Thermodynamic stability of base pairs between 2-hydroxyadenine and incoming nucleotides as a determinant of nucleotide incorporation specificity during replication

Abstract

We investigated the thermodynamic stability of double-stranded DNAs with an oxidative DNA lesion, 2-hydroxyadenine (2-OH-Ade), in two different sequence contexts (5'-GA*C-3' and 5'-TA*A-3', A* represents 2-OH-Ade). When an A*-N pair (N, any nucleotide base) was located in the center of a duplex, the thermodynamic stabilities of the duplexes were similar for all the natural bases except A (N = T, C and G). On the other hand, for the duplexes with the A*-N pair at the end, which mimic the nucleotide incorporation step, the stabilities of the duplexes were dependent on their sequence. The order of stability is T > G > C >> A in the 5'-GA*C-3' sequences and T > A > C > G in the 5'-TA*A-3' sequences. Because T/G/C and T/A are nucleotides incorporated opposite to 2-OH-Ade in the 5'-GA*C-3' and 5'-TA*A-3' sequences, respectively, these results agree with the tendency of mutagenic misincorporation of the nucleotides opposite to 2-OH-Ade in vitro. Thus, the thermodynamic stability of the A*-N base pair may be an important factor for the mutation spectra of 2-OH-Ade.

Figure 1

Oligodeoxyribonucleotides with a 5′-TA*A-3′ or 5′-GA*C-3′ sequence (underlined) used in this study. X denotes A or 2-OH-Ade; N represents A, G, C or T. A particular duplex is indicated as sequence series name followed by X–N base pair as subscript, e.g. [1]_A*T indicates 5′-GGAATATTAGCTGGT-3′/5′-ACCAGCTA*ATATTCC-3′.

Figure 2

Normalized UV melting curves. (A–D) Results for duplexes [1], [2], [3] and [4] in Figure 1, respectively. Melting behavior of control duplexes having A–T base pair as X–N are shown as curves drawn by crosses. Melting curves of duplexes with A*–A, A*–G, A*–C and A*–T are drawn by open squares, filled squares, open circles and filled circles, respectively. Each oligomer concentration was 5 µM, and measurements were done in 1 M NaCl-phosphate buffer pH 7.0.

Figure 3

Summary of stability as ΔG°₃₇ for all 20 model duplexes. (A–D) Results for duplexes [1], [2], [3] and [4], respectively. The data are aligned by the order of –ΔG°₃₇ value. Inset shows ΔΔG°₃₇ values against the ΔG°₃₇ of control duplex with A–T base pair for evaluation of the effect of trinucleotides with 2-OH-Ade.

Figure 4

Molecular modeling of a base pair of 2-OH-Ade and adenine in a tetranucleotide model of AT closing 5′-TA*A-3′ sequence, 5′-CTA*A-3′/5′TA-3′. (A) Top view of the base pair. (B) Side view of the base pair with adjacent bases.