DNA bending and unwinding due to the major 1,2-GG intrastrand cross-link formed by antitumor cis-diamminedichloroplatinum(II) are flanking-base independent

Abstract

Antitumor cisplatin [cis-diamminedichloroplatinum(II)] forms on DNA predominantly intrastrand cross-links between neighboring purine residues. Several discoveries suggested that the toxicity of cisplatin originated from these lesions. The formation of 1,2-GG intrastrand cross-link of cisplatin leads to marked conformational alterations in DNA including a directional, rigid bend toward the major groove and local unwinding. These altered structures attract various cellular proteins. This phenomenon has been postulated to mediate antitumor properties of cisplatin. Importantly, the binding affinity of several proteins that specifically recognize 1,2-GG intrastrand cross-link to platinated DNA is modulated by the nature of the base pairs that immediately flank the platinated d(GpG) site. However, the influence of sequence context on DNA bending and unwinding due to the formation of the 1,2-GG intrastrand cross-link has not been extensively investigated. In the present study we have employed electrophoretic retardation (phasing) assay to analyze bending and unwinding induced by the single, site-specific 1,2-GG intrastrand cross-link immediately flanked by various bases formed by cisplatin in nine oligodeoxyribonucleotide duplexes. The results indicate that bending and unwinding of DNA as a consequence of the formation of the major adduct of cisplatin is, in the first approximation, independent of the base pairs flanking the platinated d(GpG) site.

Figure 1

Sequences of the synthetic oligodeoxyribonucleotide duplexes used in the present study with their abbreviations. The top and bottom strands of each pair are designated top and bottom, respectively, in the text. The bold letters in the top strands of the duplexes indicate the location of the intrastrand CL after modification of the oligonucleotides by cisplatin in the way described in the Materials and Methods.

Figure 2

Autoradiogram of the ligation products of double-stranded oligonucleotides AGGA(20–23) containing a unique 1,2-GG intrastrand CL formed by cisplatin between central guanine residues in the top strand. The ligation products were separated on an 8% polyacrylamide gel (lanes Pt). NoPt, unplatinated oligomers,.

Figure 3

(A) Plots showing the relative mobility K versus sequence length curves for the oligomers AGGA(20–23) containing single 1,2-GG intrastrand CL of cisplatin, denoted as 20, 21, 22 and 23, respectively. (B) Plots showing the relative mobility K versus interadduct distance in base pairs for the duplexes AGGA(20–23) intrastrand cross-linked by cisplatin with a total length of 120 bp. The experimental points represent the average of three independent electrophoresis experiments. The curves represent the best fit of these experimental points to the equation K = ad² + bd + c (31).