Secondary structure polymorphism in Oxytricha nova telomeric DNA

Abstract

Tandem repeats of the telomeric DNA sequence d(T4G4) of Oxytricha nova are capable of forming unusually stable secondary structures incorporating Hoogsteen hydrogen bonding interactions. The biological significance of such DNA structures is supported by evidence of specific recognition of telomere end-binding proteins in the crystal state. To further characterize structural polymorphism of Oxytricha telomeric DNAs, we have obtained and interpreted Raman, ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectra of the tandem repeats d(G4T4G4) (Oxy1.5), d(T4G4)2 (Oxy2) and dT6(T4G4)2 (T6Oxy2) and related non-telomeric isomers in aqueous salt solutions. Raman markers of Oxy1.5 identify both C2'-endo/anti and C2'-endo/syn conformations of the deoxyguanosine residues and Hoogsteen hydrogen bonded guanine quartets, consistent with the quadruplex fold determined previously by solution NMR spectroscopy. Raman, UVRR and CD signatures and Raman dynamic measurements, to monitor imino NH-->ND exchanges, show that the Oxy1.5 antiparallel quadruplex fold is distinct from the hairpin structures of Oxy2 and T6Oxy2, single-stranded structures of d(TG)8 and dT6(TG)8 and previously reported quadruplex structures of d(T4G4)4 (Oxy4) and dG12. Spectral markers of the telomeric and telomere-related DNA structures are tabulated and novel Raman and UVRR indicators of thymidine and deoxyguanosine conformations are identified. The results will be useful for probing structures of Oxytricha telomeric repeats in complexes with telomere end-binding proteins.

Figure 1

(A) Hoogsteen hydrogen bonded guanine quartet. (B) Crystal structure of d(G₄T₄G₄) (Oxy1.5) incorporating guanine quartets (11). (C) Solution structure of Oxy1.5 incorporating guanine quartets (12). (D) Hairpin structure of d(T₄G₄)₂ (Oxy2) incorporating G·G pairs (10). (E) Parallel-stranded quadruplex structure of Oxy2 (9).

Figure 2

Circular dichroism spectra (240–330 nm). (A) d(TG)₈ (filled triangle) and dT₆(TG)₈ (open triangle). (B) Oxy1.5 (filled circle), Oxy2 (filled triangle) and T6Oxy2 (open square). (C) Differences: T6Oxy2 – Oxy2 (open square) and dT₆(TG)₈ – d(TG)₈ (filled square). (D) Differences: Oxy2 – dT₄ (filled circle), Oxy2 – d(TG)₈ (filled triangle) and, T6Oxy2 – dT₆(TG)₈ (open triangle). Data were obtained on a Jasco-720 spectropolarimeter (20 nm/min scan speed; 1 s response time; 1 cm path) from 3 µM DNA samples in 10 mM sodium phosphate, pH 7.5, at 20°C.

Figure 3

(A) Raman spectra of Oxy1.5, Oxy2 and T6Oxy2. (B) Raman spectra of d(TG)₈ and dT₆(TG)₈. In (A) and (B) solutions contained DNA at 30 mg/ml in 10 mM Tris pH 7.2, 1 mM EDTA and 50 mM NaCl and were maintained at 10°C. Dashed lines in (A) and (B) indicate bands discussed extensively in the text. (C) UVRR spectra of Oxy1.5, Oxy2 and T6Oxy2. (D) UVRR spectra of d(TG)₈ and dT₆(TG)₈. In (C) and (D) the DNA solutions (10– 20 µM, see text) also contained 25 mM Na₂SO₄ for use of the 981 cm^–1 band of SO₄^2– as a reference intensity and wavenumber standard. The inset at the upper left in (C) and (D) shows a 5-fold ordinate amplification of the 600–900 cm^–1 interval.

Figure 4

(Top) Time-resolved Raman spectra (600–1800 cm^–1, 532 nm excitation) of guanine exchanges in Oxy1.5 at 20°C. (A) H₂O solution (pH 7.5) prior to the onset of exchange; (B) D₂O solution (pD 7.5) following 10 min exchange; (C) D₂O solution (pD 7.5) following 4320 min exchange; (D) difference (B) – (A); (E) difference (C) – (B). (Bottom) Guanine imino exchange (N1H→N1D) monitored by the downward shift of the 1717 cm^–1 marker band, Δσ₁₇₁₇ (filled square), and intensity increase of the 980 cm^–1 marker band, ΔI₉₈₀ (open square), versus time of exchange (t). The results for both markers suggest at least two exchange regimes, each of which is approximated by an exponential fit (line) to the data points. The inset shows the data points over the period 0 < t < 300 min on an expanded scale (see text).

Figure 5

(A) Raman signature (10°C) of the hairpin fold of T6Oxy2 generated by the difference T6Oxy2 – dT₆(TG)₈. (B) UVRR signature (10°C) of the hairpin fold of Oxy2 generated by the difference Oxy2 – d(TG)₈. (C) UVRR signature of the hairpin fold of Oxy2 generated by the difference Oxy2_10°C – Oxy2_70°C. Asterisks in traces (B) and (C) indicate uncompensated intensity of the reference intensity standard (981 cm^–1 band of SO₄^2–).

Figure 6

(A) Raman signatures (10°C) of the thymidine 5′ leader generated by the differences T6Oxy2 – Oxy2 (top trace) and dT₆(TG)₈ – d(TG)₈ (bottom trace). (B) UVRR signatures (10°C) of the thymidine 5′ leader generated by the differences T6Oxy2 – Oxy2 (top trace) and dT₆(TG)₈ – d(TG)₈ (bottom trace). Asterisks indicate uncompensated intensity of the reference intensity standard (981 cm^–1 band of SO₄^2–).

Figure 7

Raman (top) and UVRR (bottom) signatures of the guanine quartet component of Oxy1.5 generated by compensating corresponding Oxy1.5 spectra for contributions from dT (see text).