Structural effect of the anticancer agent 6-thioguanine on duplex DNA

Abstract

The incorporation of 6-thioguanine (S6G) into DNA is an essential step in the cytotoxic activity of thiopurines. However, the structural effects of this substitution on duplex DNA have not been fully characterized. Here, we present the solution structures of DNA duplexes containing S6G opposite thymine (S6G.T) and opposite cytosine (S6G.C), solved by high-resolution NMR spectroscopy and restrained molecular dynamics. The data indicate that both duplexes adopt right-handed helical conformations with all Watson-Crick hydrogen bonding in place. The S6G.T structures exhibit a wobble-type base pairing at the lesion site, with thymine shifted toward the major groove and S6G displaced toward the minor groove. Aside from the lesion site, the helices, including the flanking base pairs, are not highly perturbed by the presence of the lesion. Surprisingly, thermal dependence experiments suggest greater stability in the S6G-T mismatch than the S6G-C base pair.

Figure 1

Chemical structure of 6-thioguanine and sequence of lesion containing duplexes (S represents 6-thioguanine and X represents either cytosine or thymine).

Figure 2

Base-to-H1′ region of NOESY spectra of the S6G·C (left) and S6G·T (right) duplexes, recorded in 100% D₂O buffer at 25°C. The spectra were taken at mixing times of 300 and 250 ms for the S6G·C and S6G·T duplexes, respectively. Peaks are labeled D1–D2.

Figure 3

Panel 1: one-dimensional trace labeling imino protons. Panel 2: imino–amino region. Blue boxes enclose crosspeaks due to interaction with cytosine amino protons participating in hydrogen bonds. Red boxes indicate interactions with cytosine amino protons not participating in hydrogen bonds. Green boxes enclose crosspeaks due to interaction with adenine H2 protons. Panel 3: imino–imino region. Crosspeak labeling: (A) S6GH1–G18H1, (B) S6GH1–G16H1. Blue asterisks denote crosspeaks due to stacking interactions with imino protons of adjacent bases. Panels 2 and 3 are regions of a NOESY spectrum of S6GC, recorded in 10% D₂O buffer at 3°C with a mixing time of 180 ms.

Figure 4

Panel 1: one-dimensional trace labeling imino protons. Panel 2: Imino–amino region. Blue boxes enclose crosspeaks due to interaction with cytosine amino protons participating in hydrogen bonds. Red boxes indicate interactions with cytosine amino protons not participating in hydrogen bonds. Green boxes enclose crosspeaks due to interaction with adenine H2 protons. Panel 3: Imino–imino region. Crosspeak labeling: (A) S6GH1–T17H3, (B) G18H1–T17H3, (C) G16H1–T17H3, (D) G18H1–S6GH1, (E) G16H1–S6GH1. Blue asterisks denote crosspeaks due to stacking interactions with imino protons of adjacent bases. Panels 2 and 3 are regions of a NOESY spectrum of S6G·T, recorded in 10% D₂O buffer at 3°C with a mixing time of 220 ms.

Figure 5

Energy minimized averaged structures for S6G·C (left) and S6G·T (right). Blue, S6G; green, C or T opposite S6G.

Figure 6

Base pair configurations. Top left, S6G-C; bottom left, canonical G-C (49); top right, S6G-T; bottom right, G-T (23). Hydrogen bonds are shown in yellow.

Figure 7

Stacking interactions of S6G·C (left panel) and S6G·T (right panel). The top of each panel shows the interactions with the flanking bases above the lesion site (fifth and eighteenth bases), and the bottom shows the interactions with the flanking bases below the lesion (seventh and sixteenth bases).

Figure 8

Temperature dependence experiments. Spectra (3, 15, 35 and 50°C) are depicted for each duplex as labeled. Experiments were carried out in 10% D₂O/90% H₂O buffer phosphate, pH 6.78. Base pairs in the sixth and seventeenth positions (lesion site) are marked with blue asterisks. Flanking guanine residues in the sixteenth and eighteenth positions are marked with red arrows. Terminal base pairs appear in close proximity and are marked with a single green arrow for each duplex.