Slow motions in A·T rich DNA sequence

Abstract

In free B-DNA, slow (microsecond-to-millisecond) motions that involve equilibrium between Watson-Crick (WC) and Hoogsteen (HG) base-pairing expand the DNA dynamic repertoire that could mediate DNA-protein assemblies. R_1ρ relaxation dispersion NMR methods are powerful tools to capture such slow conformational exchanges in solution using ¹³C/¹⁵ N labelled DNA. Here, these approaches were applied to a dodecamer containing a TTAAA element that was assumed to facilitate nucleosome formation. NMR data and inferred exchange parameters assign HG base pairs as the minor, transient conformers specifically observed in three successive A·T base pairs forming the TAA·TTA segment. The abundance of these HG A·T base pairs can be up to 1.2% which is high compared to what has previously been observed. Data analyses support a scenario in which the three adenines undergo non-simultaneous motions despite their spatial proximity, thus optimising the probability of having one HG base pair in the TAA·TTA segment. Finally, revisiting previous NMR data on H2 resonance linewidths on the basis of our results promotes the idea of there being a special propensity of A·T base pairs in TAA·TTA tracts to adopt HG pairing. In summary, this study provides an example of a DNA functional element submitted to slow conformational exchange. More generally, it strengthens the importance of the role of the DNA sequence in modulating its dynamics, over a nano- to milli-second time scale.

Figure 1

Chemical shifts and linewidth of H2 adenine proton resonances. (a) 1D-T1 inversion recovery spectrum of the aromatic region of the unlabelled oligomer at 25 °C in ²H₂O, after having selected the inversion recovery delay to only obtain the H2 resonances of adenines. (b) Linewidths (measured at half-width) of H2 adenine proton resonances as a function of temperature. The numbering of the DNA segment of interest is given on the right of the Figure.

Figure 2

H2–C2 region of adenines of a constant-time HSQC spectrum. The H2–C2 region of adenines of a ¹³C–¹H constant-time HSQC spectrum (600 MHz) was obtained on the labelled oligomer at 25 °C. The ¹H 1D spectrum was superposed on the proton frequencies from 1D ¹³C R_1ρ dispersion experiment at the ¹H and ¹³C frequency of A₉ signal.

Figure 3

On-resonance R_1ρ relaxation dispersion profiles of C2 atoms of adenines. R_1ρ (= R₂ + R_ex) rates of the C2 atoms of the five adenines were plotted as a function of the effective spin-lock field power (ω1/2π). The experiments were performed at 25 °C. The two-state model fits (solid lines) were obtained using the protocol described in “Materials and Methods”; (R₂ + R_ex) standard deviations were calculated from the 500 runs carried out for each fit. The averaged R_ex values are specified in each panel. Top: R_ex > 0; bottom: R_ex ~ 0.

Figure 4

On-resonance R_1ρ relaxation dispersion profiles of adenine C8 atoms. R_1ρ (= R₂ + R_ex) rates of the C8 atoms of adenines were plotted as a function of the effective spin-lock field power (ω1/2π). The experiments were performed at 25 °C. The two-state model fits (solid lines) were obtained using the protocol described in “Materials and Methods”. (R₂ + R_ex) standard deviations were calculated from the 500 runs carried out for each fit. The averaged R_ex values are specified in each panel.

Figure 5

On-resonance R_1ρ relaxation dispersion profiles of adenines C1′ atoms. R_1ρ (= R₂ + R_ex) rates of the C1′ atoms of adenines were plotted as a function of the effective spin-lock field power (ω1/2π). The experiments were performed at 25 °C. The two-state model fits (solid lines) were obtained using the protocol described in “Materials and Methods”. (R₂ + R_ex) standard deviations were calculated from the 500 runs carried out for each fit. The averaged R_ex values are specified in each panel.

Figure 6

Off-resonance R_1ρ relaxation dispersion profiles for C1′ atoms of A₇, A₈ and A₁₉. R₂ + Rex (= R_1ρ) values are given as a function of the resonance offset from the major state (Ω_off/2π). Error bars represent experimental uncertainties. The experiments were carried out at four different spin-lock powers (From 150 to 700 Hz, colour code given in the bottom right of the Figure). The fits (solid lines) were performed using Method 2, described in “Materials and Methods”. The resulting exchange parameters are reported in Tables 2 and S3.