic oligonucleotide of the corresponding sequence as the labeled strand (approx. 0.7 fmol) was added to the labeled strand, placed in a 90³C H2 O bath for 5 min, and placed in ice for 5 min. Labeled DNAs (4U104 dpm) were diluted to 10 Wl in bu¡er containing 8% glycerol, 10 mM HEPES, pH 7.5^8.5, and 1 mM EDTA. One Wl of loading dye (50% glycerol, 0.4% bromophenol blue) was added to the DNA samples prior to gel electrophoresis. Electrophoresis was per-
formed in a Hoe¡er (San Francisco, CA) SE600 series unit at various temperatures at 25 mA/gel in 45 mM Tris-borate (pH 8.5) and 1 mM EDTA (TBE). Gel plates were 14 cm (length)U16 cm (width)U1.5 mm (thickness). Electrophoresis was stopped when the bromophenol blue marker migrated approx. 10 cm. Dried gels were placed between two intensifying screens (Dupont) and exposed to Fuji RX ¢lm for 2^5 h. Temperature of the polyacrylamide gels was controlled as previously described [32]. 2.5. P1 nuclease digestion d(GAA)15 was labeled as described for the electrophoretic studies. The labeled oligonucleotide was separated from labeled vector by electrophoresis in a 2% agarose gel. Fragments were excised from gels and puri¢ed with glass beads (Mermaid Kit, Bio101, La Jolla, CA). Labeled oligonucleotide (4U103 dpm; 0.7 fmoles) was added to a synthetic oligonucleotide (1 WM) of the same sequence and placed in a 70³C bath for 3 min and placed on ice for 5 min. P1 nuclease digestions were performed in 50 mM TrisHCl (pH 7.5), 10 mM MgCl2 , and 50 mM NaCl at various temperatures essentially according to the method of Wohlrab [33] as previously described [32]. 2.6. Chemical modi¢cations 2.6.1. Diethylpyrocarbonate The GAA-containing strand was labeled as described above for the P1 nuclease analysis. Diethyl pyrocarbonate reactions were performed essentially according to the method of McCarthy et al. [34]. Modi¢cation reactions were performed at various times in bu¡er containing 50 mM Na cacodylate (pH 7.5), 2 mM EDTA, and 1 WM of synthetic oligonucleotide of the same sequence. The reaction was stopped by the addition of a 0.25 ml stop solution (0.3 M sodium acetate (pH 5.2), 0.1 mM EDTA, 25 Wg/ml tRNA) and 0.75 ml cold ethanol. The precipitated DNA was resuspended in 100 Wl 1 M piperidine and heated for 30 min at 92³C to generate strand breaks. After removal of piperidine in vacuo (U2), the DNA was resuspended in formamide loading bu¡er, placed in a boiling water bath for 2 min, immediately chilled on ice, and loaded on a DNA
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sequencing gel containing 20% polyacrylamide and 8 M urea. 2.6.2. Dimethyl sulfate The GAA-containing strand was labeled as described above for the P1 nuclease analysis. Dimethyl sulfate (DMS) reactions were performed at 21 mM with the DNAs essentially according to the method of Maxam and Gilbert [35]. 3. Results The CD spectrum of a synthetic 54-mer oligomer sequence d[AACC(GAA)15 GGATC] (referred to as d(GAA)15 ) in 50 mM NaCl was temperature-dependent, as shown in Fig. 1. The decrease in large CD bands at 248 and 272^274 nm provided evidence for the disruption of a base-paired structure as the tem-
17
perature increased. A sample immersed in boiling water and then quick-chilled in an ice bath also had a CD spectrum at 5³C (not shown) that was essentially identical to the spectrum in Fig. 1 at 6³C. The later result provided evidence that the putative base-paired structure of d(GAA)15 was unimolecular. Further, an isodichroic point at 257 nm indicated that the CD transition was two-state. The structural change was also cooperative and reversible, as shown by the CD melting and annealing data and the absorbance melting data of Fig. 2. The percent hyperchromicity at 260 nm was 15% from 25 to 50³C, which included the cooperative portion of the transition. The Tm was 40³C ( þ 1³C range) from the derivatives of three melting pro¢les. CD data for samples at di¡erent starting pH values were very similar (Fig. 2), which showed that spectral changes were not the result of temperature-dependent changes in the Tris bu¡er pH.
Fig. 1. CD spectra as a function of temperature for d(GAA)15 . Values were obtained at pH 7.5 and 6³C (999), 15.5³C (a), 25³C (8), 34³C (b), 43.5³C (R), 53³C (O), and 62³C (- - -). See Section 2 for other information.
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Fig. 2. CD values at 270 nm and absorbance values at 260 nm as a function of temperature for d(GAA)15 . Di¡erent symbols represent data from independent experiments. CD(270) values at pH 7.5 and increasing temperatures (8,R), at pH 7.5 and decreasing temperatures (b), at pH 6.5 and increasing temperatures (a); and at pH 8.5 and increasing temperatures (O). Absorbance values at pH 7.5 and increasing temperatures (-b-b-b-).
The reversible nature of the CD measurements shown in Fig. 2 suggested that d(GAA)15 formed a unimolecular structure. To further investigate this possibility, the molecular composition of d(GAA)15 , as well as its control complementary d(TTC)15 sequence was determined by assessing its electrophoretic mobility as a function of DNA concentration. To perform this and subsequent experiments, we cloned a double-stranded (ds) oligonucleotide containing d(GAAWTTC)15 into plasmid as described in Section 2. Cleavage of plasmid DNA by the appropriate enzymes results in the liberation of an unequivocally full-length sequence consisting of d[AACC(GAA)15 GGATC] (i.e., the same sequence as the synthetic oligonucleotide used for CD and UV studies). The Watson-Crick (W-C) oligonucleotide pair d(TTCWGAA)15 was ¢rst prepared such that
the 32 P label was on the TTC-containing strand only. The oligonucleotide pair was then subjected to electrophoresis before and after heating (to 70³C), thus revealing the electrophoretic positions of d(TTCWGAA)15 and d(TTC)15 (Fig. 3A). The electrophoretic mobility of d(TTC)15 , relative to its W-C oligonucleotide pair d(TTCWGAA)15 , was 0.88, a value (referred to as Mrel ) corresponding to DNA that contains no preferred secondary structure [12,13,32,36]. Addition of a 105 -fold molar excess (¢nal DNA concentration = 7 WM) of unlabeled synthetic d(TTC)15 did not result in the formation of a slower migrating complex, indicating that d(TTC)15 did not form a stable intermolecular structure. We conclude that the electrophoretic properties of d(TTC)15 are consistent with a DNA molecule that contains no preferred secondary structure. This con-
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rich triplet repeats (Mrel values at pH 8.5, 28³C range from 1.02 for d(CCG)15 [12] to 1.17 for d(CGG)15 [13]), it is higher than that of random coil singlestranded DNA (ssDNA). These results, which are consistent with the CD and UV studies described above, indicate that d(GAA)15 forms a stable intramolecular structure at or below 28³C. We note that at 6³C (data not shown) and 28³C, d(GAA)15 migrated as a single molecular species by electrophoretic analysis. This result provides evidence that at 6 or 28³C (and low strand concentration), d(GAA)15 does not £uctuate signi¢cantly from one structural conformer to another. 3.1. d(GAA)15 is refractory to digestion by P1 nuclease
Fig. 3. Structure of d(GAA)15 is concentration-independent. A ds oligonucleotide containing the Watson-Crick (W-C) oligonucleotide pair d(GAAWTTC)15 was excised from plasmid pGAA as described in Section 2. Strands end-labeled with the use of Klenow enzyme were (A) d(TTC)15 , and (B) d(GAA)15 . Prior to gel electrophoresis, the indicated amounts of unlabeled synthetic oligonucleotides d(TTC)15 or (GAA)15 were added to labeled oligonucleotides d(TTC)15 or d(GAA)15 , respectively, incubated at 70³C for 5 min, and placed on ice for 5 min. Electrophoresis was performed at 28³C in gels containing 8% polyacrylamide.
clusion was further supported by P1 nuclease studies, which revealed substantial cleavage of all phosphodiesters in d(TTC)15 (data not shown). Electrophoretic mobility experiments were performed with labeled d(GAA)15 mixed with various amounts of unlabeled synthetic oligonucleotide of the same sequence (Fig. 3B). The results were similar to those described above (Fig. 3A), with the exception that the Mrel of d(GAA)15 was 0.94 at 28³C (Fig. 3B) or 6³C (data not shown). Although this value is lower than that of hairpins containing GC-
To further investigate the possibility that the structure of d(GAA)15 contained base pairs, it was incubated with single-strand-speci¢c P1 nuclease in 50 mM NaCl at 5^65³C, pH 7.5. At 25³C, P1 nuclease failed to cleave the phosphodiester linkages in the triplet repeat region of d(GAA)15 (Fig. 4a; with the exception of moderate cleavage in triplet XV), providing evidence for formation of a secondary structure containing base pairs. Two major sites of P1 nuclease digestion were detected 3P to the triplet repeat region, both of which involved purine dinucleotides (G50pG51 and G51pA52). The observation that P1 nuclease was capable of cleaving the purine dinucleotides G50pG51 and G51pA52 provides an internal positive control (for P1 nuclease reactivity) and rules out the possibility that lack of cleavage observed in the triplet repeat region was due to the purine-rich nature of the sequence. At 5³C (Fig. 4a) and 15³C (data not shown), P1 nuclease cleavage sites were observed in the middle of the triplet repeat region (at the G26pA27, A27pA28 and A28pG29 phosphodiesters), indicating that d(GAA)15 folded into a hairpin structure at these temperatures. The amounts of cleavages of the three phosphodiesters appeared minor compared to the 3P-end of the sequence (i.e., at G50pG51 and G51pA52), providing evidence that the nucleotides in the loop were involved in extensive stacking interactions. Another interesting but subtle di¡erence was observed in the P1 nuclease cleavage pattern at the lower temperature: in contrast to results obtained at
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Fig. 4. P1 nuclease digestion of d(GAA)15 . d(GAA)15 , the complete sequence of which is shown on the left, was isolated from plasmid pGAA15 and incubated with P1 nuclease as described in Section 2 at pH 7.5. Samples were applied to a DNA sequencing gel containing 8 M urea and 20% polyacrylamide. (A) P1 nuclease analysis in 50 mM NaCl. From left to right, the amount of P1 nuclease (and time of incubation) added in each reaction were 9U1032 U (10 min), 2.2U1032 U (5 min), 2.8U1033 U (5 min), and 2.8U1033 U (5 min). Dimethyl sulfate (21 mM) modi¢cation was performed at 25³C in 50 mM NaCl as described in Section 2. A hairpin structure that is consistent with the P1 nuclease data at 5³C is shown to the left. Arrows indicate phosphodiester bonds cleaved by P1 nuclease. Lines between bases indicate formation of presumed H-bonds. Roman numerals correspond to triplet repeats. Conventional Arabic numerals indicate the position of a nucleotide with respect to the 5P-end. (B) P1 nuclease analysis in 150 mM NaCl. Where indicated, 9U1032 U of P1 nuclease was incubated with d(GAA)15 for 10 min at 5³C. Control lane contained no P1 nuclease. Dimethyl sulfate (21 mM) modi¢cation was performed at 5³C in 150 mM NaCl. Amount of P1 nuclease = 0.09 U, 10 min.
25³C, the A49pG50 phosphodiester bond was completely protected from digestion. The signi¢cance of this result is discussed below. Cleavages of the three central phosphodiesters
were also observed at 5³C at pH 6.5 or 8.5 (data not shown). The result at the higher pH provides evidence that the putative hairpin structure of d(GAA)15 does not require protonation of the adenine residues, which is necessary for a hairpin containing Aanti W+Gsyn base pairs [37] or for a helical single-stranded structure containing alternating As and Gs [38^40]. At 5³C, 150 mM Na , pH 7.5, the sites of P1 nuclease cleavage were similar to those generated in 50 mM Na , with the exception that the cleavage at the A28pG29 phosphodiester was more pronounced (Fig. 4b). At 45 and 65³C, minor and uniform cleavage of the triplet repeat region of d(GAA)15 was observed (Fig. 4a). However, the relative amount of cleavage was reduced with respect to the phosphodiesters at the 3P-end of the sequence. These results at 45 and 65³C are consistent with a DNA structure in which (i) no loop is present, and (ii) the bases are partially stacked. Incubation of d(GAA)15 with DMS in 50 mM Na at 25³C (Fig. 4a) or 5³C (Fig. 4b) resulted in uniform modi¢cation of all guanines. This result provides evidence that the guanine N7s of the d(GAA)15 structure are not involved in H-bond formation. 3.2. The central adenines of d(GAA)15 are hypersensitive to modi¢cation by diethylpyrocarbonate at 5³C To further investigate the possibility that d(GAA)15 contained secondary structure, it was incubated with diethylpyrocarbonate (DEPC). This compound preferentially alkylates the N7 of adenine residues that are not involved in stacking interactions [41]. At 5³C, the adenines in triplet VIII were extensively modi¢ed by DEPC (Fig. 5), providing evidence that a loop was contained in the central region of d(GAA)15 (i.e., the sequence formed a hairpin). The adenines in triplet VII, as well as the adenines at nucleotide positions 6 and 9 were also highly susceptible to modi¢cation (Fig. 5). Of the remaining adenines, those that were immediately 3P to the nearest guanine appeared to be more reactive compared to those that were 5P (Fig. 5). This result provides evidence that at least some of the 5P adenines were well stacked in a DNA structure. At 15^45³C, the reactivity of the adenines in the central portion of the
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Fig. 5. Diethylpyrocarbonate modi¢cation of d(GAA)15 . d(GAA)15 , the complete sequence of which is shown on the left, was isolated from plasmid pGAA15 and incubated with 0.46 M diethylpyrocarbonate at the indicated temperatures as described in Section 2. Times of incubation were 60 min (5 and 15³C), 15 min (25 and 35³C), 4 min (45³C), and 2 min (55³C). Arrows point to adenines that were extensively modi¢ed by DEPC at 5³C. A hairpin structure that is consistent with the modi¢cation data is shown to the left.
triplet repeat decreased (Fig. 5), providing evidence that either the structure of d(GAA)15 did not contain a loop, or alternatively, that the adenines in the loop were not hypersensitive to DEPC. 4. Discussion CD and absorption data gave one cooperative transition of d(GAA)15 from an ordered state below 25³C having large CD bands to a hyperchromic state having small CD bands above 50³C. The latter state
21
is probably a stacked single-stranded state since there was a further gradual increase in hyperchromicity as the temperature increased above 50³C. Our data (discussed below) suggest that the structure of the ordered (`self-complexed') form of the d(GAA)15 is a hairpin. Self-complexes with similarly large CD bands have been observed for other A- and G-containing polymers and oligomers. Lee et al. [42] reported CD spectra for the RNA polymer poly[r(GAA)] in 10 mM Tris-HCl, pH 8, 0.1 M EDTA, and 0.25 M NaCl. At 15³C, the CD spectrum of this RNA analogue, with a large positive CD band (OL 3OR W9 M31 cm31 ) at 280 nm, is similar to the spectra of d(GAA)15 at low temperatures shown in Fig. 1. Self-complexes of poly[d(GA)] and oligo[d(AG)n ] have been studied by Lee et al. [43] and Antao et al. [44]. Oligo[d(AG)n ] (10 6 n 6 30) was shown to form a self-complex with a large positive CD band (OL 3OR W8 M31 cm31 ) at 260 nm) at neutral pH and a Na concentration above 0.2 M [44]. An ordered structure of oligo[d(AG)30 ] could also be induced by lowering the pH to 4 at a lower Na concentration of 0.01 M [44]. The self-complex of poly[d(GA)] has a Tm of about 38³C over the pH range of 5^8 in a bu¡er of 10 mM sodium acetate, 0.1 mM EDTA, and the Tm of the polymer increases with increasing ionic strength [43]. At low strand concentration (6U1037 M), neutral pH, low temperature (5^15³C) and physiologic salt concentration (150 mM Na ), Fresco and colleagues observed that d(GA)10 adopted a hairpin conformation as evidenced by relative rapid electrophoretic mobility and cleavage in the central portion of the sequence by S1 nuclease [45]. Similar to the results obtained by Fresco and colleagues [45], we observed that at 5³C, pH 7.5, 50 or 150 mM NaCl, d(GAA)15 adopted a hairpin conformation. Data supporting this conclusion were P1 nuclease digestion of three phosphodiester linkages in the central portion of the triplet repeat sequence (Fig. 4), and hypersensitivity of the adenines in triplets VII and VIII to modi¢cation by DEPC (Fig. 5). Based primarily on the P1 nuclease cleavage pattern at the 3P-end of the d(GAA)15 sequence (Fig. 4), and to a lesser extent on the modi¢cation pattern by DMS (Fig. 4) and DEPC (Fig. 5), we have tentatively assigned a base-pairing arrangement of the antiparallel strands in the d(GAA)15 hairpin such that a 5P-GAA-3P re-
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Fig. 6. Potential conformers of d(GAA)15 . The schema depicts potential structures formed by the d(GAA)15 sequence as a function of temperature. The hairpin structure on the left represents the one shown in Fig. 4. The hairpin at the top portion of the ¢gure is speculative. The di¡erence in asymmetries of the two hairpins implies di¡erences in base-pairing arrangements, as discussed in the text.
peat on the 5P-side is paired to a 5P-AGA-3P repeat on the 3P side (i.e., a hairpin containing AWG and AWA pairs in a 2:1 ratio; Figs. 4 and 5). In an interesting study on a 20-mer, d(GATA)5 , Vorlickova¨ et al. [46] found that there were two types of self-complexes. One type, in a bu¡er of 10 mM sodium phosphate, 0.3 mM EDTA, pH 7, plus 0.7 M NaCl, had a CD spectrum characteristic of alternating AT base pairs, as if the G were taking the place of T. At a higher salt concentration of 5 M NaCl, another form of the 20-mer, called the `GA duplex', had a CD spectrum very much like that of d(GAA)15 shown in Fig. 1. d(GATA)5 also melted in a twostate fashion with an isodichroic point at 255.5 nm [46]. While the base pairing is not known for the GA form of the 20-mer, the pairing appears to be between two strands (from concentration dependence and gel mobility shifts) and to be of a type such that T is not involved or it takes the place of G. The d(GATA)5 could have the same base pairing as our d(GAA)15 if the T looped out of the former sequence at high salt concentrations. Results from three independent studies (CD (Figs. 1 and 2), UV absorbance (Fig. 2), and electrophoretic mobility (data not shown)) suggested a single structural transition of d(GAA)15 between the temperatures of 5 and 45³C. Our P1 nuclease data suggested that the transition at these temperatures was from a hairpin in which the G50pG51 and G51pA52 phosphodiesters were extensively cleaved (Fig. 4), to an unknown structure that retained a similar cleavage pattern at the 3P-end. However, at 25³C (Fig. 4)
or 37³C (data not shown), P1 nuclease was not able to cleave the phosphodiesters located in the central portion of the helix. Although the structure of d(GAA)15 at 25 or 37³C is unknown, a close analysis of the P1 nuclease pattern at the 3P-end perhaps provides some clues to the identity of the structure. As described above, cleavage of the A49G50 phosphodiester was observed at 25 or 37³C, but not at 5³C. Possible explanations for these somewhat paradoxical results are: (1) Although the base-pairing arrangements of the hairpins formed at 5 and 37³C are the same, the loop region is better stacked at the higher temperature. (2) At the higher (25^37³C) temperatures, a hairpin is formed in which the basepairing arrangement is di¡erent compared to 5³C; stacking interactions of the loop increase, while those at the 3P-end decrease (Fig. 6). (3) At the higher temperatures, a well-de¢ned stacked structure is formed that lacks a loop region. We favor possibility 1, since the conformational change of the loop region must be minor enough so that it is not detected as a separate transition by CD. In support of possibility 1, studies have shown that when GdAGA [47], d(GAAA) [48] or d(GNA) [49] sequences are present within the loops of hairpin structures, they are stabilized by extensive stacking interactions. In summary, the d(GAA)15 can form a self-complex that has a CD spectrum that is most like that of the self-complex of poly[r(GAA)] and the `GA form' of d(GATA)5 . It melts in a two-state fashion, so that any intermediate form that is resistant to P1 digestion likely re£ects a minor perturbation on the base
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pairing or loop structure. The type of base pairing is not known, but from the work by Vorlickova¨ et al. [47] it would appear not to involve GWA pairs of a type that could stack and coexist with AWT pairs. Acknowledgements We thank Drs. X. Gao and G. Gupta for helpful discussions, U. Melcher for critical review of this manuscript. P1 nuclease, electrophoretic mobility, and chemical modi¢cation studies were supported by the Oklahoma Center for the Advancement of Science and Technology (Project HN6-019), by the Oklahoma Agricultural Experiment Station at OSU, and from an OSU Wentz Project awarded to M.C. CD and UV absorbtion studies were supported by Grant AT-503 from the Robert A Welch Foundation and funds from Cytoclonal Pharmaceutics, Inc., of Dallas, TX. J.N.R. was a Clark Undergraduate Scholar at UT-Dallas. References [1] J.C.T. Ashley, S.T. Warren, Annu. Rev. Genet. 29 (1995) 703^728. [2] L.T. Timchenko, C.T. Caskey, FASEB J. 10 (1996) 1589^ 1597. [3] A.R. La Spada, Brain Pathol. 7 (1997) 943^963. [4] M. Mitas, Nucleic Acids Res. 25 (1997) 2245^2253. [5] J.J. Miret, L. Pessoa-Brandao, R.S. Lahue, Mol. Cell. Biol. 17 (1997) 3382^3387. [6] J.K. Schweitzer, D.M. Livingston, Hum. Mol. Genet. 7 (1998) 69^74. [7] C.H. Freudenreich, S.M. Kantrow, V.A. Zakian, Science 279 (1998) 853^856. [8] R.D. Wells, J. Biol. Chem. 271 (1996) 2875^2878. [9] G.M. Samadashwily, G. Raca, S.M. Mirkin, Nat. Genet. 17 (1997) 298^304. [10] S. Schumacher, R.P. Fuchs, M. Bichara, J. Mol. Biol. 279 (1998) 1101^1110. [11] R.D. Wells, P. Parniewski, A. Pluciennik, A. Bacolla, R. Gellibolian, A. Jaworski, J. Biol. Chem. 273 (1998) 19532^ 19541. [12] A. Yu, M.D. Barron, R.M. Romero, M. Christy, B. Gold, J. Dai, D.M. Gray, I.S. Haworth, M. Mitas, Biochemistry 36 (1997) 3687^3699. [13] M. Mitas, A. Yu, J. Dill, I.S. Haworth, Biochemistry 34 (1995) 12803^12811. [14] K. Usdin, K.J. Woodford, Nucleic Acids Res. 23 (1995) 4202^4209.
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