Thermodynamic signature of DNA damage: characterization of DNA with a 5-hydroxy-2'-deoxycytidine·2'-deoxyguanosine base pair

Abstract

Oxidation of DNA due to exposure to reactive oxygen species is a major source of DNA damage. One of the oxidation lesions formed, 5-hydroxy-2'-deoxycytidine, has been shown to miscode by some replicative DNA polymerases but not by error prone polymerases capable of translesion synthesis. The 5-hydroxy-2'-deoxycytidine lesion is repaired by DNA glycosylases that require the 5-hydroxycytidine base to be extrahelical so it can enter into the enzyme's active site where it is excised off the DNA backbone to afford an abasic site. The thermodynamic and nuclear magnetic resonance results presented here describe the effect of a 5-hydroxy-2'-deoxycytidine·2'-deoxyguanosine base pair on the stability of two different DNA duplexes. The results demonstrate that the lesion is highly destabilizing and that the energy barrier for the unstacking of 5-hydroxy-2'-deoxycytidine from the DNA duplex may be low. This could provide a thermodynamic mode of adduct identification by DNA glycosylases that requires the lesion to be extrahelical.

Figure 1

CD spectra of: (a) ODN-1 (black line, ④), ODN-2 (③) and ODN-1 (▲) heated to 90 °C; (b) ODN-3 (④), ODN-4 (③), ODN-5 (■) and ODN-3 (▲) heated to 90 °C.

Figure 2

(a) UV melting curves of ODN-1 (③) and ODN-2 (④); (b) ODN-3 (④), ODN-4 (③) and ODN-5 (■) in 10 mM sodium phosphate buffer (pH 7.0) at ~ 10 μM strand concentration.

Figure 3

(a) T_M dependence on strand concentration of ODN-1 (④) and ODN-2 (③); (b) ODN-3 (④), ODN-4 (③) and ODN-5 (■) in 10 mM sodium phosphate buffer (pH 7.0) in ~ 4-75μM strand concentration.

Figure 4

Differential scanning calorimetry (DSC) curves in 10 mM sodium phosphate buffer (pH 7.0) at ~ 150-200 μM strand concentration for (a) ODN-1 (④) and ODN-2 (③); (b) at ~ 124–150 μM strand concentration for ODN-3 (④), ODN-4 (③) and ODN-5 (■).

Figure 5

¹H-¹H NMR NOESY spectrum showing resonances for the thymine and guanine imino protons and sequential NOE connectivity for the imino protons of the base pairs G²·C¹¹ to A⁶·T⁷ for (a) unmodified ODN-1 and (b) 5-HO-dC modified ODN-2.

Figure 6

Expansion of the ¹H-¹H NOESY spectrum for 5-OH-dC modified ODN-2, showing the conservation of Watson-Crick base pairing and base stacking.

Figure 7

(a) ¹H-NMR of imino proton resonances as a function of temperature for (a) unmodified ODN-1 and (b) 5-HO-dC modified ODN-2. (b) Temperature dependence of line widths of the imino proton resonances of (a) unmodified ODN-1 and (b) 5-HO-dC modified ODN-2.

Figure 8

(a) T_M dependence on salt concentration for ODN-1 (④) and ODN-2 (③) in 10 mM sodium phosphate buffer (pH. 7.0); (b) ODN-3 (④), ODN-4 (③) at ~ 8 μM strand concentration.

Figure 9

(a) T_M dependence on osmolyte concentration (function of ethylene glycol) for for ODN-1 (④) and ODN-2 (③); (b) ODN-3 (④), ODN-4 (③) in 10 mM sodium phosphate buffer (pH. 7.0) at ~ 8 μM strand concentration.