Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Nov 15;29(22):4684-90.
doi: 10.1093/nar/29.22.4684.

The fragile X chromosome (GCC) repeat folds into a DNA tetraplex at neutral pH

P Fojtík  1 , M Vorlícková
Affiliations

The fragile X chromosome (GCC) repeat folds into a DNA tetraplex at neutral pH

P Fojtík et al. Nucleic Acids Res. .

Abstract

UV absorption and CD spectroscopy, along with polyacrylamide gel electrophoresis, were used to study conformational properties of DNA fragments containing the trinucleotide repeat (GCC)(n) (n = 4, 8 or 16), whose expansion is correlated with the fragile X chromosome syndrome. We have found that the conformational spectrum of the (GCC)(n) strand is wider than has been shown so far. (GCC)(n) strands adopt the hairpin described in the literature under a wide range of salt concentrations, but only at alkaline (>7.5) pH values. However, at neutral and slightly acid pH (GCC)(4) and (GCC)(8) strands homodimerize. Our data suggest that the homodimer is a bimolecular tetraplex formed by two parallel-oriented hairpins held together by hemi-protonated intermolecular C.C(+) pairs. The (GCC)(16) strand forms the same tetraplex intramolecularly. We further show that below pH 5 (GCC)(n) strands generate intercalated cytosine tetraplexes, whose molecularity depends on DNA strand length. They are tetramolecular with (GCC)(4), bimolecular with (GCC)(8) and monomolecular with (GCC)(16). i-Tetraplex formation is a complex and slow process. The neutral tetraplex, on the other hand, arises with fast kinetics under physiological conditions. Thus it is a conformational alternative of the (GCC)(n) strand duplex with a complementary (GGC)(n) strand.

PubMed Disclaimer

Figures

Figure 1
Figure 1
The pH dependence of migration in polyacrylamide gels of (GCC)4, (GCC)8 and (GCC)16 and of heteroduplexes with their complementary strands (which serve as markers). The gels were run at 0°C in 0.15 M Na+ (Robinson–Britton buffer + NaCl) at (A) pH 8.5, (B) pH 7, (C) pH 6.5 and (D) pH 6.
Figure 2
Figure 2
pH-induced changes in the (A) CD spectra and (C) UV absorption spectra of (GCC)4 dissolved in 0.15 M Na+ (Robinson–Britton buffer + NaCl) measured at 0°C. The pH values were: pH 8.5 (open circles), pH 7.2 (dashes), pH 6.9 (dash-dot) and pH 6 (filled circles). These CD and absorption spectra were measured in a 0.1 cm path length cell with a DNA concentration of 0.7 mM; thin line in (A), (GCC)4 in the same solvent at pH 6 measured in a 0.01 cm path length cell with a DNA concentration of 7 mM. (B) Temperature-induced changes in CD spectra of (open circles) the (GCC)4 hairpin in 0.15 M Na+, pH 8.5, and (full circles) of (GCC)4 homodimer in 0.15 M Na+, pH 6.
Figure 3
Figure 3
pH-induced changes in the CD and absorption spectra of (GCC)4 (circles), (GCC)8 (triangles) and (GCC)16 (squares) dissolved in 0.15 M Na+ (Robinson–Britton buffer + NaCl) measured at 0°C in a 0.1 cm path length cell (conditions as for Fig. 2): (upper) the ratio of absorption at 295 and 258 nm; (middle and bottom) CD changes monitored at 284 and 209 nm, respectively. All points in the dependences correspond to equilibrium states.
Figure 4
Figure 4
Ferguson plots of the (GCC)8 hairpin (full circles) at pH 8.5 and of the hairpin dimer (open circles) at pH 6 compared with the heteroduplex (GCC)8·(GGC)8 at pH 8.5 (full squares) and pH 6 (opens squares), respectively. The logarithm of relative mobility [with respect to that of the (CAG)4 hairpin] is plotted against the concentration of acrylamide in the gel. The gels were run at 0°C in 0.15 M Na+ (Robinson–Britton buffer + NaCl).
Figure 5
Figure 5
pH-induced changes in the (A) CD spectra and (B) UV absorption spectra of (GCC)4. The sample was dissolved in 0.15 M Na+ (Robinson–Britton buffer + NaCl), pH 6 (dots). The latter three spectra were measured 4 days after adjusting the pH to: pH 5.1 (dashes), pH 4.9 (dash-dot) and pH 4.4 (solid trace). The spectra were measured at 0°C in a 0.1 cm path length cell at a DNA concentration of 0.7 mM; (thin line) (GCC)4 in the same solvent, pH 5, measured in a 0.01 cm path length cell at a DNA concentration of 6 mM, after 4 days equilibration at pH 5 and 0°C. (C) Temperature-induced changes in the CD spectra of (GCC)4 dissolved in 0.15 M Na+, pH 4.4 [conditions as for solid trace in (A) and (B)]. The filled circle corresponds to the CD value obtained immediately after cooling the sample from 85 to 0°C. No time-dependent changes in CD spectra were observed in the temperature interval 0–20°C; the spectra changed with time in the temperature interval 20–40°C, so the spectra were taken after 70 min equilibration at the particular temperatures at which all the samples reached equilibrium. No changes in CD spectra with time were again observed at temperatures >40°C.
Figure 6
Figure 6
Acid PAGE of (GCC)4, (GCC)8 and (GCC)16 and of heteroduplexes with their complementary strands. The gel was run at 0°C in 0.15 M Na+ (Robinson–Britton buffer + NaCl), pH 5. The samples were loaded onto the gel in two sets: at pH 5, immediately after dissolving the oligonucleotides in electrophoresis buffer (lanes 1, 3 and 5); at pH 4.3, after 2 days equilibration at 0°C (lanes 2, 4 and 6); (GCC)16 loaded onto the gel after 5 days equilibration at pH 4.2 and 0°C (lane 7). Duplexes (GCC)4·(GGC)4, (GCC)8·(GGC)8 and (GCC)16·(GGC)16 were loaded immediately after mixing at pH 5 (lanes 8, 10 and 12) or pH 4.3 (lanes 9, 11 and 13) after 2 days equilibration of particular strands at 0°C.
Figure 7
Figure 7
Schematic models of (GCC)n structures. (A) A hairpin of (GCC)4 existing at pH > 8. (B) (Top) Neutral tetraplex of (GCC)4 arising in the course of decreasing the pH from 8 to ∼6.5, as a result of homodimerization of two parallel oriented hairpins; (bottom) model of the intramolecular neutral tetraplex of (GCC)16. (C) (Top) i-Tetraplex of (GGC)4 formed by four strands at pH < 5; (bottom) model of the intramolecular acidic tetraplex of (GCC)16.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ashley C.T.J. and Warren,S.T. (1995) Trinucleotide repeat expansion and human disease. Annu. Rev. Genet., 29, 703–728. - PubMed
    1. Oostra B.A. and Willems,P.J. (1995) A fragile gene. Bioessays, 17, 941–947. - PubMed
    1. Jin P. and Warren,S.T. (2000) Understanding the molecular basis of fragile X syndrome. Hum. Mol. Genet., 9, 901–908. - PubMed
    1. Mitas M. (1997) Trinucleotide repeats associated with human disease. Nucleic Acids Res., 25, 2245–2254. - PMC - PubMed
    1. Darlow J.M. and Leach,D.R.F. (1998) Secondary structures in d(CGG) and d(CCG) repeat tracts. J. Mol. Biol., 275, 3–16. - PubMed

Publication types