©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Fragile X Syndrome Single Strand d(CGG) Nucleotide Repeats Readily Fold Back to Form Unimolecular Hairpin Structures (*)

(Received for publication, July 5, 1995; and in revised form, August 30, 1995)

Yotvat Nadel Pnina Weisman-Shomer Michael Fry (§)

From the Unit of Biochemistry, Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, P. O. Box 9649, Haifa 31096, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Expansion of a d(CGG) run within the 5`-untranslated region of the X-linked human gene FMR1 blocks FMR1 transcription, delays its replication, and precipitates the fragile X syndrome. We showed previously that d(CGG) tracts aggregate into interstrand tetrahelical complexes (Fry, M., and Loeb, L. A.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4950-4954). Here we show that these sequences also form under physiological conditions in in vitro unimolecular hairpin structures. Folding is demonstrated by temperature-dependent mobility of d(CGG) oligomers in a nondenaturing polyacrylamide gel, by UV-hyperchromicity of thermally denaturing oligomers, and by UV cross-linking of compact forms of d(CGG) runs interspersed by thymidine clusters. That the compact d(CGG) structures are unimolecular is suggested by their zero-order kinetics of formation. Diethyl pyrocarbonate modification reveals a single, 4-5 residue-long central or epicentral unpaired loop in folded d(CGG) oligomers. The position of this loop remains unchanged by insertion of thymidine clusters into 15- or 33-mer d(CGG) tracts as indicated by KMnO(4) probing of unpaired thymidines. The presence of a single loop in folded d(CGG) oligomers and the accessibility of every guanine to dimethyl sulfate modification suggest that they are hairpin and not tetraplex structures. Modeling indicates that different d(CGG)hairpins are stabilized by guanine-guanine Hoogsteen hydrogen bonds or by Hoogsteen and Watson-Crick bonds. If formed in vivo, d(CGG) hairpins could entail slippage and trinucleotide expansion during replication and could obstruct FMR1 gene transcription and replication.


INTRODUCTION

Fragile X syndrome is an inherited, X-linked dominant mental retardation disorder affecting about one male in 1500 and one female in 2500 (Webb, 1989; Richards and Sutherland, 1992). This syndrome is frequently associated with a folate-sensitive fragile site, Xq27.3, on chromosome X of cells of affected males (Sutherland, 1977). The fragile X syndrome is characterized by a substantial expansion of a d(CGG) trinucleotide repeat located in the 5`-untranslated region of a housekeeping gene, FMR1, which was identified at a locus coincident with the Xq27.3 breakpoint (Verkek et al., 1991). Whereas normal individuals have 2-50 copies of the d(CGG) sequence, the trinucleotide is amplified in affected subjects to >200-2000 copies (Fu et al., 1991; Kremer et al., 1991; Oberléet al., 1991; Nakhahori et al., 1991; Verkek et al., 1991; Yu et al., 1991). Expansion of the d(CGG) repeat is accompanied by methylation of the FMR1 promoter and of the amplified trinucleotide tract (Bell et al., 1991; Vincent et al., 1991; Pieretti et al., 1991; Luo et al., 1993). Subsequent to d(CGG) expansion and hypermethylation, the FMR1 gene becomes transcriptionally silent (Pieretti et al., 1991; Hansen et al., 1992; Sutcliffe et al., 1992), and the replication of a chromosomal segment spanning geq150 kb 5` and geq34 kb 3` from the d(CGG) stretch is delayed (Hansen et al., 1993).

The molecular mechanisms that govern d(CGG) amplification and hypermethylation and that link d(CGG) expansion to the suppressed transcription of FMR1 and to its delayed replication are not known. In addressing these problems, we showed recently that model d(CGG) oligomers form under physiological conditions a tetramolecular four-stranded structure in a time-dependent and DNA concentration-dependent kinetics. We also found that the multimolecular tetraplex structure of short d(CGG)tracts is stabilized by 5-methylation of the cytosine residues (Fry and Loeb, 1994). The facile formation of tetramolecular complexes by d(CGG) raised the hypothesis that single stranded stretches of this tract may also generate unimolecular hairpin or tetraplex structures (Fry and Loeb, 1994). In this communication we use gel mobility analysis and chemical probing to demonstrate that d(CGG) oligodeoxynucleotides can fold back at a physiological range of salt concentrations, temperatures, and pH values to form hairpin structures. In contrast to the second-order kinetics of formation of tetramolecular quadruplex d(CGG) (Fry and Loeb, 1994), hairpin structures of this sequence are formed at a zero-order kinetics. Our evidence for hairpin formation by d(CGG) is sustained by reports by Gacy et al.(1995) and Chen et al.(1995), which were published as this work was completed and that provide NMR evidence to also show the formation of hairpin structures by d(CGG) and by other trinucleotide tracts.

The folding of exposed expanded single strand runs of d(CGG)during the replication of fragile X cell DNA could entail slippage and trinucleotide expansion. Furthermore, guanine-rich tracts of RNA or DNA that fold back to generate a hairpin (Christiansen et al., 1994) or tetraplex structure (Christiansen et al., 1994; Woodford et al., 1994) were shown to arrest DNA synthesis in vitro. If formed in vivo along single strand stretches, back-folded d(CGG) structures may also obstruct DNA transcription and replication. These structures might, therefore, contribute to the observed blocking of FMR1 transcription and to its delayed replication in fragile X cells.


EXPERIMENTAL PROCEDURES

Oligodeoxynucleotides

DNA oligomers listed under Table 1were prepared by Operon Technologies, Alameda, CA. To purify these oligodeoxynucleotides, they were electrophoresed through a 15% polyacrylamide denaturing sequencing gel, and following detection by UV absorption, DNA bands were cut out and the gel slices were extracted by slow agitation overnight in 0.5 M ammonium acetate, 1 mM magnesium acetate, 0.1 mM EDTA. To remove gel residues, the extracts were passed through a SepPak cartridge (Waters, Milford, MA) as instructed by the manufacturer. The purified oligomers were dried, resuspended to a concentration of 5 µg/ml in 10 mM Tris-HCl buffer, pH 8.0, 1 mM EDTA (TE buffer) and labeled at their 5`-end with P using T4 bacteriophage polynucleotide kinase and [-P]ATP (Sambrook et al., 1989).



Gel Electrophoresis

Intramolecular folding or unfolding of 5` P-labeled oligodeoxynucleotides were followed by electrophoresis of the DNA in a nondenaturing polyacrylamide gel at 4 or at 75 °C, respectively (Williamson et al., 1989; Zahler et al., 1991; Murchie and Lilley, 1994). Temperature-controlled electrophoresis was performed in a Polar Bear isothermal electrophoresis apparatus (Owl Scientific, Cambridge, MA) attached to a thermostatic circulator bath (Neslab Instruments, Newington, NH). The 5` end-labeled oligodeoxynucleotides were boiled for 3 min in TE buffer, pH 8.0, in the presence or absence of salt as indicated. For electrophoresis at 75 °C, the boiled oligomers were promptly loaded onto a preheated 12% polyacrylamide gel (19:1, acrylamide/bisacrylamide), in 89 mM Tris borate buffer, pH 8.3, 1 mM EDTA (TBE buffer) with or without salt as indicated, and electrophoresis was conducted in the presence or absence of salt, at 4.5 or 13-17 V/cm, respectively. For electrophoresis at 4 °C, DNA oligomers were boiled, annealed on ice for 20 min, loaded onto a gel precooled to 4 °C, and electrophoresed at this temperature in the presence or absence of salt, at 6.7 or 20-31 V/cm, respectively. Electrophoresis was stopped when the bromphenol blue dye marker reached a distance of 22.0 cm. The gels were dried, and positions of the labeled oligomers were detected and recorded by phosphor imaging (FujiX BAS 1000 bio-imaging analyzer). The electrophoretic mobility of the d(CGG)(n) oligomers was determined relative to the following marker oligodeoxynucleotides: Tet, a Tetrahymena telomeric sequence 5`-d(T(2)G(4))(4)-3`; bacteriophage M13 universal primer 5`-d(TCCCAGTCACGACGT)-3`; and cross-linked H3T2 oligomer that was generated by UV-light irradiation and purified by sequencing gel electrophoresis as described below. Resolution of oligodeoxynucleotides according to size was performed by electrophoresis through a 12% polyacrylamide, 8 M urea sequencing gel (Fry and Loeb, 1992).

Absorbance Thermal Denaturation Analysis

Unlabeled oligodeoxynucleotides in TE buffer, pH 8.0, 100 mM KCl were boiled for 3 min, annealed at 4 °C for 30 min, and then subjected to thermal denaturation. DNA denaturation was monitored by following the change in absorption at 257 mµ as a function of temperature using a Varian DMS 200S UV spectrophotometer attached to a thermostatic circulator water bath.

UV Cross-linking of DNA

Cross-linking of DNA by UV irradiation was performed with oligomers H3T2 or H3T3, which contain d(CGG)(n) runs interrupted by three clusters of two or three thymidines each (Table 1). These sequences were folded by first denaturing for 2 min at 95 °C 0.5 µg of gel-purified 5` P-labeled oligomer in TE with or without added salt, followed by annealing on ice for 20 min. The folded oligomers were irradiated at 254 mµ for 5 min at 4 °C and at a distance of 5 cm by a UV light source (UVP, San Gabriel, CA; 580 microwatts/cm^2 at 6 inches). The irradiated DNA was denatured for 2 min at 90 °C in a sequencing gel loading buffer and resolved by 12% polyacrylamide, 8.0 M urea denaturing gel electrophoresis. To isolate the cross-linked form of H3T2, 0.1 µg of end-labeled oligomer, mixed with 8 µg of unlabeled oligomer was UV irradiated and then resolved by denaturing gel electrophoresis as detailed above. The cross-linked forms of the oligomers, which migrated ahead of the main oligomer band, were excised from the gel, extracted, and purified according to the standard procedure employed for gel purification of DNA fragments (see above).

DEPC^1and KMnO(4)Probing of d(CGG) Oligomers

DEPC detects unpaired purine residues in DNA (Herr, 1985; Furlong and Lilley, 1986; Huertas et al., 1993; Murchie and Lilley, 1994). To probe unpaired guanines in folded d(CGG)(n), 0.2-0.25 µg/µl 5` P-labeled oligomer in 40 µl of TBE buffer, pH 8.3, 100 mM KCl, was denatured at 90 °C for 2 min and reannealed on ice for 30 min. Following annealing, 0.1 volume of DEPC (Sigma) was added, and the DNA samples were incubated for 3 h at 4 °C. To probe unpaired guanine residues in fully unfolded DNA, labeled oligomers were denatured, incubated at 80 °C for additional 5 min and then exposed to 0.1 volume of DEPC for 4.5 min at 80 °C. Reaction with the longer oligomers (33-mer, H3T1, H3T2, and H3T3) was terminated by the addition of an equal volume of 3.0 M sodium acetate, pH 7.0, 2.0 M beta-mercaptoethanol, and 200 µg of yeast tRNA carrier, and DEPC was removed by twice precipitating the DNA by 2 volumes of ethanol. With all of the remaining oligomers, the reaction was terminated by the addition of water to a final volume of 100 µl, and DEPC was removed by centrifugation through a Sephadex G-25 minicolumn. The isolated DNA was dried, resuspended in 40 µl of freshly diluted 1.0 M piperidine, and incubated for 5 min at 90 °C. The piperidine-treated DNA samples were dried, resuspended in sequencing gel loading buffer and electrophoresed through a 20% polyacrylamide, 8.0 M urea sequencing gel. Patterns of oligomer fragmentation were detected by autoradiography.

Thymidine residues in oligomers that contain thymidine clusters were probed by KMnO(4), which selectively modifies unpaired thymidine residues in folded nucleotide sequences (Burton and Reilly, 1966; Hayatsu and Ukita, 1967; Friedman and Brown, 1978). DNA oligomers, folded or denatured as described above, were exposed to KMnO(4) at a final concentration of 0.5 mM for either 30 min at 4 °C (folding conditions) or for 5 min at 80 °C (denaturation conditions). Termination of the KMnO(4) reaction, isolation of the DNA, its treatment by piperidine and electrophoresis in a 20% polyacrylamide sequencing gel were as described above for DEPC.

Methylation-protection Analysis of d(CGG)(n) Oligomers

The accessibility to methylation of guanine N7 groups in folded or denatured d(CGG)(n) tracts was assessed by reacting oligomers with dimethyl sulfate (DMS). A folded DNA structure was formed by heating 5` P-labeled oligomer for 2 min at 90 °C in 40 µl of TBE buffer, pH 8.3, 100 mM KCl followed by 30 min annealing at 4 °C. After adding 5 µl of 1 mg/ml dG carrier, the folded oligomer was alkylated by the addition of 5 µl of 0.5% DMS freshly diluted in TBE buffer, pH 8.3, 100 mM KCl. The reaction mixture was incubated for 15 min at 4 °C, and methylation was terminated by the addition of 25 µl of 0.9 M sodium acetate pH 7.0, 0.3 M beta-mercaptoethanol, and 300 µg/ml yeast tRNA carrier. The DNA was twice precipitated at 4 °C by 2.0 volumes of ethanol, dried, and hydrolyzed by 1.0 M piperidine as described for DEPC above. Denatured oligomers were similarly methylated except that carrier dG and DMS were added immediately after the labeled d(CGG)(n) oligomer was denatured for 2 min at 90 °C, and modification by DMS was conducted for 45 s at 90 °C. Termination of the methylation reaction, cleavage by piperidine, and sequencing gel electrophoresis were performed as described for DEPC above.


RESULTS

Folding of d(CGG)(n) Oligomers

To examine whether or not d(CGG)(n) oligomers are capable of forming folded structures, their electrophoretic migration in a nondenaturing polyacrylamide gel was compared at 4 and 75 °C. Mobilities of d(CGG)(n) oligomers of different length were assessed relative to each other and to the migration of the following marker DNA fragments: Tet, which forms a unimolecular folded quadruplex structure at 4 °C and unfolds at 75 °C (Williamson et al., 1989); bacteriophage M13 universal primer, which lacks an intrastrand complementarity necessary for folding; and UV cross-linked H3T2 oligomer, which maintains a folded form under both native or denaturing conditions (see Fig. 3below). As shown in Fig. 1, d(CGG)(n) oligomers display different mobilities at 4 and 75 °C. The oligomers migrate at 75 °C at rates inversely proportional to their length, both in the presence of salt (Fig. 1D) or in its absence (Fig. 1B). This mobility pattern is similar to the pattern of migration in a denaturing sequencing gel (Fig. 1E). By contrast, at 4 °C and with (Fig. 1C) or without salt (Fig. 1A), every tested d(CGG)(n) oligomer displays an increased mobility. In contrast to the progressively slowed mobility of oligomers of increasing length at 75 °C (Fig. 1, B and D), 8-mer, 11-mer, and 15-mer d(CGG) sequences display comparable mobilities at 4 °C, indicating an accelerated migration of the 15- and perhaps the 11-mer oligonucleotides (Fig. 1, A and D). The temperature dependence of the mobility of d(CGG)(n) oligomers is also underscored by the similar electrophoretic behavior of 24-mer d(CGG) and of the telomeric Tet sequence, which forms a compact, rapidly migrating unimolecular tetraplex in the presence of a monovalent alkali cation (Williamson et al., 1989). As seen in Fig. 1, A and B, both the mobilities of the 24-mer and of Tet are strongly affected by temperature. At 4 °C, the two DNA oligomers migrate at a rate similar to that of the M13 universal primer (Fig. 1A), but at 75 °C, both sequences migrate at a significantly slower rate than the M13 primer (Fig. 1B). Note, however, that whereas the migration at 75 °C of the 24-mer is retarded in the presence of potassium, Tet retains a high mobility despite the elevation in temperature (Fig. 1D). It appears, therefore, that unlike quadruplex Tet, which requires potassium for stabilization (Sen and Gilbert, 1991; Williamson, 1992), the compact form of the 24-mer is less stabilized by this cation (see below). The strong temperature dependence of the electrophoretic mobility of the d(CGG)(n) oligomers is also apparent for the 33-mer and for H3T2. These oligomers migrate at 4 °C ahead of the folded UV cross-linked H3T2 marker (Fig. 1, A and C) but display a slower relative migration at 75 °C (Fig. 1, B and D).


Figure 3: Formation by UV-irradiation of covalently cross-linked forms of the H3T3 oligomer. End-labeled H3T3 DNA was heat-denatured, annealed, and irradiated by UV light in the absence or presence of 200 mM of the indicated salt as described under ``Experimental Procedures.'' Shown is an autoradiogram of 12% polyacrylamide denaturing gel electrophoresis of control unirradiated and UV irradiated H3T3 oligomer samples. Arrows mark the band of rapidly migrating cross-linked DNA.




Figure 1: Electrophoretic migration of d(CGG)oligomers in a nondenaturing polyacrylamide gel at 4 or 75 °C. A, electrophoregram of d(CGG) oligomers run under nondenaturing conditions at 4 °C and in the absence of salt. End-labeled oligomer or DNA marker, 500 pg of DNA in 2.5 µl of TE buffer pH 8.0, was heat-denatured for 3 min at 90 °C, annealed for 30 min at 4 °C, and electrophoresed at 4 °C through a nondenaturing 12% polyacrylamide gel (see ``Experimental Procedures''). B, electrophoregram of d(CGG)oligomers run under nondenaturing conditions at 75 °C and in the absence of salt. End-labeled oligomer or DNA marker, 500 pg of DNA in 2.5 µl TE buffer pH 8.0, was heat-denatured for 3 min at 90 °C, incubated for additional 30 min at 75 °C, and electrophoresed at 75 °C through a nondenaturing 12% polyacrylamide gel (see ``Experimental Procedures''). C, electrophoregram of d(CGG) oligomers run under nondenaturing conditions at 4 °C and in the presence of salt. DNA sample preparation and electrophoresis were performed as described for A above, except that DNA annealing and electrophoresis were conducted in buffers contained 100 mM KCl. D, electrophoregram of d(CGG) oligomers run under nondenaturing conditions at 75 °C and in the presence of salt. DNA sample preparation and electrophoresis were performed as in B above except that DNA annealing and electrophoresis were conducted in buffers contained 100 mM KCl. E, electrophoregram of d(CGG) oligomers run through a 12% polyacrylamide, 8.0 M urea denaturing gel. DNA sample preparation and electrophoresis were conducted as described under ``Experimental Procedures.'' Marker DNA designations were as follows: Univ., bacteriophage M13 universal primer; Tet, Tetrahymena telomeric sequence 5`-d(T(2)G(4))(4)-3`; H3T2 CL, UV-light cross-linked oligomer H3T2.



The rapidly migrating forms of all of the examined d(CGG)(n) oligomers are found to form within 5-7 min, which is the shortest testable annealing time (data not presented). Furthermore, these structures are generated at an identical efficiency over a range of DNA concentrations of 1.0-500.0 µg/ml (results not shown). Thus, in contrast to the second-order kinetics of formation of a multimolecular d(CGG)(n) tetraplex (Fry and Loeb, 1994) the observed compact forms of d(CGG)(n) oligomers are generated at a zero-order kinetics, suggesting that these structures are unimolecular. It is notable that when incubated for >30 min at 4 °C in the presence of 100 mM KCl, the 24-mer d(CGG) sequence displays in a nondenaturing gel a major rapidly migrating band and a minor electrophoretically retarded band that presumably represents a multimolecular tetraplex (Fry and Loeb, 1994) (results not shown). It thus appears that an equilibrium between a folded unimolecular form of d(CGG)(n) and a multistranded tetraplex form of such a sequence is determined by the concentration of DNA, the presence of salt, and the time of incubation.

That d(CGG)(n) oligomers unfold at elevated temperatures was substantiated by absorbance thermal denaturation analysis. As shown in Fig. 2, heating of every examined d(CGG)(n) oligomer elicits hyperchromicity at 257 mµ, indicating the melting of these DNA fragments.


Figure 2: Absorbance thermal denaturation of d(CGG) oligomers. Oligomer DNA, 2.0-4.0 µg in 1.0 ml of TE buffer pH 8.0, 100 mM KCl, was boiled for 3 min and annealed for additional 30 min at 4 °C. Changes in the absorbance of the DNA at 257 mµ as a function of temperature were measured as described under ``Material and Methods.'' The curve depicts the increase in absorbance of each oligomer relative to its absorption at 20 °C normalized to 1.0.



To further demonstrate the formation of compact forms of d(CGG)(n) variants, we irradiated the annealed DNA with UV light to form covalently cross-linked folded species. A unimolecular tetrahelical form of the Tetrahymena telomeric sequence Tet, d(T(2)G(4))(4) was shown to become cross-linked by UV-irradiation through the generation of thymidine dimers (Williamson et al., 1989). To examine the formation of cross-linked folded d(CGG)(n) structures, oligomers H3T2 and H3T3 were constructed that contain d(CGG)(n) tracts interspersed by thymidine runs (Table 1). End-labeled H3T2 or H3T3 oligodeoxynucleotides were UV irradiated, and the formation of covalently cross-linked compact forms of these oligomers was detected by discerning species that retain rapid migration in a denaturing gel (see ``Experimental Procedures''). As shown in Fig. 3, H3T3 that was UV-irradiated in a buffer solution with no added salt or in the presence of 200 mM chloride salts of Na, K, Cs, or Rb displays similar amounts of a form that migrates in a denaturing sequencing gel ahead of the main band of denatured oligomer. This form is generated at a lower efficiency when H3T3 is UV-irradiated in the presence of 200 mM NH(4)Cl (Fig. 3), and it is undetectable when this oligomer is irradiated in the presence of 10 mM Mg (not shown). Comparable results are obtained by UV irradiation of H3T2 oligomer, except that a rapidly migrating cross-linked form of this sequence is generated efficiently in the presence of NH(4) ions (data not presented). Hence, d(CGG)(n) tracts interspersed by runs of thymidines form compact structures that can be cross-linked by UV irradiation. Unlike quadruplex DNA, which requires specific cations for its formation and stabilization (Sen and Gilbert, 1991; Williamson, 1992) the compact forms of d(CGG)(n) appear to be generated independently of the type or the presence of a cation.

Chemical Probing of Folded Forms of d(CGG)(n) Oligomers

To investigate the structure of the folded forms of d(CGG)(n) oligomers, we first probed them with DEPC, which detects unpaired purine residues in DNA (Herr, 1985; Furlong and Lilley, 1986; Huertas et al., 1993; Murchie and Lilley, 1994). Oligomers were incubated with DEPC in a TBE buffer solution, pH 8.3, containing 100 mM KCl, at either 4 °C (folding conditions) or at 80 °C (denaturing conditions). After the reaction was terminated and DEPC was removed, the DNA was exposed to piperidine to cleave DEPC-modified unpaired guanine residues, and product DNA fragments were resolved by sequencing gel electrophoresis (see ``Experimental Procedures''). In defining the reaction conditions, we found that bacteriophage M13 universal primer is modified and cleaved to the same extent when treated by DEPC at 4 or 80 °C, indicating a comparable reactivity of DEPC under the nondenaturing and denaturing temperatures (results not shown). Similarly, the patterns of DEPC modification of the shorter 8-mer and 11-mer d(CGG)(n) oligodeoxynucleotides (Fig. 4), reveal that all of the guanine residues in these DNA fragments are cleaved to a similar extent at 4 and 80 °C. It thus appears that these shorter oligomers fold only minimally or randomly at 4 °C. By contrast, only selected clusters of guanine residues are modified by DEPC in the 15-, 24-, and 33-mer oligodeoxynucleotides (Fig. 4A). A reproducible pattern of DEPC modification of these oligomers identifies within each d(CGG)(n) tract a central or epicentral cluster of four to five residues, which is modified by DEPC and thus constitutes an unpaired loop. Conversely, all the other guanine residues remain protected and are located, therefore, in paired regions (Fig. 4B). These results sustain the previous indications for the formation of folded structures by d(CGG)(n) tracts (Fig. 1, Fig. 2, and Fig. 3). The presence of a single DEPC-modified region in the compacted oligomers indicates that these tracts probably fold back to form a hairpin-like structure with a central or epicentral single strand loop.


Figure 4: Modification by DEPC of d(CGG)oligomers. A, denaturing polyacrylamide gel electrophoregram of DEPC-treated d(CGG) oligomers. End-labeled 8-, 11-, 15-, 24- and 33-mer oligodeoxynucleotides, 0.20-0.25 µg of DNA in 40 µl of TE buffer, pH 8.0, containing 100 mM KCl, were heat-denatured as described under ``Experimental Procedures.'' The oligomers were either annealed and treated with DEPC at 4 °C or heated and exposed to DEPC at 80 °C. The DEPC-treated DNA was cleaved by piperidine and electrophoresed through a denaturing 12% polyacrylamide, 8.0 M urea gel as detailed under ``Experimental Procedures.'' Bck, piperidine cleavage of oligomers that were not exposed to DEPC; solid arrowheads, strong cleavage; open arrowheads, weak cleavage. B, allocation of DEPC-modified nucleotide tracts within the d(CGG) sequence. The solid-line frame marks a strongly cleaved region, and the broken line demarcates the weakly cleaved nucleotides.



Telomeric DNA fragments contain clusters of thymidines (Guo et al., 1993; Murchie and Lilley, 1994) or of thymidine and adenine residues (Murchie and Lilley, 1994) interspersed between tracts of contiguous guanines. Folding of these sequences into tetraplex structures involves the looping-out of the thymidine or thymidine-adenine runs. Adenine residues were also found to be interspersed within the d(CGG)(n) sequences of the FMR1 gene in normal subjects (Kunst and Warren, 1994). We inquired, therefore, whether or not the introduction of thymidine clusters into a d(CGG)(n) stretch alters its pattern of folding. Annealed or denatured 15-mer and H1T2 oligomers were modified by DEPC or KMnO(4), which detect unpaired guanine or thymidine residues, respectively. As seen in Fig. 5, the region that loops-out at 4 °C is located at a similar position in the two oligomers. However, the two thymidine residues introduced into H1T2 remain unpaired at 4 °C and merge with the unpaired guanines to form a larger loop. To broaden this inquiry, the 33-mer, H3T1, H3T2, and H3T3 oligomers were similarly probed by DEPC and KMnO(4) for regions of unpaired guanine and thymidine residues, respectively. Patterns of DEPC-modification shown in Fig. 6A indicate that the introduction of three runs of one, two, or three thymidine residues each into the 33-mer DNA does not alter the position of the primary unpaired loop. Note that the patterns of KMnO(4) modification demonstrate that whereas the central thymidine cluster merges with the unpaired guanines to form a larger loop, the remaining two thymidine runs form two additional unpaired regions that do not include guanine residues (Fig. 6B). However, all three unpaired regions in H3T3 contain thymidine residues with only a negligible participation of guanines in the looped-out tracts (Fig. 6, A and B and the scheme in Fig. 6C). It appears, therefore, that the symmetry of the primary fold in d(CGG)(n) remains unaltered whether or not thymidine residues are introduced. Yet, as additional tracts of thymidine are added, they may form auxiliary loops that do not include guanine residues.


Figure 5: Modification by DEPC and KMnO(4) of the 15-mer and H1T2 oligomers. A, denaturing polyacrylamide gel electrophoregram of DEPC- and KMnO(4)-treated oligomers. End-labeled oligomers, 0.20-025 µg of DNA in 40 µl of TE buffer, pH 8.0, 100 mM KCl, were heat-denatured, annealed at 4 °C, or heated at 80 °C and treated by DEPC or KMnO(4) at these respective temperatures as described under ``Experimental Procedures.'' Piperidine cleavage and 12% polyacrylamide, 8.0 M urea denaturing gel electrophoresis were conducted as described for Fig. 4. Bck, piperidine cleavage of oligomers not treated by DEPC or KMnO(4); solid arrowheads, strong cleavage; open arrowheads, weak cleavage. B allocation of DEPC- and KMnO(4)-modified nucleotide tracts within the 15-mer and H1T2 sequences. The solid line frame marks a strongly cleaved region, and a broken line demarcates the weakly cleaved nucleotides.




Figure 6: Modification by DEPC and KMnO(4) of d(CGG) oligomers containing thymidine tracts of different length. A, denaturing polyacrylamide gel electrophoregram of DEPC-treated oligomers. End-labeled 33-mer, H3T1, H3T2, and H1T2 oligomers, 0.20-0.25 µg of DNA in 40 µl of TE buffer, pH 8.0, 100 mM KCl were denatured, annealed at 4 °C, or heated at 80 °C and reacted with DEPC at these respective temperatures as described under ``Experimental Procedures.'' The oligomers were cleaved by piperidine and electrophoresed through a 12% polyacrylamide, 8.0 M urea denaturing gel as described in the legend to Fig. 4. Bck, piperidine cleavage of oligomers not treated by DEPC; solid arrowheads, strong cleavage; open arrowheads, weak cleavage. B, denaturing polyacrylamide gel electrophoregram of KMnO(4)-treated oligomers. End-labeled 33-mer, H3T1, H3T2, and H1T2 oligomers were treated with KMnO(4), cleaved by piperidine, and electrophoresed through a 12% polyacrylamide denaturing gel under conditions detailed under ``Experimental Procedures.'' Bck, piperidine cleavage of oligomers not treated by KMnO(4); solid arrowheads, strong cleavage; open arrowheads, weak cleavage. C, allocation of DEPC- and KMnO(4)-modified nucleotide tracts within the examined sequences. A solid line frame marks the strongly cleaved region, and the broken line demarcates the weakly cleaved nucleotides.



Guanine Residues in Folded d(CGG)(n) Oligomers Are Not Protected against Methylation

By acting as both donors and acceptors of Hoogsteen hydrogen bonds that involve the N-7 position of each residue, four guanines can be arrayed cyclically to form a guanine quartet. This N-7 hydrogen bonding renders the guanine residues in the quartet resistant to methylation by DMS (Sen and Gilbert, 1991; Williamson, 1992). However, guanine pairs in a Hoogsteen-bonded double-stranded structure are readily alkylated as the N-7 position of only one residue participates in a hydrogen bond, whereas the other remains free and accessible to DMS. To examine whether or not the compact forms of different d(CGG)(n) oligomers represent unimolecular tetrahelices, methylation protection analysis of the folded structures was conducted. We probed the 33-mer DNA fragment, which displays a single unpaired region ( Fig. 4and Fig. 6) and H3T2 and H3T3, which have three unpaired regions each (Fig. 6). The oligomers were exposed to DMS at 4 °C (folding conditions) or at 90 °C (denaturing conditions) and subsequently treated with piperidine as detailed under ``Experimental Procedures.'' As Fig. 7shows, the compact form of the telomeric sequence Ter that folds at 4 °C and in the presence of potassium ions to form a unimolecular tetraplex (Balagurumoorthy et al., 1992; Lee and Lee, 1993; Murchie and Lilley, 1994) remains largely protected against methylation. By clear contrast, all of the guanine residues in folded 33-mer, H3T2, and H3T3 become methylated (Fig. 7). It appears, therefore, that the compact forms of these oligomers are not tetrahelices but rather back-folded hairpin structures that are stabilized by either Watson-Crick or by Hoogsteen hydrogen bonds (see ``Discussion'').


Figure 7: Methylation-protection analysis of d(CGG) oligomers. Oligomers, 0.20-0.25 µg of DNA in 40 µl of TBE buffer, pH 8.3, 100 mM KCl were heat-denatured for 3 min at 90 °C and annealed for 30 min at 4 °C. After the addition of 0.1 volume of 1.0 mg/ml dG carrier DNA and 0.1 volume of 0.5% DMS freshly diluted in TBE buffer, pH 8.3, 100 mM KCl, the samples were either incubated for 15 min at 4 °C or for 45 s at 90 °C. Termination of the methylation reaction, piperidine cleavage, and denaturing gel electrophoresis were conducted as described under ``Experimental Procedures.''




DISCUSSION

The substantial expansion of a d(CGG) trinucleotide repeat within the 5` exon of the FMR1 gene in fragile X syndrome cells (Fu et al., 1991; Kremer et al., 1991; Oberléet al., 1991; Nakahori et al., 1991; Verkerk et al., 1991; Yu et al., 1991) and the hypermethylation of this tract and of the FMR1 gene promoter (Bell et al., 1991; Verkerk et al., 1991; Yu et al., 1991), entail transcriptional silencing of FMR1 (Pieretti et al., 1991; Hansen et al., 1992; Sutcliffe et al., 1992) and a delay in the replication of a DNA region that encompasses FMR1 (Hansen et al., 1993). Illumination of the molecular basis for the expansion of the d(CGG)(n) tract in fragile X cells and the ensuing blocking of FMR1 transcription and its delayed replication, requires a better understanding of the alternative structures of the d(CGG)(n) sequence. We reported previously that d(CGG)(n) oligomers readily form in vitro a multistrand Hoogsteen-bonded tetrahelical structure that is stabilized by cytosine C-5 methylation (Fry and Loeb, 1994). This observation led us to speculate that single strand tracts of d(CGG)(n), which become exposed during replication or transcription, might also fold back to form Hoogsteen-bonded unimolecular hairpin or tetraplex domains. Such structures could act in vivo to precipitate slippage during replication that could result in d(CGG)(n) expansion, and they may also block the progression of the transcription or replication machineries.

In this communication, we demonstrate that short stretches of d(CGG)(n) readily loop back under physiological conditions in vitro to form hairpin structures. Unimolecular hairpin or tetraplex structures of telomeric DNA display an increased electrophoretic mobility in a nondenaturing gel and conversely, their electrophoretic migration slows when they become thermally denatured (Williamson et al., 1989). In analogy, we find that whereas heat-denatured d(CGG)(n) oligomers migrate in a nondenaturing gel at rates inversely proportional to their length, their relative mobility becomes anomalously accelerated under annealing conditions (Fig. 1). Taken as evidence for the folding of d(CGG)(n) into more compact forms, this observation is strengthened by the demonstration of UV hyperchromicity of thermally denaturing d(CGG)(n) oligomers (Fig. 2). Similar hyperchromicity is displayed by unfolding telomeric DNA sequences (Scaria et al., 1992). Finally, as was shown in the past for telomeric DNA (Williamson et al., 1989), compact forms of d(CGG)(n) runs interspersed by thymidine clusters can be covalently cross-linked by UV light (Fig. 3).

That the compact structures of d(CGG)(n) are unimolecular is indicated by their faster mobility relative to the unfolded oligomers ( Fig. 1and Fig. 3) and by the zero-order kinetics of their formation (see ``Results''). Such monomolecular compact forms may represent either fold-back hairpin structures or an intrastrand tetrahelix. Formation and stabilization of tetraplex DNA require Na or K cations (Sen and Gilbert, 1991; Williamson, 1992). As the compact forms of d(CGG)(n) are generated and maintained to a comparable extent with or without various cations ( Fig. 1and Fig. 3), it is unlikely that they represent quadruplex structures. The modification of all of the guanines in folded d(CGG)(n) oligomers by DMS (Fig. 7) further argues against their arrangement in a tetraplex conformation. Finally, DEPC modification provides direct evidence for the folding of d(CGG)(n) into a hairpin structure rather than into a tetrahelix. Had the folded structures been unimolecular quadruplexes, three unpaired loops that serve as hinges should have been observed. Instead, a single region of unpaired nucleotides is discerned at a central or epicentral location within several d(CGG)(n) oligomers (Fig. 4). Insertion of three thymidine clusters consisting of one, two, or three residues each into a d(CGG)(n) tract does not change the location of this primary looped-out region ( Fig. 5and Fig. 6). Insertion of three thymidine clusters into the d(CGG)(n) sequence introduces two additional regions of unpaired thymidines, which do not include contiguous guanines ( Fig. 5and Fig. 6). These oligomers remain fully accessible to DMS (Fig. 7), and thus it appears that these oligomers also maintain a hairpin structure rather than a tetraplex conformation. The weight of the evidence provided here points, therefore, at a similarity between the d(CGG)(n) tracts and telomeric DNA sequences, which are capable of forming back-folded hairpin structures at a physiological range of salt concentrations, temperatures and pH values (Sundquist and Klug, 1989; Sen and Gilbert, 1990; Balagurumoorthy and Brahmachari, 1994; Choi and Choi, 1994).

The gel mobility and chemical probing evidence provided here, which indicates the facile formation of hairpin structures by d(CGG)(n) tracts, is corroborated by the works of Gacy et al.(1995) and Chen et al.(1995), which were published as this work was completed. Employing NMR analysis, these authors demonstrated that d(CGG)(n), as well as the complementary d(CCG)(n) sequence (Chen et al., 1995) and other trinucleotide sequences that expand in human disease (Gacy et al., 1995), form hairpin structures. Calculations indicate that the stability of d(CGG)(n) hairpin increases linearly with length and that a threshold stability is attained at lengths conforming with those of expanded d(CGG)(n) sequence in individuals afflicted with the fragile X syndrome (Gacy et al., 1995).

Based on the location of the looped-out sector in d(CGG)(n) oligomers (Fig. 4) or in tracts interspersed with thymidine clusters ( Fig. 5and Fig. 6), we constructed models for the structure of d(CGG)(n) fold-back hairpins. The scheme presented in Fig. 8A suggests that the 24-mer DNA folds back asymmetrically to form a hairpin structure that is stabilized by five guanine-guanine Hoogsteen base pairs. By contrast, the 33-mer folds back with a nearly perfect symmetry to form a hairpin structure that is maintained by four guanine-guanine Hoogsteen base pairs and/or 10 guanine-cytosine Watson-Crick pairs (Fig. 8A). Modification by DEPC of purines in the folded 33-mer d(CGG) tract cannot reveal whether or not the cytosine residue at position 14 from the 5` end (marked *C in Fig. 8A), is paired or not. Hence, an alternative structure of folded 33-mer can be envisioned in which this residue remains unpaired and the guanine at position 19 pairs with a guanine at position 13. A resulting hairpin structure is less symmetric than the one depicted in Fig. 8A, having a dinucleotide 3` unpaired tail and being stabilized by eight Watson-Crick and/or five Hoogsteen hydrogen bonds (model not shown). Since the electrophoregram shown in Fig. 8A fails to distinguish between a single- or dinucleotide 3` overhang, it cannot be determined which alternative folded form exists. A generally similar model for the hairpin structure of a d(CGG)(n) oligomer, based on NMR analysis was presented recently by Gacy et al.(1995). A stereo model of d(CGG)(9) constructed on the basis of NMR analysis implicates both GbulletC and GbulletG in the stabilization of the stem of the hairpin (Chen et al., 1995). Hence, it appears that the schematic representation of the hairpin structures in Fig. 8A is sterically plausible as attested by independent NMR analysis and model construction.


Figure 8: Models for the back-folded hairpin forms of d(CGG) oligomers. A, schemes of the hairpin forms of the 24- and 33-mer d(CGG) fragments. The model is based on the identification by DEPC-modification of looped regions of unpaired guanines in the annealed oligomers (Fig. 4). Watson-Crick and non-Watson-Crick bonds are marked by three dashes and two Xs, respectively. The absence of a bridging notation indicates lack of base pairing. The marking of pairing in the scheme does not indicate that any specific bond is actually formed. The notation *C in the model of folded 33-mer marks a cytosine residue that may or may not be paired (see ``Discussion''). B, schemes of alternative folded forms of the H3T3 oligomer. The models are based on the identification by DEPC and KMnO(4) modification of unpaired guanines and thymidines, respectively, in the annealed oligomers (Fig. 6).



As indicated in Fig. 8B, the H3T3 oligomer can assume either one of two alternative folded structures. Both possible hairpin forms are maintained by eight guanine-guanine pairs. However, whereas conformation I is linear, the central loop in form II serves as a hinge for a parallel arrangement of two duplex arms (Fig. 8B). Form II is perhaps less likely to exist than conformation I since the former could easily convert into a tetraplex that, as evidence indicates (Fig. 7), is not generated.

The propensity of the d(CGG)(n) sequence to form Hoogsteen-bonded tetrahelix (Fry and Loeb, 1994) or back-folded hairpin structures (Gacy et al.(1995) and this communication) may have a biological significance. First, the exposure of long stretches of single-stranded d(CGG)(n) during DNA replication and the formation of a stable hairpin structure by this tract could ensue slippage and trinucleotide expansion. Second, hairpin formation could block replication, transcription, and perhaps translation. It was shown recently that the progression in vitro of AMV reverse transcriptase along insulin-like growth factor II mRNA is blocked at a guanine-rich region that forms a quadruplex domain and two stable hairpins (Christiansen et al., 1994). Similarly, the progression in vitro of a variety of DNA polymerases along a guanine-rich region in the beta globin promoter DNA is arrested at an intrastrand tetraplex-forming sequence (Woodford et al., 1994). Last, expansion of the trinucleotide (CGG) repeat stalls the progression of the 40 S ribosomal subunit during the translation of FMR1 mRNA (Feng et al., 1995). Hence, it may be argued that the exposure of stretches of expanded (CGG)(n) tracts in DNA or mRNA and their folding into hairpin structures might block replication and transcription or translation, respectively. The formation of (CGG)(n) secondary structures in FMR1 DNA or mRNA could thus contribute to the observed delay of FMR1 replication in fragile X cells (Hansen et al., 1993), to the silencing of its transcription (Pierreti et al., 1991; Hansen et al., 1992; Sutcliffe et al., 1992), and to the interrupted translation of its mRNA (Feng et al., 1995). Last, evidence indicates that cytosine methylation by methyltransferase is enhanced by hairpins formed by human c-Ha-ras telomere-like sequence and d(CCG) trinucleotide repeat (Smith et al., 1994). Thus, the folding of d(CGG)(n) may also be instrumental in the hypermethylation of the d(CGG)(n) region in DNA of fragile X syndrome cells.

Folding of d(CGG)(n) and the resulting inhibition of DNA synthesis and transcription may not occur in cells of normal and ``premutation'' individuals that have a shorter and unstable d(CGG)(n) stretch (Gacy et al., 1995). These shorter tracts may also be stabilized in a rigid conformation by single strand d(CGG)(n) binding proteins of the type that was recently identified in HeLa cells (Richards et al., 1993). The expansion of the d(CGG)(n) stretch in individuals afflicted with the fragile X syndrome could, however, defeat the capacity of such binding proteins to coat it, and the naked d(CGG)(n) stretches may fold-back to form stable hairpin domains.


FOOTNOTES

*
This study was supported in part by grants (to M. F.) from the United States-Israel Binational Science Fund, the Israel Science Foundation administered by the Israel Academy of Science and Humanities, the Council for Tobacco Research, and the Fund for Promotion of Research in the Technion. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 972-429-5328; Fax: 972-451-0375.

(^1)
The abbreviations used are: DEPC, diethyl pyrocarbonate; DMS, dimethyl sulfate.


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