Laser-induced photo-cross-linking of cisplatin-modified DNA to HMG-domain proteins.
Laser-induced photo-cross-linking was investigated for DNA, modified with cisplatin at specific sites, bound to structure-specific recognition domains of proteins in the high-mobility group (HMG) class. The efficiency of photo-cross-linking depends on the wavelength and power of the laser, the nature of the protein domain, and the oligodeoxyribonucleotide sequences flanking the platinated site. Introduction of 5-iodouridine at thymine sites of the oligodeoxyribonucleotide as an additional photoreactive group did not increase the photo-cross-linking yield. Formation of platinum-mediated DNA-DNA interstrand cross-linking observed previously upon irradiation with 302 nm light [Kane, S. A., and Lippard, S. J. (1996) 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 platinum in the major groove. Studies with proteins containing specifically mutated amino acids, and with DNA probes in which the sequences flanking the platinum cross-link site were varied, suggest that the most effective photo-cross-linking occurs for protein domains bound symmetrically and flexibly to cisplatin-modified DNA. The thermodynamic equilibrium between the protein-platinated DNA complex and its components, revealed in gel electrophoretic mobility shift assays (EMSAs), is significantly shifted to the right upon irreversible 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 an effective tool for investigating the interaction of cisplatin-modified DNA with damage-recognition proteins under heterogeneous conditions such those in cell extracts or living cells.
Mikata
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Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, USA.
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Lippard
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D020029:Base Pairing; D001483:Base Sequence; D002945:Cisplatin / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D018736:DNA Adducts / Q000737:chemistry* / Q000528:radiation effects*; D006609:High Mobility Group Proteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*; D007065:Idoxuridine / Q000031:analogs & derivatives* / Q000737:chemistry; D007834:Lasers; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000528:radiation effects; D009944:Organoplatinum Compounds / Q000737:chemistry / Q000528:radiation effects; D010777:Photochemistry; D017434:Protein Structure, Tertiary / Q000528:radiation effects
D003432:Cross-Linking Reagents; D018736:DNA Adducts; D006609:High Mobility Group Proteins; D015345:Oligonucleotide Probes; D009944:Organoplatinum Compounds; C035991:5-iodouridine; D007065:Idoxuridine; D002945:Cisplatin