Solution structures of a DNA dodecamer duplex with and without a cisplatin 1,2-d(GG) intrastrand cross-link: comparison with the same DNA duplex containing an oxaliplatin 1,2-d(GG) intrastrand cross-link

Abstract

Proteins that discriminate between cisplatin-DNA adducts and oxaliplatin-DNA adducts are thought to be responsible for the differences in tumor range, toxicity, and mutagenicity of these two important chemotherapeutic agents. However, the structural basis for differential protein recognition of these adducts has not been determined and could be important for the design of more effective platinum anticancer agents. We have determined high-resolution NMR structures for cisplatin-GG and undamaged DNA dodecamers in the AGGC sequence context and have compared these structures with the oxaliplatin-GG structure in the same sequence context determined previously in our laboratory. This structural study allows the first direct comparison of cisplatin-GG DNA and oxaliplatin-GG DNA solution structures referenced to undamaged DNA in the same sequence context. Non-hydrogen atom rmsds of 0.81 and 1.21 were determined for the 15 lowest-energy structures for cisplatin-GG DNA and undamaged DNA, respectively, indicating good structural convergence. The theoretical NOESY spectra obtained by back-calculation from the final average structures showed excellent agreement with the experimental data, indicating that the final structures are consistent with the NMR data. Several significant conformational differences were observed between the cisplatin-GG adduct and the oxaliplatin-GG adduct, including buckle at the 5' G6.C19 base pair, opening at the 3' G7.C18 base pair, twist at the A5G6.T20C19 base pair step, slide, twist, and roll at the G6G7.C19C18 base pair step, slide at the G7C8.C18G17 base pair step, G6G7 dihedral angle, and overall bend angle. We hypothesize that these conformational differences may be related to the ability of various DNA repair proteins, DNA binding proteins, and DNA polymerases to discriminate between cisplatin-GG and oxaliplatin-GG adducts.

FIGURE 1

Duplex DNA sequence used in this study. The underlined bases show the position of the platinum adduct. Chemical structures of cisplatin and oxaliplatin.

FIGURE 2

HPLC–UV–MS chromatogram of digestion products obtained from an undamaged 12-mer duplex (I) and from the same 12-mer duplex containing the CP–GG intrastrand cross-link. The spectra include (I) the HPLC–UV elution profile of the digestion products obtained from the undamaged 12-mer duplex, (II)A the HPLC–UV elution profile of the digestion products obtained from the 12-mer duplex containing the CP–GG adduct [the peak with the asterisk corresponds to the elution position of a CP–d(GpG) standard], and (II)B MS-positive mass spectrum of the peak identified with the asterisk in Figure 2(II)A.

FIGURE 3

Expanded nonexchangeable proton regions taken from the 2D NOESY (200 ms) spectrum acquired in D₂O for the CP–GG 12-mer duplex. The H6/H8–H2′/H2″ (top) and H6/H8–H1′ (bottom) regions are shown. The left and right regions correspond to the GG (the strand containing the Pt–GG adduct) and CC (complementary) strands, respectively.

FIGURE 4

Effect of platination on chemical shifts of selected base and sugar proton chemical shifts. The values were determined by using experimental chemical shifts (Tables S1 and S2 and ref 26).

FIGURE 5

Expanded imino region from 1D ¹H NMR spectra of the CP–DNA (top) and OX–DNA (bottom) (26) duplexes recorded in H₂O buffer at various temperatures. The positions of the nucleotides in the 12-mer duplexes that give rise to the resonances are indicated. The asterisk in the 1D ¹H NMR spectra of the CP–DNA adduct indicates the terminal guanine (G24). The experimental temperatures are shown at the left.

FIGURE 6

Expanded region of the DQF-COSY spectra showing the H1′–H2′ and H1′–H2″ cross-peaks of the undamaged DNA and CP–DNA duplex at 25 °C in H₂O. Assigned peaks are labeled.

FIGURE 7

(A) Stereoviews of the average solution structures of the CP–DNA duplex (left) and undamaged DNA (right) in the AGGC sequence context. The platinum atom in the CP–DNA adduct is shown as a yellow sphere. (B) Comparison of the central 4 bp of the CP–DNA adduct (green) with those of the undamaged DNA (blue) and OX–DNA adduct (red) (26).