Laser-induced photo-cross-linking of cisplatin-modified DNA to HMG-domain proteins

Laser-induced photo-cross-linking was investigated for DNA, modified with c isplatin at specific sites, bound to structure-specific recognition domains of proteins in the high-mobility group (HMG) class. The efficiency of phot o-cross-linking depends on the wavelength and power of the laser,. the natu re of the protein domain, and the oligodeoxyribonucleotide sequences flanki ng the platinated site. Introduction of 5-iodouridine at thymine sites of t he oligodeoxyribonucleotide as an additional photoreactive group did not in crease the photo-cross-linking yield. Formation of platinum-mediated DNA-DN A interstrand crosslinking observed previously upon irradiation with 302 nm light [Kane, S. A., and Lippard, S. J. (1995) Biochemistry 35, 2180-2188] was significantly reduced with laser irradiation. HMG1 domain B is superior to domain A for platinum-mediated photo-cross-linking, a result attributed to the different positioning of the proteins with respect to the platinum adduct and the greater ability of domain B to access photolabilized platinu m in the major groove. Studies with proteins containing specifically mutate d;amino acids, and with DNA probes in which the sequences flanking the plat inum cross-link site were varied, suggest that the most effective photo-cro ss-linking occurs for protein domains bound symmetrically and flexibly to c isplatin-modified DNA. The thermodynamic equilibrium between the protein-pl atinated DNA complex and its components, revealed in gel electrophoretic mo bility shift assays (EMSAs), is significantly shifted to the right upon irr eversible photo-cross-linking. Thus, only upon photo-cross-linking can the interaction of cisplatin-DNA 1,3-intrastrand d(GpTpG) or interstrand cross- links with HMG1 domain B protein be detected. Photo-cross-linking is thus a n effective tool for investigating the interaction of cisplatin-modified DN A with damage-recognition proteins under heterogeneous conditions such thos e in cell extracts or living cells.