Bases in the opposite strands of DNA crosslinked by dinicaily ineffective trans-diamminedichloroplatinum(ll) {trans-[Pt(NH3)2C121} have been identified by means of three experimental approaches. These include HPLC analysis of enzymatic digests of synthetic oligonucleotide duplexes containing the interstrand cross-link, footprinting experiments on the interstrand cross-linked oligonucleotide duplexes, and termination of the duplex transcription on trans-[Pt(NH3)2C12J- However, it binds to DNA and forms intrastrand and interstrand cross-links (1-4). Stereochemical limitations preclude trans-[Pt(NH3)2Cl2] from forming intrastrand cross-links between adjacent base residues. Therefore, it has been speculated that the differences in antitumor activity of the two platinum(II) isomers may arise from the different nature of distortions induced in DNA by the intrastrand lesions.Both isomers form DNA interstrand cross-links (ICLs). Although these lesions make up only a small fraction of all adducts, correlation between interstrand cross-linking by cis-[Pt(NH3)2C12] and its cytotoxicity has been reported (5, 6). Recently, it was shown (7) that after cis-[Pt(NH3)2Cl2] treatment several genes in the cells resistant to this drug and parental cells had similar initial contents of intrastrand and interstrand cross-links. However, the ICLs were removed more efficiently in the resistant than in the parental cell lines. These results suggest that acquired cellular resistance to cis-[Pt(NH3)2CI2] may be associated with an increased DNA The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.repair efficiency of the ICLs (7). The ICLs of cis-[Pt(NH3)2C12] are preferentially formed between guanine residues at the 5'-GC-3' sites (8, 9). They bend the double helix by --55°toward the major groove, and the distortion is localized at the platinated sequence d(GC/GC) (10 MATERIALS AND METHODSThe synthesis and purification of the single-stranded oligodeoxyribonucleotides (the top and bottom strands in the duplexes shown in Fig. 1) have already been described (11). Plasmid pSP73KB was prepared as described (9). SP6 and T7 RNA polymerases, ribonucleotide triphosphates, and RNasin ribonuclease inhibitor were from Promega. 3'-Deoxynucleotide triphosphates were purchased from Pharma-Waldhof (Dusseldorf, Germany). Restriction enzymes, Klenow fragment of DNA polymerase I, T4 DNA polymerase, and T4 polynucleotide kinase were from Boehringer Mannheim and Bethesda Research Laboratories. All radioactive products were from Amersham. Electrophoresis-grade acrylamide, N,N'-methylenebisacrylamide, agarose, sodium cyanide, thiourea, and dimethyl sulfate (DMS) were from Merck. cisand trans-[Pt(NH3)2Cl2] were from Lachema (Brno, Czech Republic). Monoaquamonochloro derivatives were generated by allowing cis-and trans-[Pt(NH3)2Cl2] to react with AgNO3 (12).The oligonucleotide duplexes containing the IC...

A DNA restriction fragment with convergent SP6 and T7 promoters has undergone reaction with cisdiamminedichloroplatinum(II) (cis-DDP) and was then used as a template for RNA synthesis in vitro. The T7 and SP6 RNA polymerases generate fragments of defmed sizes. Analysis of the RNA fragments shows that the polymerases are mainly blocked at the level of the d(GG) and d(AG) sites and to a lesser extent at the level of the d(GC) sites. The adducts at the d(GC) sites are more resistant to cyanide ion attack than those at the major sites and are identified as interstrand cross-links. The formation of an interstrand cross-link between the N-7 atoms of two guanine residues at the d(GC) sites was further confirmed by chemical modifications.cis-Diamminedichloroplatinum(II) (cis-DDP) is an antitumor agent of major clinical importance. Much evidence suggests that the cytotoxic action of the drug is related to its ability to react with DNA even though the mechanism of action is still unknown (for general reviews, see refs. 1-5 and references therein). Like many chemicals used in cancer chemotherapy, cis-DDP is a bifunctional agent that forms in vivo and in vitro intrastrand and interstrand cross-links on DNA. The major lesions are d(GG) and d(AG) 1,2-intrastrand cross-links, representing 65% and 25% of the total adducts, respectively (1-5). Although the interstrand cross-links represent a minor portion (<10% of total adducts), several experiments in tissue culture systems have correlated the DNA interstrand cross-linking reaction with cytotoxicity (for general reviews, see refs. 6 and 7).DNA interstrand cross-linking occurs predominantly between two guanine N-7 atoms on opposite strands (2,8,9).A distance of -3 A is required for the cis-DDP cross-linking reaction (10, 11). Thus, two adjacent guanine residues on the opposite strands, either in the 5'-CG-3' or 5'-GC-3' sequences, are the most probable reaction sites on DNA. However, in both cases, formation of the cross-link in B-DNA implies a large distortion of the double helix since the two N-7 atoms in d(CG) and d(GC) sequences are separated by about 9 and 7 A (12), respectively. Recently, manipulation of three-dimensional molecular models inferred that the d(CG) sequence is more able to match the interstrand crosslinking requirement (2,8).In vitro studies have shown that DNA synthesis by DNA polymerases of different origins acting on cis-DDP-modified DNAs was arrested at the level of the adducts (1-5, 13-16). This finding has been extensively used to map the sites of platination on DNA. However, in these studies, the interstrand adducts were not detected because either the platinated DNA was single stranded or the assay was not sensitive enough.We show here that the in vitro RNA synthesis by bacteriophage RNA polymerase acting on platinated DNA is blocked at the level of the adducts. The ability of the adducts to terminate transcription has been used to map the cis-DDP binding sites on a double-stranded DNA. Furthermore, analysis of the modification patterns shows ...

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cis-diamminedichloroplatinum (II) (cisplatin) is a powerful anti-tumor drug whose target is cellular DNA. In the reaction between DNA and cisplatin, covalent intrastrand and interstrand cross-links (ICL) are formed. Two solution structures of the ICL have been published recently. In both models the double-helix is bent and unwound but with significantly different angle values. We solved the crystal structure at 100K of a double-stranded DNA decamer containing a single cisplatin ICL, using the anomalous scattering (MAD) of platinum as a unique source of phase information. We found 47 degrees for double-helix bending and 70 degrees for unwinding in agreement with previous electrophoretic assays. The crystals are stabilized by intermolecular contacts involving two cytosines extruded from the double-helix, one of which makes a triplet with a terminal G.C pair. The platinum coordination is nearly square and the platinum residue is embedded into a cage of nine water molecules linked to the cross-linked guanines, to the two amine groups, and to the phosphodiester backbone through other water molecules. This water molecule organization is discussed in relation with the chemical stability of the ICL.