NMR structure of duplex DNA containing the alpha-OH-PdG.dA base pair: a mutagenic intermediate of acrolein

Abstract

Acrolein, a cell metabolic product and main component of cigarette smoke, reacts with DNA generating alpha-OH-PdG lesions, which have the ability to pair with dATP during replication thereby causing G to T transversions. We describe the solution structure of an 11-mer DNA duplex containing the mutagenic alpha-OH-PdG.dA base pair intermediate, as determined by solution nuclear magnetic resonance (NMR) spectroscopy and retrained molecular dynamics (MD) simulations. The NMR data support a mostly regular right-handed helix that is only perturbed at its center by the presence of the lesion. Undamaged residues of the duplex are in anti orientation, forming standard Watson-Crick base pairs alignments. Duplication of proton signals at and near the damaged base pair reveals the presence of two enantiomeric duplexes, thus establishing the exocyclic nature of the lesion. The alpha-OH-PdG adduct assumes a syn conformation pairing to its partner dA base that is protonated at pH 6.6. The three-dimensional structure obtained by restrained molecular dynamics simulations show hydrogen bond interactions that stabilize alpha-OH-PdG in a syn conformation and across the lesion containing base pair. We discuss the implications of the structures for the mutagenic bypass of acrolein lesions.

Figure 1

Chemical structure of acrolein adducts and sequence composition of the α-OH-PdG•dA duplex.

Figure 2

Expanded region of a 600 MHz NOESY (300 ms mixing time) spectrum recorded at 25°C with the sample dissolved in ‘100%’ D₂O phosphate buffer, pH 6.6, containing 50 mM NaCl and 1 mM EDTA. The figure displays the ‘finger print’ region of the spectrum showing the sequential interactions between base (6.94–8.40 ppm) and the sugar H1’ (5.12–6.34 ppm) protons, from T3 through T9 on the damaged strand (left) and from A14 through A20 on unmodified strand (right). Residue labels indentify intraresidue base-H1’ NOEs, with X6 being that of α-OH-PdG, and asterisks indicate cytosine H6-H5 interactions. Blue and red colors differentiate resolved NOE peaks originated from the S and R enantiomers, respectively. Other labels are assigned as follows: A, X6(Hα)-C5(H6); B, X6(H8)-C7(H1’); C, X6(H8)-C7(H5); D, A4(H2)-C5(H1’); E, A8(H2)-T9(H1’); F, A14(H2)-A14(H1’); G, A14(H2)-G10(H1’); H, A14(H2)-T9(H1’); I, A14(H2)-T15(H1’); J, A17(H2)-A17(H1’); K, A17(H2)-G18(H1’); L, A17(H2)-C7(H1’); M, A20(H2)-A20(H1’); N, A20(H2)-A4(H1’); O, A20(H2)-T3(H1’); P, A20(H2)-C21(H1’); Q, A4(H8)-C5(H5); R, G10(H8)-C11(H5); S, G12(H8)-C13(H5); T, A20(H8)-C21(H5).

Figure 3

Expanded contour plots of regions of a 600 MHz NOESY (300 ms mixing time) spectrum recorded at 25°C with the sample dissolved in ‘100%’ D₂O phosphate buffer, pH 6.6, containing 50 mM NaCl and 1 mM EDTA, depicting NOE interactions of the exocyclic α-OH-PdG protons. Blue and red labels differentiate peaks originated from the S or R enantiomer, respectively. Labels are assigned as follows: A, X6(Hα)-X6(Hγ/γ'); B, X6(Hα)-X6(Hβ/β'); C, X6(Hα)-X6(Hγ/γ'); D, X6(Hα)-X6(Hβ/β'); E, C5(H3’)-X6(Hα); F, C5(H5)-X6(Hγ/γ'); G, C5(H5)-X6(Hγ/γ'); H, X6(Hγ)-X6(Hγ'); I, X6(Hγ)-X6(Hγ'). The geminal β and γ protons of α-OH-PdG were not stereo specifically assigned.

Figure 4

Expanded regions of a 600 MHz NOESY (220 ms mixing time) spectrum recorded at 5°C with the α-OH-PdG•dA duplex dissolved in 10% D₂O phosphate buffer, pH 6.6, containing 10 mM phosphate, 50 mM NaCl and 1 mM EDTA, and a 1D trace shown on top. The figure depicts NOE interactions involving the exchangeable protons. Blue and red labels differentiate peaks originated from the S and R enantiomers, respectively. Labeled peaks are assigned as follows: A, T9(N3H)-A14(H2); B, T3(N3H)-A20(H2); C, T15(N3H)-A8(H2); D, T19(N3H)-A4(H2); E, G18(N1H)-C5(N4H)_hb; E’, G18(N1H)-C5(N4H)_nhb; F, G2(N1H)-C21(N4H)_hb; F’, G2(N1H)-C21(N4H)_nhb; G, G10(N1H)-C13(N4H)_hb; G’, G10(N1H)-C13(N4H)_nhb; H, G16(N1H)-C7(N4H)_hb; H’, G16(N1H)-C7(N4H)_nhb; I, G18(N1H)-A4(H2); J, G18(N1H)-A17(N6H)_hb; K, G2(N1H)-A20(H2); L, G10(N1H)-A14(H2); M, G16(N1H)-A17(H2); N, G18(N1H)-A8(H2); O, A17(N4H)_hb-A17(N4H)_nhb; P, A17(N4H)_hb-C5(N4H)_hb; P’, A17(N4H)_nhb-C5(N4H)_hb; Q, A17(N4H)_hb-C5(N4H)_nhb; Q’, A17(N4H)_nhb-C5(N4H)_nhb; R, X6(N2H)-X6(Hα). Hb and nhb stand for hydrogen bonded and non-hydrogen-bonded.

Figure 5

Stick-ribbon representation of isomeric α-OH-PdG•dA duplex structures shown with the major groove prominent. Atoms are colored by type with the backbone in green.

Figure 6

Close up view of the central three base pair fragment depicting hydrogen bond interactions that stabilize the syn conformation of α-OH-PdG. For clarity, the chain orientation in this figure is different from that in Figure 5.

Figure 7

Hydrogen bonds and staking interactions at the lesion site of the α-OH-PdG•dA duplex.