Interstrand cross-links are preferentially formed at the d(GC) sites in the reaction between cis-diamminedichloroplatinum (II) and DNA.

Abstract

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 ...

X-ray structure and mechanism of RNA polymerase II stalled at an antineoplastic monofunctional platinum-DNA adduct

“…These rules are clearly violated by cationic, monofunctional platinum compounds such as pyriplatin (4,5). Other monofunctional platinum complexes, including ½PtðdienÞCl þ , ½PtðNH 3 Þ 3 Cl þ , and trans-½PtðNH 3 Þ 2 ðpyÞCl þ , are inactive and do not arrest pol II transcription, whereas the cis-fPtðNH 3 Þ 2 ðpyÞg 2þ unit bound to guanosine blocks pol II transcription and has significant anticancer properties in mice when administered as pyriplatin (4,5,8,(24)(25)(26)(27)(28)(29)(30)(31)(32).…”

Identification of genomic DNA sequences bound by mutant p53 protein (Gly245→Ser) in vivo

“…An outline of the strategy applied is shown in Figure 1a. To ensure that only DNA directly in contact with p53 would be cross-linked to mut p53, we used cisplatin as a cross-linker, as cisplatin is known to form DNA-DNA, and DNA-protein cross-links, but no proteinprotein cross-links (Lemaire et al, 1991). After crosslinking, the cells were lysed with detergent and treated with 2 M NaCl to remove chromatin proteins not cross-linked to DNA.…”

A new anticancer platinum compound, (–)-(R)-2-aminomethyl-pyrrolidine(1,1-cyclobutanedicarboxylato) platinum(II): DNA interstrand crosslinking, repair and lethal effects in normal human, Fanconi's anaemia and xeroderma pigmentosum cells

“…Amounts of MC and Pt(II) interstrand crosslinks in the cell DNA are 2-10%, compared to 90% or more intrastrand crosslinks. Therefore, the XPA cells can tolerate a great amount of monoadducts and intrastrand crosslinks by the replication-dependent or postreplication repair pathway, even though the major d(GpG) intrastrand crosslinks effectively block replication in vitro (Ciccarelli et al, 1985;Heiger-Bernays et al, 1990;Lemaire et al, 1991;Iwata et al, 1991). In consequence, interstrand crosslinks are more potentially lethal than intrastrand adducts.…”

“…Thus, DWA2114R appears to be a promising new analog. Both Pt(II) intra-and interstrand crosslinks formed at the specific DNA sequences block DNA synthesis in vitro (Heiger-Bernays et al, 1990;Lemaire et al, 1991;Iwata et al, 1991) and in vivo (Roberts & Friedlos, 1987), and inhibit RNA transcription in vitro (Corda et al, 1991) at damaged sites. Some studies have indicated that the main cytotoxic lesions are abundant Pt(II) intrastrand crosslinks (HeigerBernays et al, 1990;Lepre & Lippard, 1990), while the others have indicated that the minor Pt(II) interstrand crosslinks are more lethal (Knox et al, 1986;Roberts & Friedlos, 1987).…”

“…Total DNA platinations by CDDP consist of 90% or more intrastrand crosslinks in the sequences of 5'-d(GpG)-3' (>65%), 5'-d(ApG)-3' (20-25%) and 5'-d(ApNpG)-3'(-4%; N for any base) after in vitro reaction (Eastman et al, 1985;1987;Lepre & Lippard, 1990), and < 2% interstrand cross-links in DNA in vivo (Fichtinger-Schepman et al, 1985;Knox et al, 1986;Roberts & Friedlos, 1987). Interstrand crosslinking occurs between two dG residues of the complementary 5'-d(GpC)-3' sequences (Lemaire et al, 1991). A rate of in vitro DNA platination by carboplatin possessing a hydrolysisresistant CBDCA group is 100 times slower than that by CDDP (Knox et al, 1986).…”