The major adduct of the antitumor drug cis-diamminedichloroplatinum(II) with DNA bends the duplex by approximately equal to 40 degrees toward the major groove.

Abstract

We used gel electrophoresis methods to show that reaction of DNA with cis-diamminedichloroplatinum (II) results in a substantial (--40°) bend in the double helix at the intrastrand crosslink between the N7 atoms of adjacent guanosine nucleosides, which bond to the platinum(II) complex with loss of two chloride ions. Multimers of a 22-base-pair (bp) oligonucleotide platinated at a single site show strong anomalies in their electrophoretic mobilities as a consequence of coherent addition of in-phase platinum-induced bends. Increase of the sequence repeat of the platinated site to 27 bp yields multimers of nearly normal mobility because the bends are out of phase. MATERIALS AND METHODS Oligonucleotides were synthesized on an Applied Biosystems model 380B DNA synthesizer and purified by reversephase HPLC. The strand (Fig. 1) containing adjacent guanines was platinated with cis-[Pt(NH3)2(H2O)2 ' by standard methods (16). After the crude reaction mixture was purified by reverse-phase HPLC, the single major product was shown by flameless atomic absorption spectroscopy to have 0.98 platinum atom per strand. From the sequence, which was chosen to contain two adjacent guanosines and no other purines, and from the known chemistry of cisplatin with oligonucleotides (5), this adduct may be safely concluded to be the intrastrand d(pGpG) crosslink. Complementary strands were labeled with 32P at the 5' terminus, hybridized, and ligated as described (14).RESULTS AND DISCUSSION Multimers of the platinated 22-base-pair (bp) duplex oligonucleotide shown in Fig. 1, designated P, were used to investigate Pt-induced DNA bending. Since cisplatin unwinds the DNA double helix (17)(18)(19), an 11-bp repeat (slightly more than the normal helix repeat of 10.5 bp) was selected to optimize the match between the sequence repeat and the expected helix screw. The ligation reaction yielded a mixture of circular and linear oligomers; these products separated into two intersecting ladders in nondenaturing polyacrylamide electrophoresis gels (Fig. 2). The smallest circular product (CS) was excised from the gel, denatured, and run on a 20%6 (20:1, acrylamide/methylenebisacrylamide) denaturing gel containing 7 M urea. The sample contained predominantly intact and oncenicked single strands (data not shown); the mobility of the latter corresponded to a length of 110 bp, representing a 5-mer of the original monomer sequence. The predominance of circular products for multimers larger than 100 bp is consistent with strong DNA bending and confirms that there is a close match between the 22-bp sequence repeat and the helix screw (20, 21).The comparative mobilities of different-length fragments generated by self-ligation of the 22-bp platinated monomer P are plotted in Fig. 3. The ratio RL of apparent size (determined by comparison with the BamHI ladder) to true size increases dramatically with DNA length, resembling the electrophoretic behavior ofbent DNA molecules in which the sequence repeat closely matches the double helix screw repeat (14). ...

Crystal Structure of the Anticancer Drug Cisplatin Bound to Duplex DNA

“…This work confirmed the conversion of the 5' platinated guanosine from C2'-endo to C3'-endo found in numerous earlier NMR studies of the cis-{Pt(NH 3 ) 2 } 2 + 1,2-intrastrand d(GpG) cross-link (Sherman & Lippard, 1987). The bend angle was 580, in agreement with molecular mechanics studies (Kozelka & Chottard, 1990), but considerably larger than the 32°-40' angle estimated by gel electrophoresis studies (Bellon & Lippard, 1990;Rice et al, 1988) and the 2.6 A crystal structure (Takahara et al, 1995). The NMR solution structure has many features in common with the present crystal structure determination, The crystal structure also reveals a potential for drug design with this complex.…”

Cisplatin-DNA damage recognition proteins in human tumour extracts

“…It has been further suggested that these proteins do not recognise specific adducts, but bind to sites where damage has caused a conformational change in the DNA (Donahue et al, 1990;Bruhn et al, 1992). An example of such conformational change may be the 40°bend produced by an intrastrand crosslink between adjacent guanines (Rice et al, 1988). Support for the role of damage recognition proteins in DNA repair has come from the demonstration that a protein which binds specifically to UV-damaged DNA can restore the DNA repair capacity of cell extracts from repair deficient xeroderma pigmentosum cell lines of complementation group A (Robins et al, 1991).…”

Human REV1 Modulates the Cytotoxicity and Mutagenicity of Cisplatin in Human Ovarian Carcinoma Cells

“…Most mutations induced by UV irradiation are generated when DNA containing residual unrepaired damage is replicated during S phase of the cell cycle. Such lesions perturb the structure of DNA (Rice et al, 1988;Park et al, 2002) and are likely to block replicative DNA polymerases that have stringent base-pairing requirements. In Saccharomyces cerevisiae, mutagenic bypass of DNA damage is equivalent to error-prone translesion replication (Broomfield et al, 2001).…”