Photoreactivity of platinum(II) in cisplatin-modified DNA affords specific cross-links to HMG domain proteins

Abstract

Cisplatin-modified DNA forms specific complexes with proteins that contain the DNA binding motif known as the high-mobility group (HMG) domain. As a tool for investigating the role of these proteins in mediating the cytotoxic effects of cisplatin, a set of cisplatin analogs was prepared in which one of the ammine ligands was replaced with a photoreactive tethered aryl azide ligand. The ability of DNA modified by these platinum complexes to photo-cross-link to HMG1 was investigated. During this study, it was discovered that DNA modified with cisplatin itself can undergo photoinduced cross-linking to HMG1 when irradiated with 300 nm light. The covalent complexes resulting from this latter cross-linking reaction are completely reversed by the addition of sodium cyanide and can be degraded by proteinase K. These results confirm the presence of a protein-DNA cross-link and demonstrate that the platinum atom itself forms the point of attachment. By contrast, DNA modified with transdiamminedichloroplatinum(II), [Pt(dien)Cl]Cl, or [Pt(NH3)3Cl]Cl does not cross-link to HMG1 upon irradiation. The photochemistry was exploited to cross-link a 15-base pair oligonucleotide containing a single, site-specific cis-[Pt(NH3)2{d(GpG)-N7(1),-N7(2)}] intrastrand adduct to domain B of HMG1. Following proteolytic digestion of the resulting covalent complex, the site of attachment to the protein was determined by Edman degradation of the resulting peptide-DNA complex to be a single residue on HMG domain B, Lys-6. The data further suggest that this amino acid binds to platinum at a site made available by photolabilization of a purine ligand. These results afford the first structural information about the interaction of HMG domain proteins with cisplatin-modified DNA.